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New research suggests that gingival stem cells can be used in tissue regeneration

Posted: July 18, 2013 at 6:49 pm

Public release date: 18-Jul-2013 [ | E-mail | Share ]

Contact: Ingrid L. Thomas ithomas@iadr.org 703-299-8084 International & American Associations for Dental Research

Alexandria, Va., USA Today, the International and American Associations for Dental Research (IADR/AADR) published a paper titled "Gingivae Contain Neural-crest- and Mesoderm-derived Mesenchymal Stem Cells." The paper, written by lead author Songtao Shi, Center for Craniofacial Molecular Biology, Ostrow School of Dentistry, University of Southern California, Los Angeles, USA, is published in the OnlineFirst portion of the IADR/AADR Journal of Dental Research.

Gingivae represent a unique soft tissue that serves as a biological barrier to cover the oral cavity side of the maxilla and mandible. Recently, the gingivae were identified as containing mesenchymal stem cells (GMSCs). However, it is unknown whether the GMSCs are derived from cranial neural crest cells (CNCC) or the mesoderm.

In this study, Shi and his team of researchers demonstrated that around 90 percent of GMSCs are derived from CNCC and 10 percent from the mesoderm. In comparison with mesoderm MSCs (M-GMSCs), CNCC-derived GMSCs (N-GMSCs) show an elevated capacity to differentiate into neural cells and chondrocytes as well as to modulate immune cells. When transplanted into mice with dextran sulfate sodium-induced colitis, N-GMSCs showed superior effects in ameliorating inflammatory-related disease phenotype in comparison with the M-GMSC treatment group.

Further research is required to understand the interaction between the neural crest cell derived and mesoderm derived gingivae mesenchymal stem cells (N-GMSCs and M-GMSCs) in terms of their functional roles in gingival immune defense and wound healing.

"The tooth and surrounding tissues are a rich source of stem cells, and this JDR manuscript demonstrates that gingivae contain highly proliferative stem cells from two different embryonic origins and that these cells exhibit distinct behaviors," said JDR Associate Editor Jacques Nr. "These results suggest that gingivae, an easily accessible tissue, are an attractive source for stem cells that can be used in tissue regeneration."

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Visit http://jdr.sagepub.com/content/early/recent to read the JDR manuscript titled "Gingivae Contain Neural-crest- and Mesoderm-derived Mesenchymal Stem Cells" or contact Ingrid L. Thomas at ithomas@iadr.org to request the PDF.

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New research suggests that gingival stem cells can be used in tissue regeneration

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Dental research: Gingival stem cells can be used in tissue regeneration

Posted: July 18, 2013 at 6:49 pm

July 18, 2013 Gingivae represent a unique soft tissue that serves as a biological barrier to cover the oral cavity side of the maxilla and mandible. Recently, the gingivae were identified as containing mesenchymal stem cells (GMSCs). However, it is unknown whether the GMSCs are derived from cranial neural crest cells (CNCC) or the mesoderm.

Today, the International and American Associations for Dental Research (IADR/AADR) published a paper titled "Gingivae Contain Neural-crest- and Mesoderm-derived Mesenchymal Stem Cells." The paper, written by lead author Songtao Shi, Center for Craniofacial Molecular Biology, Ostrow School of Dentistry, University of Southern California, Los Angeles, USA, is published in the OnlineFirst portion of the IADR/AADR Journal of Dental Research.

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Dental research: Gingival stem cells can be used in tissue regeneration

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TAYSHA GENE THERAPIES, INC. Management’s Discussion and Analysis of Financial Condition and Results of Operations. (form 10-Q) – Marketscreener.com

Posted: August 14, 2022 at 2:02 am

The following discussion and analysis of our financial condition and results ofoperations should be read in conjunction with our unaudited condensedconsolidated financial statements and related notes included in this QuarterlyReport on Form 10-Q and the audited financial statements and notes thereto as ofand for the year ended December 31, 2021 and the related Management's Discussionand Analysis of Financial Condition and Results of Operations, included in ourAnnual Report on Form 10-K for the year ended December 31, 2021, or AnnualReport, filed with the Securities and Exchange Commission, or the SEC, on March31, 2022. Unless the context requires otherwise, references in this QuarterlyReport on Form 10-Q to "we," "us," and "our" refer to Taysha Gene Therapies,Inc. together with its consolidated subsidiaries.

Forward-Looking Statements

The information in this discussion contains forward-looking statements andinformation within the meaning of Section 27A of the Securities Act of 1933, asamended, or the Securities Act, and Section 21E of the Securities Exchange Actof 1934, as amended, or the Exchange Act, which are subject to the "safe harbor"created by those sections. These forward-looking statements include, but are notlimited to, statements concerning our strategy, future operations, futurefinancial position, future revenues, projected costs, prospects and plans andobjectives of management. The words "anticipates," "believes," "estimates,""expects," "intends," "may," "plans," "projects," "will," "would" and similarexpressions are intended to identify forward-looking statements, although notall forward-looking statements contain these identifying words. We may notactually achieve the plans, intentions, or expectations disclosed in ourforward-looking statements and you should not place undue reliance on ourforward-looking statements. Actual results or events could differ materiallyfrom the plans, intentions and expectations disclosed in the forward-lookingstatements that we make. These forward-looking statements involve risks anduncertainties that could cause our actual results to differ materially fromthose in the forward-looking statements, including, without limitation, therisks set forth in Part II, Item 1A, "Risk Factors" in this Quarterly Report onForm 10-Q and Part II, Item 1A, "Risk Factors" in our Annual Report. Theforward-looking statements are applicable only as of the date on which they aremade, and we do not assume any obligation to update any forward-lookingstatements.

Note Regarding Trademarks

All brand names or trademarks appearing in this report are the property of theirrespective holders. Unless the context requires otherwise, references in thisreport to the "Company," "we," "us," and "our" refer to Taysha Gene Therapies,Inc.

Overview

We are a patient-centric gene therapy company focused on developing andcommercializing AAV-based gene therapies for the treatment of monogenic diseasesof the central nervous system, or CNS, in both rare and large patientpopulations. We were founded in partnership with The University of TexasSouthwestern Medical Center, or UT Southwestern, to develop and commercializetransformative gene therapy treatments. Together with UT Southwestern, we areadvancing a deep and sustainable product portfolio of gene therapy productcandidates, with exclusive options to acquire several additional developmentprograms at no cost. By combining our management team's proven experience ingene therapy drug development and commercialization with UT Southwestern'sworld-class gene therapy research capabilities, we believe we have created apowerful engine to develop transformative therapies to dramatically improvepatients' lives. In March 2022, we announced strategic pipeline prioritizationinitiatives focused on GAN and Rett syndrome. We will conduct smallproof-of-concept studies in CLN1 disease and SLC13A5 deficiency. Development ofthe CLN7 program will continue in collaboration with existing partners withfuture clinical development to focus on the first-generation construct.Substantially all other research and development activities have been paused toincrease operational efficiency.

In April 2021, we acquired exclusive worldwide rights to TSHA-120, aclinical-stage, intrathecally dosed AAV9 gene therapy program for the treatmentof giant axonal neuropathy, or GAN. A Phase 1/2 clinical trial of TSHA-120 isbeing conducted by the National Institutes of Health, or NIH, under an acceptedinvestigational new drug application, or IND. We reported clinical safety andfunctional MFM32 data from this trial for the highest dose cohort of 3.5E14total vg in January 2022, where we saw continued clinically meaningful slowingof disease progression similar to that achieved with the lower dose cohorts,which we considered confirmatory of disease modification. We recently completeda commercially representative GMP batch of TSHA-120 which demonstrated that thepivotal lots from the commercial grade material were generally analyticallycomparable to the original clinical trial material. Release testing for thisbatch is currently underway and expected to be completed in September 2022.Additional discussions with Health Authorities are planned to discuss thesecomparability data and a potential registration pathway with feedbackanticipated by the end of 2022. For Rett syndrome, we submitted a Clinical TrialApplication, or CTA, filing to Health Canada in November 2021 and announcedinitiation of clinical development of TSHA-102 under the approved CTA in March2022. We expect to report preliminary clinical data for TSHA-102 in Rettsyndrome by year-end 2022. We recently executed an exclusive option from UTSouthwestern to license worldwide rights to a clinical-stage CLN7 program. TheCLN7 program is currently in a Phase 1 clinical

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proof-of-concept trial run by UT Southwestern, and we reported preliminaryclinical safety data for the first patient in history to be intrathecally dosedat 1.0x1015 total vg with the first-generation construct in December 2021.Development of the CLN7 program will continue in collaboration with existingpartners with future clinical development to focus on the first-generationconstruct. We will conduct small proof-of-concept studies in CLN1 disease andSLC13A5 deficiency that we believe can further validate our platform.

We have a limited operating history. Since our inception, our operations havefocused on organizing and staffing our company, business planning, raisingcapital and entering into collaboration agreements for conducting preclinicalresearch and development activities for our product candidates. All of our leadproduct candidates are still in the clinical or preclinical developmentstage. We do not have any product candidates approved for sale and have notgenerated any revenue from product sales. We have funded our operationsprimarily through the sale of equity, raising an aggregate of $319.0 million ofgross proceeds from our initial public offering and private placements of ourconvertible preferred stock as well as sales of common stock pursuant to ourSales Agreement (as defined below). In addition, we drew down $30.0 million and$10.0 million in term loans on August 12, 2021 and December 29, 2021,respectively.

On August 12, 2021, or the Closing Date, we entered into a Loan and SecurityAgreement, or the Term Loan Agreement, with the lenders party thereto from timeto time, or the Lenders and Silicon Valley Bank, as administrative agent andcollateral agent for the Lenders, or the Agent. The Term Loan Agreement providesfor (i) on the Closing Date, $40.0 million aggregate principal amount of termloans available through December 31, 2021, (ii) from January 1, 2022 untilSeptember 30, 2022, an additional $20.0 million term loan facility available atthe Company's option upon having three distinct and active clinical stageprograms, determined at the discretion of the Agent, at the time of draw, (iii)from October 1, 2022 until March 31, 2023, an additional $20.0 million term loanfacility available at our option upon having three distinct and active clinicalstage programs, determined at the discretion of the Agent, at the time of drawand (iv) from April 1, 2023 until December 31, 2023, an additional $20.0 millionterm loan facility available upon approval by the Agent and the Lenders, or,collectively, the Term Loans. We drew $30.0 million in term loans on the ClosingDate and drew an additional $10.0 million term loan on December 29, 2021. Theloan repayment schedule provides for interest only payments until August 31,2024, followed by consecutive monthly payments of principal and interest. Allunpaid principal and accrued and unpaid interest with respect to each term loanis due and payable in full on August 1, 2026.

Since our inception, we have incurred significant operating losses. Our netlosses were $84.0 million for the six months ended June 30, 2022 and $73.0million for the six months ended June 30, 2021. As of June 30, 2022, we had anaccumulated deficit of $319.6 million. We expect to continue to incursignificant expenses and operating losses for the foreseeable future. Weanticipate that our expenses will increase significantly in connection with ourongoing activities, as we:

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Our Pipeline

We are advancing a deep and sustainable product portfolio of gene therapyproduct candidates for monogenic diseases of the CNS in both rare and largepatient populations, with exclusive options to acquire several additionaldevelopment programs at no cost. Our portfolio of gene therapy candidatestargets broad neurological indications across three distinct therapeuticcategories: neurodegenerative diseases, neurodevelopmental disorders and geneticepilepsies. Our current pipeline, including the stage of development of each ofour product candidates, is represented in the table below:

TSHA-120 for Giant Axonal Neuropathy (GAN)

In March 2021, we acquired the exclusive worldwide rights to a clinical-stage,intrathecally dosed AAV9 gene therapy program, now known as TSHA-120, for thetreatment of giant axonal neuropathy, or GAN, pursuant to a license agreementwith Hannah's Hope Fund for Giant Axonal Neuropathy, Inc., or HHF. Under theterms of the agreement, HHF received an upfront payment of $5.5 million and willbe eligible to receive clinical, regulatory and commercial milestones totalingup to $19.3 million, as well as a low, single-digit royalty on net sales uponcommercialization of TSHA-120.

GAN is a rare autosomal recessive disease of the central and peripheral nervoussystems caused by loss-of-function gigaxonin gene mutations. There are anestimated 5,000 affected GAN patients in addressable markets.

Symptoms and features of children with GAN usually develop around the age offive years and include an abnormal, wide based, unsteady gait, weakness and somesensory loss. There is often associated dull, tightly curled, coarse hair, giantaxons seen on a nerve biopsy, and spinal cord atrophy and white matterabnormality seen on MRI. Symptoms progress and as the children grow older theydevelop progressive scoliosis and contractures, their weakness progresses to thepoint where they will need a wheelchair for mobility, respiratory musclestrength diminishes to the point where the child will need a ventilator (usuallyin the early to mid-teens) and the children often die during their late teens orearly twenties, typically due to respiratory failure. There is an early- andlate-onset phenotype associated with the disease, with shared physiology. Thelate-onset phenotype is often categorized as Charcot-Marie-Tooth Type 2, orCMT2, with a lack of tightly curled hair and CNS symptoms with relatively slowprogression of disease. This phenotype represents up to 6% of all CMT2diagnosis. In the late-onset population, patients have poor quality of life butthe disease is not life-limiting. In early-onset disease, symptomatic treatmentsattempt to maximize physical development and minimize the rate of deterioration.Currently, there are no approved disease-modifying treatments available.

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TSHA-120 is an AAV9 self-complementary viral vector encoding the full lengthhuman gigaxonin protein. The construct was invented by Dr. Steven Gray and isthe first AAV9 gene therapy candidate to deliver a functional copy of the GANgene under the control of a JeT promoter that drives ubiquitous expression.

We have received orphan drug designation and rare pediatric disease designationfrom the U.S. Food and Drug Administration, or the FDA, for TSHA-120 for thetreatment of GAN. In April 2022, we received orphan drug designation from theEuropean Commission for TSHA-120 for the treatment of GAN.

There is an ongoing longitudinal prospective natural history study being led bythe NIH, that has already identified and followed a number of patients with GANfor over five years with disease progression characterized by a number ofclinical assessments. The GAN natural history study was initiated in 2013 andincluded 45 patients with GAN, aged 3 to 21 years. Imaging data from this studyhave demonstrated that there are distinctive increased T2 signal abnormalitieswithin the cerebellar white matter surrounding the dentate nucleus of thecerebellum, which represent one of the earliest brain imaging findings inindividuals with GAN. These findings precede the more widespread periventricularand deep white matter signal abnormalities associated with advanced disease. Inaddition, cortical and spinal cord atrophy appeared to correspond to moreadvanced disease severity and older age. Impaired pulmonary function in patientswith GAN also was observed, with forced vital capacity correlating well withseveral functional outcomes such as the MFM32, a validated 32-item scale formotor function measurement developed for neuromuscular diseases. Nocturnalhypoventilation and sleep apnea progressed over time, with sleep apnea worseningas ambulatory function

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deteriorated. Total MFM32 score also correlated with ambulatory status, whereindependently ambulant individuals performed better and had higher MFM32 scoresthan the non-ambulant group, as shown in the graph below.

Patients also reported significant autonomic dysfunction based on the COMPASS 31self-assessment questionnaire. In addition, nerve conduction functiondemonstrated progressive sensorimotor polyneuropathy with age. As would beexpected for a neurodegenerative disease, younger patients have higher baselineMFM32 scores. However, the rate of decline in the MFM32 scores demonstratedconsistency across patients of all ages, with most demonstrating an average8-point decline per year regardless of age and/or baseline MFM32 score, as shownin the natural history plot below.

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A 4-point score change in the MFM32 is considered clinically meaningful,suggesting that patients with GAN lose significant function annually. To date,we have up to eight years of robust data from this study.

Preclinical Data

TSHA-120 performed well across in vitro and in vivo studies, and demonstratedimproved motor function and nerve pathology, and long-term safety across severalanimal models. Of note, improved dorsal root ganglia, or DRG, pathology wasdemonstrated in TSHA-120-treated GAN knockout mice. These preclinical resultshave been published in a number of peer-reviewed journals.

Additional preclinical data from a GAN knockout rodent model that had receivedAAV9-mediated GAN gene therapy demonstrated that GAN rodents treated at 16months performed significantly better than 18-month old untreated GAN rodentsand equivalently to controls. These rodents were evaluated using a rotarodperformance test which is designed to evaluate endurance, balance, grip strengthand motor coordination in rodents. The time to fall off the rotarod, known aslatency, was also evaluated and the data below demonstrated the clear differencein latency in treated versus untreated GAN rodents.

A result is considered statistically significant when the probability of theresult occurring by random chance, rather than from the efficacy of thetreatment, is sufficiently low. The conventional method for determining thestatistical significance of a result is known as the "p-value," which representsthe probability that random chance caused the result (e.g., a p-value = 0.01means that there is a 1% probability that the difference between the controlgroup and the treatment group is purely due to random chance). Generally, ap-value less than 0.05 is considered statistically significant.

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With respect to DRG inflammation, a topic of considerable interest within thegene therapy arena, the DRG have a significantly abnormal histologicalappearance and function as a consequence of underlying disease pathophysiology.Treatment with TSHA-120 resulted in considerable improvements in thepathological appearance of the DRG in the GAN knockout mice. Shown below istissue from a GAN knockout mouse model with numerous abnormal neuronalinclusions containing aggregates of damaged neurofilament in the DRG asindicated by the yellow arrows. On image C, tissue from the GAN knockout micetreated with an intrathecal (IT) injection of TSHA-120 had a notable improvementin the reduction of these neuronal inclusions in the DRG.

When a quantitative approach to reduce inclusions in the DRG was applied, it wasobserved that TSHA-120 treated mice experienced a statistically significantreduction in the average number of neuronal inclusions versus the GAN knockoutmice that received vehicle as illustrated below.

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Additionally, TSHA-120 demonstrated improved pathology of the sciatic nerve inthe GAN knockout mice as shown below.

Results of Ongoing Phase 1/2 Clinical Trial

A Phase 1/2 clinical trial of TSHA-120 is being conducted by the NIH under anaccepted IND. The ongoing trial is a single-site, open-label, non-randomizeddose-escalation trial, in which patients are intrathecally dosed with one of 4dose levels of TSHA-120 - 3.5E13 total vg, 1.2E14 total vg, 1.8E14 total vg or3.5E14 total vg. The primary endpoint is to assess safety, with secondaryendpoints measuring efficacy using pathologic, physiologic, functional, andclinical markers. To date, 14 patients have been intrathecally dosed and twelvepatients have at least three years' worth of long-term follow up data.

At 1-year post-gene transfer, a clinically meaningful and statisticallysignificant slowing or halting of disease progression was seen with TSHA-120 atthe highest dose of 3.5E14 total vg (n=3). The change in the rate of decline inthe MFM32 score improved by 5 points in the 3.5E14 total vg cohort compared toan 8-point decline in natural history.

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Although the change in the MFM32 score was clinically meaningful, we might haveexpected a greater change in the MFM32 score compared to natural history in thefirst year but one patient in the high dose cohort was a delayed responder. Atthe 12-month follow-up visit, the patient had a 7-point decline in the MFM32total score that was similar to the slope of the natural history curve as shownbelow. Notably, from Year 1 post gene transfer to Year 2, this patient's changein the MFM32 score remained unchanged suggesting stabilization of disease at 2years post-treatment. At that 2-year post treatment timepoint, there was a9-point improvement in the patient's MFM32 score compared to the estimatednatural history decline of 16 points. The annualized estimate of natural historyover time assumes the same rate of decline as in Year 1.

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An additional analysis was performed to examine the change in the rate ofdecline in the MFM32 score of all therapeutic doses combined (n=12). As shownbelow, the change in the rate of decline in the MFM32 score improved by 7 pointsby Year 1 compared to the natural history decline in the MFM32 score of 8points. This result was clinically meaningful and statistically significant.

A Bayesian analysis was conducted on the 1.2E14 total vg, 1.8E14 total vg and3.5E14 total vg dose cohorts at Year 1 to assess the probability of clinicallymeaningful slowing of disease progression as compared to natural history. Thistype of statistical analysis enables direct probability statements to be madeand is both useful and accepted by regulatory agencies in interventional studiesof rare diseases and small patient populations. As shown in the table below, forall therapeutic dose cohorts, there was nearly 100% probability of any slowingof disease and a 96.7% probability of clinically meaningful slowing of 50% ormore following treatment with TSHA-120 compared to natural history data.

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There remained consistent improvement in TSHA-120's effect over time on the meanchange from baseline in the MFM32 score for all patients in the therapeutic dosecohorts compared to the estimated natural history decline over the years. ByYear 3, as depicted below, there was a 10-point improvement in the mean changefrom baseline in MFM32 score for all patients in the therapeutic dose cohorts.

In addition to the compelling three-year data, there was one patient at Year 5whose MFM32 change from baseline improved by nearly 26-points in the 1.2E14total vg dose cohort compared to the estimated natural history decline of 40points by this timepoint.

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Below is an additional analysis of the mean change from baseline in MFM32 scorefor the therapeutic dose cohorts compared to natural history at patients' lastvisit. As shown, TSHA-120 demonstrated increasing improvement in the mean changein MFM32 score from baseline over time.

Sensory nerve action potential, or SNAP, was assessed through nerve conductionstudies in patients with GAN. Natural history data from the NIH suggest rapidand irreversible decline in sensory function early in life in patients with GAN.SNAPs are within normal limits early in life and rapid reduction in SNAPamplitude occurs around the age of symptom presentation. As demonstrated below,all patients with classic GAN have an abnormally low SNAP by the age of 4,reflective of compromised sensory neuronal function. By age 9, all patients hadan irreversibly absent SNAP. The results from these nerve conduction studiesreflect the clinical progression of patients with GAN.

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TSHA-120-treated patients demonstrated a durable improvement in SNAP responsecompared to natural history. Five of the twelve patients treated demonstrated aresponse. One patient demonstrated near complete recoverability to normal fromzero at the time of treatment.

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Once SNAP reaches zero, natural history suggests sensory function is presumednon-recoverable. Among patients treated with 1.2E14 total vg or greater ofTSHA-120, the three patients with a positive value at baseline maintained apositive SNAP at last study visit with the longest span of 3 years to date andcontinue to improve.

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Below are individual patient SNAP change from baseline from treated patients whoshowed a positive response including their run-in natural history.

Biopsies of TSHA-120-treated patients confirmed presence of regenerative nerveclusters. Below is pathology data from biopsies of the superficial radialsensory nerve in 11 out of 11 patient samples analyzed. The remaining twosamples were unable to be assessed due to biopsy limitations. Peripheral nervebiopsies from the superficial radial sensory nerve were obtained at baseline andat 1 year post gene therapy transfer. Data consistently generated an increase inthe number of regenerative clusters observed at Year 1 compared to baseline,indicating active regeneration of nerve fibers following treatment withTSHA-120. Data also indicated improvement in disease pathology, providingevidence that the peripheral nervous system can respond to treatment.

Loss of vision has been frequently cited by patients and caregivers as a symptomthey find particularly debilitating and would like to see improvement infollowing treatment. Patients were analyzed for visual acuity using a standardLogarithm of the Minimum Angle of Resolution, or LogMAR. An increase in LogMARscore represents a decrease in visual acuity. A LogMAR score of 0 means normalvision, approximately 0.3 reflects the need for eyeglasses, and a score value of1.0 reflects blindness. Based on natural history,

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individuals with GAN experience a progressive loss in visual function asindicated by an increase in the LogMAR score. Ophthalmologic assessmentsfollowing treatment with TSHA-120 demonstrated preservation of visual acuityover time compared to the loss of visual acuity observed in natural history.Stabilization of visual acuity was observed following treatment with TSHA-120 asdemonstrated below.

The thickness of the retinal nerve fiber layer or RNFL was also examined as anobjective biomarker of visual system involvement and overall nervous systemdegeneration in GAN. Treatment with TSHA-120 resulted in stabilization of RNFLthickness and prevention of axonal nerve degeneration compared to diffusethinning of RNFL observed in natural history as measured by optical coherencetomography, or OCT. Analysis by individual dose groups, as seen on the graphbelow, demonstrated relatively stable RNFL thickness which is in contrast to thenatural history of GAN, where RNFL decreases. Overall, these data provide newevidence of TSHA-120's ability to generate nerve fibers and preserve visualacuity.

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As of January 2022, there were 53 patient-years of clinical data to supportTSHA-120's favorable safety and tolerability profile. TSHA-120 has beenwell-tolerated at multiple doses with no signs of significant acute or subacuteinflammation, no sudden sensory changes and no drug-related or persistingtransaminitis. Adverse events related to immunosuppression or study procedureswere similar to what has been seen with other gene therapies and transient innature. There was no increase in incidence of adverse events with increaseddose. Importantly, TSHA-120 was safely dosed in the presence of neutralizingantibodies as a result of the combination of route of administration, dosing andimmunosuppression regimen.

We currently have up to six years of longitudinal data in individual patientswith GAN and collectively 53-patient years of clinical safety and efficacy datafrom our ongoing clinical study. Treatment with TSHA-120 was well-tolerated withno significant safety issues. There was no increase in incidence of adverseevents with increased dose, no dose-limiting toxicity, no signs of acute orsubacute inflammation, no sudden sensory changes and no drug-related orpersistent elevation of transaminases. Adverse events related toimmunosuppression or study procedures were similar to what was seen with othergene therapies and transient in nature.

We believe the comprehensive set of evidence generated across diseasemanifestations, depicted in the table below, support a robust clinical packagefor TSHA-120 in GAN.

In order to deliver a robust chemistry, manufacturing, and controls, or CMC,data package to support licensure discussions, we have successfully completedsix development and GMP lots of TSHA-120 with our contract development andmanufacturing organization, or CDMO, partner. We have also completed acomprehensive side-by-side biochemical and biophysical analysis of

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current and previous clinical lots. Our CDMO utilizes the same Pro10TMmanufacturing platform used to produce the original GAN lots, therefore reducingwhich is intended to reduce comparability risk. Five development lots rangingfrom 2L to 250L scale and one full-scale 500L GMP lot were analyzed side-by-sidewith the current TSHA-120 clinical lot using a comprehensive analytical panelthat meets current regulatory requirements including assays for criticalattributes such as product and process residuals, empty/full ratio, geneticintegrity, potency and strength.

The side-by-side analysis demonstrated that the newly produced TSHA-120 lotswere generally comparable to the original clinical trial material in impurityprofile including host cell contaminants, residual plasmid, empty particlecontent, aggregate content and genomic integrity. These results supported ourbiophysical and biochemical comparability of the newly produced lots.Furthermore, we developed product-specific GAN potency methods which have alsodemonstrated that the previous and current clinical lots were functionallyindistinguishable. Validation of our potency release assay is now underway.

We have applied our panel of release assays for side-by-side testing of theoriginal clinical trial material and our commercial grade lots. Shown below areeight of the most critical attributes of TSHA-120.

First, all results demonstrated that both the clinical and commercial grade lotswere of a high purity and lacked significant levels of host cell or processcontaminants such as protein and, DNA or and aggregated species. Vector puritywas in excess of 95% for all three lots and host cell protein contamination wasbelow detection. In addition, and aggregation of all lots was very low. Hostcell and plasmid DNA contamination are also important attributes to discuss withregulatory agencies since carryover represents a theoretical immunogenicity oroncogenicity risk. Residual plasmid and host cell DNA were similar for all lots,indicating a similar safety profile for both products. Empty capsids are a keyattribute for AAV vectors since empty capsids can stimulate immune responses tothe vector and reduce potency. All three lots were highly enriched in fullparticles. Potency of AAV vectors is a key measure that correlates with clinicalefficacy. We developed a number of product-specific potency assays to measurethe functional activity of our product which is reported relative to a referencestandard. These assays recapitulated the biological activity of TSHA-120starting with transduction of GAN knockout cell lines. Activity is measured byquantitation of transgene RNA or protein expression as two independent andcomplimentary readouts. We observed good agreement with both readouts and highactivity of all three lots against our reference suggesting that the lots are ofhigh and comparable activity.

Overall, these results support that our early clinical and pivotal lots arebiochemically and biophysically similar and based on these results we believethey should perform identically in a clinical study.

Recently, regulators have encouraged sponsors to conduct deeper analysis ofproduct contaminants not covered by standard release assays to better assessproduct safety and comparability. To comply with this guidance, we have addedPac-Bio next generation sequencing to our product characterization panel tobetter understand the nature of nucleic acid contaminants in our products. Thismethod not only allows us to identify the source of the nucleic acid, but alsothe fragment size, and sequence variability, which also needs to be consideredwhen assessing AAV safety and efficacy. Our analysis of the clinical trial lotand commercial grade pivotal batches demonstrated that the source andcomposition of transgene and contaminating host and plasmid

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DNA are nearly identical and provided further support that for a conclusion thatthe nature of our product is unchanged between our early clinical and pivotalbatches as noted in the below pie charts.

The TSHA-120 pivotal lot, which yielded over 50 patient doses of TSHA-120 at thehighest dose cohort of 3.5E14 total vg, is expected to complete quality releasetesting by end of the third quarter of 2022. This material positions us forfuture BLA-enabling activities and commercial production. These lots were alsoplaced on stability to provide critical shelf-life data in support of our BLAfiling.

In September 2021, we submitted a request for a Scientific Advice meeting forTSHA-120 to the United Kingdom's Medicines and Healthcare products RegulatoryAgency, or MHRA, and were granted a meeting in January 2022. MHRA agreed on ourcommercial manufacturing and release assay testing strategy including potencyassays and we plan to dose a few additional patients with commercial gradematerial, which will be released in September 2022. Finally, MHRA was supportiveof our proposal to perform validation work on MFM32 for GAN as a key clinicalendpoint and for us to explore the MFM32 items with patients and families aspart of this process. Given the positive comparability data for TSHA-120 that werecently received, additional discussions with Health Authorities to discussthese data and potential registration pathway are planned with regulatoryfeedback anticipated by year-end 2022.

TSHA-102 for Rett Syndrome

TSHA-102, a neurodevelopmental disorder product candidate, is being developedfor the treatment of Rett syndrome, one of the most common genetic causes ofsevere intellectual disability, characterized by rapid developmental regressionand in many cases caused by heterozygous loss of function mutations in MECP2, agene essential for neuronal and synaptic function in the brain. The estimatedprevalence of Rett syndrome is 350,000 patients worldwide and the disease occursin 1 of every 10,000 female births worldwide. We designed TSHA-102 to preventgene overexpression-related toxicity by inserting microRNA, or miRNA, targetbinding sites into the 3' untranslated region of viral genomes. Thisoverexpression of MECP2 is seen in the clinic in patients with a condition knownas MECP2 duplication syndrome, where elevated levels of MECP2 result in aclinical phenotype similar to Rett syndrome both in terms of symptoms andseverity. TSHA-102 is constructed from a neuronal specific promoter, MeP426,coupled with the miniMECP2 transgene, a truncated version of MECP2, andmiRNA-Responsive Auto-Regulatory Element, or miRARE, our novel miRNA targetpanel, packaged in self-complementary AAV9. Currently, there are no approvedtherapies for the treatment of Rett syndrome, which affects more than 350,000patients worldwide, according to the Rett Syndrome Research Trust.

In May 2021, preclinical data from the ongoing natural history study forTSHA-102 were published online in Brain, a highly esteemed neurological sciencepeer-reviewed journal. The preclinical study was conducted by the UTSouthwestern Medical Center laboratory of Sarah Sinnett, Ph.D., and evaluatedthe safety and efficacy of regulated miniMECP2 gene transfer, TSHA-102(AAV9/miniMECP2-miRARE), via IT administration in adolescent mice between fourand five weeks of age. TSHA-102 was compared to unregulated full length MECP2(AAV9/MECP2) and unregulated miniMECP2 (AAV9/miniMECP2).

TSHA-102 extended knockout survival by 56% via IT delivery. In contrast, theunregulated miniMECP2 gene transfer failed to significantly extend knockoutsurvival at either dose tested. Additionally, the unregulated full-length MECP2construct did not demonstrate a significant extension in survival and wasassociated with an unacceptable toxicity profile in wild type mice.

In addition to survival, behavioral side effects were explored. Mice weresubjected to phenotypic scoring and a battery of tests including gait, hindlimbclasping, tremor and others to comprise an aggregate behavioral score. miRAREattenuated

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miniMECP2-mediated aggravation in wild type aggregate phenotype severity scores.Mice were scored on an aggregate severity scale using an established protocol.AAV9/MECP2- and AAV9/miniMECP2-treated wild type mice had a significantly highermean (worse) aggregate behavioral severity score versus that observed forsaline-treated mice (p <0.05; at 6-30 and 7-27 weeks of age, respectively).TSHA-102-treated wild type mice had a significantly lower (better) meanaggregate severity score versus those of AAV9/MECP2- and AAV9/miniMECP2-treatedmice at most timepoints from 11-19 and 9-20 weeks of age, respectively. Nosignificant difference was observed between saline- and TSHA-102-treated wildtype mice.

miRARE-mediated genotype-dependent gene regulation was demonstrated by analyzingtissue sections from wild type and knockout mice treated with AAV9 vectors givenintrathecally. When knockout mice were injected with a vector expressing themini-MECP2 transgene with and without the miRARE element, miRARE reduced overallminiMECP2 transgene expression compared to unregulated miniMECP2 in wild typemice as shown below.

TSHA-102 demonstrated regulated expression in different regions of the brain. Asshown in the graph and photos below, in the pons and midbrain, miRARE inhibitedmean MECP2 gene expression in a genotype-dependent manner as indicated bysignificantly fewer myc(+) cells observed in wild type mice compared to knockoutmice (p<0.05), thereby demonstrating that TSHA-102 achieved MECP2 expressionlevels similar to normal physiological parameters.

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In preclinical animal models, intrathecal myc-tagged TSHA-102 was not associatedwith early death and did not cause adverse behavioral side effects in wild typemice demonstrating appropriate downregulation of miniMECP2 protein expression ascompared to unregulated MECP2 gene therapy constructs. In addition, preclinicaldata demonstrated that miRARE reduced overall expression of miniMECP2 transgeneexpression and regulated genotype-dependent myc-tagged miniMECP2 expressionacross different brain regions on a cell-by-cell basis and improved the safetyof TSHA-102 without compromising efficacy in juvenile mice. Pharmacologicactivity of TSHA-102 following IT administration was assessed in the MECP2knockout mouse model of Rett syndrome across three dose levels and three agegroups (n=252). A one-time IT injection of TSHA-102 significantly increasedsurvival at all dose levels, with the mid to high doses improving survivalacross all age groups compared to vehicle-treated controls. Treatment withTSHA-102 significantly improved body weight, motor function and respiratoryassessments in MECP2 knockout mice. An additional study in neonatal mice isongoing, and preliminary data suggest normalization of survival. Finally, anIND/CTA-enabling 6-month Good Laboratory Practice, or GLP, toxicology study(n=24) examined the biodistribution, toxicological effects and mechanism ofaction of TSHA-102 when intrathecally administered to Non-Human Primates, orNHPs, across three dose levels. Biodistribution, as reflected by DNA copynumber, was observed in multiple areas of the brain, sections of spinal cord andthe DRG. Importantly, mRNA levels across multiple tissues were low, indicatingmiRARE regulation is minimizing transgene expression from the construct in thepresence of endogenous MECP2 as expected, despite the high levels of DNA thatwere delivered. No toxicity from

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transgene overexpression was observed, confirmed by functional andhistopathologic evaluations demonstrating no detrimental change inneurobehavioral assessments and no adverse tissue findings on necropsy.

In neonatal knockout Rett mice, treatment with TSHA-102 resulted in nearnormalization of survival as shown below. A single intracerebroventricular, orICV, injection of TSHA-102 at a dose of 8.8E10 vg/mouse (Human Equivalent Doseof 2.86E14 vg/participant) within 48 hours after birth in Mecp2-/Y male micesignificantly extended the survival of the animals as shown below. All cohorts,including vehicle, were sacrificed at 34 weeks. Preliminary data demonstratedapproximately 70% of the treated Mecp2-/Y males survived to 34 weeks of agecompared to 9 weeks in the vehicle-treated Mecp2-/Y male.

In addition, neonatal knockout Rett mice demonstrated normalization of behaviorfollowing treatment with TSHA-102 as assessed by the Bird Score, a compositemeasure of six different phenotypic abilities. Knockout animals were initiallyassessed at 4 weeks of age with a mean Bird Score of 4. Over the course of thestudy, TSHA-102 improved the behaviors (as assessed by the Bird aggregate score)of TSHA-102 treated mice as shown below.

In summary, we believe the totality of preclinical data generated to date,specifically including the mouse pharmacology study to ascertain minimallyeffective dose, the two toxicology studies (wild type rat and wild type NHP) andthe recent mouse neonatal data, represents the most robust package supportingclinical advancement of TSHA-102 in Rett syndrome as shown below.

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Safety and biodistribution assessments in NHPs were presented in May 2022 at theInternational Rett Syndrome Foundation (IRSF) meeting along with the caregiverperspective on Rett syndrome in adulthood. At the ASCEND National Summit, therewas an oral presentation on "Putting Patients at the Center." Finally, mousepharmacology, rat and NHP toxicology data were presented at the 25th AnnualMeeting of the American Society of Gene & Cell Therapy (ASGCT).

We submitted a CTA for TSHA-102 in November 2021 and announced initiation ofclinical development under a CTA approved by Health Canada in March 2022. We areadvancing TSHA-102 in the REVEAL Phase 1/2 clinical trial which is anopen-label, dose escalation, randomized, multicenter study that will examine thesafety and efficacy of TSHA-102 in adult female patients with Rett syndrome. Upto 18 patients will be enrolled. In the first cohort, a single 5E14 total vgdose of TSHA-102 will be given intrathecally. The second cohort will be given a1E15 total vg dose of TSHA-102. Key assessments will include Rett-specific andglobal assessments, quality of life, biomarkers, and neurophysiology and imagingassessments. Sainte-Justine Mother and Child University Hospital Center inMontreal, Quebec, Canada has been selected as the initial clinical trial siteunder the direction of Dr. Elsa Rossignol, Assistant Professor Neuroscience andPediatrics, and Principal Investigator. We expect to report preliminary clinicaldata for TSHA-102 in Rett syndrome by year-end 2022.

We have received orphan drug designation and rare pediatric disease designationfrom the FDA and orphan drug designation from the European Commission forTSHA-102 for the treatment of Rett syndrome.

TSHA-121 for CLN7 Disease

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Effect of tensile frequency on osteogenic differentiation | IJGM – Dove Medical Press

Posted: July 3, 2022 at 2:26 am

Introduction

Mechanical stress enhances bone metabolism and periodontal tissue remodeling.1 During orthodontic tooth movement (OTM), bone remodeling is initiated via the periodontal ligament.2 As the main mesenchymal stem cells (MSCs) in the periodontal ligament, periodontal ligament stem cells (PDLSCs) play an important role in mechanical signal transduction. Currently, a consensus has been reached that cyclic mechanical tension is a strong driver of the differentiation of PDLSCs into the osteoblast lineage.24 Mechanical tension activates calcium channels,5 which activate the ERK1/2 and P38 MAPK pathways through integrin-FAK or protein kinase (PKC)-SR signaling6 and induce the phosphorylation of Runt-associated transcription factor 2 (Runx2),7 promoting osteogenic precursor cell synthesis and the transcription of mineralizable proteins.8 At present, the TGF-, BMP, MAPK, Notch, Wnt, Hedgehog, FGF, and Hippo signaling pathways have been found to be involved in this process.

Force parameters (including magnitude, frequency, and duration) are crucial for well-regulated tissue remodeling. However, numerous in vitro studies performed to date show enormous heterogeneity in tensile force parameters.9 In different studies, cyclic tension was applied with magnitudes ranging from 1% to 24%, frequencies ranging from 0.1 Hz to 1.0 Hz, and stimuli duration ranging from 1 hour to 6 days, thereby reducing comparability between different studies.9 To establish strategies to optimize tensile force parameters, it is of particular importance to understand how different tensile force parameters affect the osteogenic differentiation of PDLSCs.

The effects of different tensile force magnitudes and durations have been investigated in some studies. Among the magnitudes, a magnitude of 10% generally led to a lower level of inflammation and a higher level of osteogenesis,10 whereas a magnitude of 12% was found to correlate well with strain conditions at the mid-root under physiological loading conditions11,12 and to induce optimal effects in both the proliferation and osteogenesis of PDLSCs.13 Cyclic tension alone at 3000 strain significantly enhanced SATB Homeobox 2 (Satb2) after 3 h of loading and significantly upregulated Runx2 after 6 h.14 The synthesis of BMP9 increased under 6-h continuously applied cyclic tension.15 In addition, 12% cyclic tensile force gradually upregulated the expression of Runx2, alkaline phosphatase (ALP), and osteocalcin (OCN) with force durations of 6 h, 12 h, and 24 h, respectively.16,17 The protein level of osterix increased stepwise following 3 h, 6 h, 12 h, and 24 h of exposure to tensile strain.14 Recently, temporal gene expression patterns were delineated.17

Tensile frequency varies largely among different studies. The ROCKTAZ pathway and its interaction with Cbf1 were found to be essential for the cyclic tension (12% elongation, 0.1 Hz)-induced osteogenic differentiation in PDLSCs.18 Cyclic tension (10% elongation, 0.5 Hz) stimulated the osteogenic differentiation of PDLSCs by inhibiting miR-129-5p expression and activating the BMP2/Smad pathway.17 LncRNAs-miRNAs-mRNAs networks in PDLSCs were depicted under cyclic tension (10% elongation, 1.0 Hz).19 However, there have been rare studies examining the impact of different cyclic tensile frequencies on osteogenesis of PDLSCs and the expression of relevant genes thus far. The low-magnitude high-frequency (LMHF) vibration approach was excluded because it is used to simulate a masticatory force, while cyclic tension is used to simulate an orthodontic force, and the two methods of force application are completely different.2,20 Previous animal studies on long bone distraction osteogenesis have shown that loading frequency affects the osteogenic response of bone tissue.21 The mechano-regulation of trabecular bone adaptation is logarithmically dependent on the loading frequency.22 Therefore, we hypothesized that tensile frequency would affect osteogenesis of PDLSCs, in which some tensile frequency-sensitive genes may play an important role. To test our hypotheses, human PDLSCs were subjected to cyclic mechanical tension at different frequencies of 0.10.7 Hz to examine the osteoblastic differentiation of PDLSCs, and high-throughput sequencing was performed to characterize the frequency-course expression patterns of mRNA during the osteogenic differentiation of PDLSCs. This study aimed to investigate the effects of tensile frequency on the osteogenic differentiation of PDLSCs as well as the relevant molecular mechanisms.

Healthy periodontal ligament tissues were scraped from the middle third of tooth roots, which were extracted for orthodontic reasons, with informed consents. All donors were aged from 14 to 16 years and had no systemic or oral diseases. The periodontal ligament tissues were cut into small pieces and enzymatically digested for 40 min at 37C with collagenase I (3 mg/mL, Sigma-Aldrich, St. Louis, MO, USA) and dispase II (4 mg/mL; Sigma-Aldrich). The cells were seeded in 25 cm2 flasks (Falcon, BD Biosciences, Franklin Lakes, NJ, USA) with -minimal essential medium (-MEM; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS, Gibco, Life Technologies Co., Grand Island, NY, USA) and antibiotics (100 U/mL penicillin and 100 g/mL streptomycin, Hyclone, Logan, Utah, USA), and incubated in a humidified atmosphere (37C, 5% CO2). The medium was changed every 3 days. After reaching 80% of confluence, the cells were detached with 0.25% trypsin/EDTA (Gibco, Life Technologies Co., Grand Island, NY, USA), and single-cell suspensions were cloned with the limiting-dilution method to purify the stem cells.17 Cell clusters from the colony were trypsinized and serially sub-cultured.

The third-passage PDLSCs were sub-cultured into six-well plates until confluent. The culture medium was then removed, and cells were fixed with 4% formaldehyde (Zhonghuihecai, Xian, CN) for 20 min, permeabilized with 0.3% Triton X-100 (Zhonghuihecai) for 5 min, and incubated with primary antibodies (anti-pan-cytokeratin, 1:300, Abcam, Cambridge, MA, USA; anti-vimentin, 1:500, Abcam; anti-STRO-1, 1:200, Abcam; anti-CD146, 1:200, Abcam) overnight at 4C. The cells were then washed with PBS and incubated with CY3/FITC-conjugated secondary antibodies (1:500, Zhuangzhi, Xian, CN) in darkness for 30 min, and then washed with PBS. Finally, the nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI, Zhuangzhi), and fluorescent images were captured with a fluorescence microscope (Olympus, Japan).

For osteogenic and adipogenic differentiation, PDLSCs were seeded into six-well plates at a density of 2105 cells/well, and after reaching 80% confluence, the medium was replaced with an osteogenic or adipogenic inductive medium (Osteogenesis or Adipogenesis Differentiation Kit, Cyagen, USA). Seven days after osteogenic induction, the cells were stained with ALP (ALP staining kit, Solarbio, CN), and 14 days after osteogenic incubation, the cells were stained with Alizarin Red (ARS staining kit, Cyagen, USA). After 21 days of adipogenic incubation, the cells were stained with Oil Red O (Cyagen, USA).

Flexcell FX-5000T Tension Plus System (Flexcell International Corporation, Hillsborough, NC, USA) was used to mimic the tensile force exerted on PDLSCs during OTM, according to previous studies.23 Cyclic tensile loading experiments were performed on the fourth-passage PDLSCs from four different healthy donors in triplicate. PDLSCs were seeded onto six-well type I collagen (COL-I)-coated silicone culture plates (Flexcell International Corporation) at a density of 2105 cells/well. Upon reaching 80% confluence, the cells were serum-starved overnight, and the medium was changed to osteogenic medium (Cyagen, USA). Cyclic tensile force (12% bottom membrane elongation) was applied to different plates at different frequencies of 0.1 Hz, 0.5 Hz, and 0.7 Hz. Control cells were cultured under identical culture condition but without mechanical stimulation.

After 10 h of cyclic tensile force, the cells were collected. Proteins were isolated, electrophoretically separated, and immunoblotted as previously described.23 Briefly, PDLSCs were lysed with RIPA buffer containing 1% phenylmethanesulfonyl fluoride (PMSF, proteinase inhibitor, Zhonghuihecai) and 1% phosphatase inhibitor (Zhonghuihecai). After centrifugation, the supernatant was collected and measured quantitatively using a BCA Protein Assay Kit (Absin, Shanghai, CN). Total protein from cell lysates (20 g/lane) was separated by SDS-PAGE gels (Beyotime, Hangzhou, CN) and then transferred onto a polyvinylidene difluoride (PVDF) membrane (EMD Millipore, Billerica, MA, USA). After blocking with 5% skimmed milk in tris-buffered saline tween-20 (TBST) for 2 h at room temperature, the membranes were incubated overnight at 4C with primary antibodies (runt-related transcription factor 2 (Runx2), 1:500, ImmunoWay, USA; COL-I, 1:1000, Proteintech, USA; Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 1:10000, Proteintech, USA), followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000, Proteintech, USA) for 2 h at room temperature. The protein expression was visualized using ChemiDocTM XRS+ (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with an enhanced chemiluminescence (ECL) kit (Millipore, Billerica, MA, USA). GAPDH was used as an internal control for normalization.

The fourth-passage PDLSCs from three donors were used for RNA sequencing after tension loading. Total RNA was extracted from the four groups of cells (normal PDLSCs and PDLSCs tensioned at frequencies of 0.1 Hz, 0.5 Hz, and 0.7 Hz for 6 h) using the Trizol (Sigma-Aldrich), according to the manufacturers protocols. After digestion with DNase, rRNA were depleted using a Ribo-Zero magnetic kit, and sequencing libraries were constructed as previously described.24 The sequencing of the cDNA library was carried out by Gene Denovo Biotechnology Co. (Guangzhou, China). The gene expression level was evaluated by reads per kilobase transcriptome per million mapped reads (RPKM). Requirements for filtering differentially expressed genes (DEGs) were as follows: (1) |log2 (fold-change)| 1; (2) p value < 0.05. DESeq2 (differential gene expression analysis based on the negative binomial distribution)25 was used to calculate p values and adjusted p (adj. p) values. Heatmaps and volcano plots analyses were used to visualize these DEGs using the Complex Heatmap package and ggplots2 package of R software. The online tool Venny 2.1 (http://bioinfogp.cnb.csic.es/tools/venny/index.html) was applied to identify the common DEGs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used for annotation visualization and integrated discovery. Raw data of the performed RNA sequencing (RNA-seq) were recorded in the SRA database with the SRA accession: PRJNA665587.

Gene expression pattern analysis is used to cluster genes of similar expression patterns for multiple samples in a tensile frequency order. To examine the expression pattern of DEGs in different frequencies, the expression data of each sample (in the order of treatment) were normalized to 0, log2 (v1/v0), and log2 (v2/v0), and then clustered using the Short Time-series Expression Miner software (STEM).26 The parameters were set as follows: (1) maximum unit change in model profiles between frequency points was 1; (2) maximum output profiles number was 20 (similar profiles will be merged; and (3) minimum ratio of fold change of DEGs was no less than 2.0. The clustered profiles with p value < 0.05 were considered as significant profiles. Then, the DEGs in all profiles or in each profile were subjected to GO and KEGG pathway enrichment analysis. STRING (https://string-db.org/) was used for PPI network analysis. The DEGs were mapped onto the PPI network with a minimum interaction score of 0.4. Cytoscape v3.7.1 software was used to visualize the PPI network. Gene network clustering analysis was performed to identify the key PPI network modules, using the MCODE and cytoHuba app from the Cytoscape software suite. Adj. p value < 0.05 was set as the significance threshold.

Total RNA was extracted using Trizol (Sigma-Aldrich) according to the manufacturers protocols. Quantitative real-time PCR (RTqPCR) was performed in triplicate using the Power SYBR Green PCR Mastermix (Applied Biosystems, Foster City, CA, USA). Sequences of the primers used are shown in Table S1. The mean expression values were calculated relative to GAPDH, which was used as an internal control for normalization.

For differential gene expression analysis, a likelihood ratio test was used assuming an underlying zero-inflated negative binomial distribution.25,27 FDR-corrected p-values were calculated using the Benjamini-Hochberg procedure.28 K-means cluster method29 was used for frequency cluster expression pattern analysis by the STEM software. P and Q values of GO/KEGG functional analysis and frequency cluster analysis were calculated based on a hypergeometric distribution.30 Values of the relative protein and mRNA expression were expressed as mean SD within each group. One-way ANOVA followed by SNK post hoc tests was used in the Western blotting assay and RTqPCR assay. The significance threshold was set at 0.05.

Immunofluorescent staining revealed that the isolated cells were positive for CD146, vimentin, and STRO-1 (Figure 1AC, respectively) but negative for pan-cytokeratin (Figure 1D), confirming that the cells were mesenchymal stem cells of mesodermal origin. The osteogenic potential of PDLSCs was determined by positive ALP staining after 7 days of osteogenic induction (Figure 1E and F) and red mineralized matrix nodules in Alizarin Red staining after 14 days of osteogenic induction (Figure 1G and H). The presence of red lipid droplets in Oil Red staining after 21 days of adipogenic induction (Figure 1I) indicated the adipogenic differentiation of PDLSCs.

Figure 1 Identification of PDLSCs. The Immunofluorescence showed that the cells were positive for CD146 (A, red), vimentin (B, green), and STRO-1 (C, red) but negative for pan-cytokeratin (D). Scale bar = 50 m. ALP staining was positive both visually (E, black) and under the microscope (F, black, Scale bar = 100 m), after 7 days of osteogenesis induction culture. After 14 days of osteogenesis induction, ARS staining was observed to be positive by the naked eye (G, red), and mineralized nodules were obvious under the microscope (H, red, Scale bar = 100 m). After 21 days of adipogenic induction, oil red O-positive lipid clusters were observed microscopically (I, red, Scale bar = 100 m).

PDLSCs were exposed to 12% cyclic tension, which contributed to the cellular reorientation, including an irregular arrangement at the central region and a parallel arrangement at the peripheral region of the plate (Figure 2A). As shown in Figure 2B and C, after consecutive 10-h cyclic tensile force loading, the protein expression levels of Runx2 and COLI increased with increasing tensile frequency from 0.1 Hz to 0.7 Hz and were remarkably higher than that in the group without tension application (p < 0.05). The result suggested that mechanical tension (12% deformation) upregulated the osteogenesis of PDLSCs in a frequency-dependent manner. Higher frequencies of cyclic tension were associated with higher osteogenic differentiation of PDLSCs.

Figure 2 Cyclic tension promoted the osteogenic differentiation of PDLSCs. Under cyclic equibiaxial tension, PDLSCs reoriented in parallel alignment at the peripheral region of the plate, while in random orientation at the center of the plate (A, Scale bar = 100 m). Western blotting was used to detect protein levels of osteogenesis-related genes, COL-I and Runx2, at different tensile frequencies (B and C). **p < 0.01, ***p < 0.001, vs control group.

It has been previously shown that cyclic mechanical tensile stress can improve osteogenesis of PDLSCs, and that consecutive 46 h of tension can significantly upregulate the mRNA expression of osteogenesis-related genes.16,17 To gain insight into the molecular mechanisms by which mechanical tension stimulates osteoblast differentiation of PDLSCs, total RNA was extracted from PDLSCs to conduct RNA-seq after 6 h of cyclic tension. The mRNA expression profiles of the PDLSCs at different tensile frequencies (0.1 Hz, 0.5 Hz, and 0.7 Hz) were detected. Comparative expression analyses were performed according to the different frequencies of the tensile stress (0.1 Hz vs control, 0.5 Hz vs control, and 0.7 Hz vs control). Heat maps of the top 40 DEGs (Figure 3AC) and volcano plots (Figure 3DF) were depicted. In total, 50 mRNAs were upregulated, and 261 mRNAs were downregulated at 0.1 Hz. At 0.5 Hz, 656 mRNAs were upregulated, and 1474 mRNAs were downregulated. At 0.7Hz, 139 mRNAs were upregulated, and 194 mRNAs were downregulated. A Venny analysis (Figure 3G and H) showed that 78 genes were simultaneously upregulated and 118 were simultaneously downregulated among the 0.1 Hz, 0.5 Hz, and 0.7 Hz groups. The GO analysis (Figure 4A) demonstrated that changes in biological processes (BPs) were mainly enriched in metabolic process, response to stimulus, biological regulation, signaling, and localization. Changes in Cellular Components (CCs) were mainly enriched in organelle, membrane, macromolecular complex, and membrane-enclosed lumen. Moreover, binding, catalytic activity, and nucleic acid binding transcription factor activity emerged as the highest-ranked Molecular Function (MF) groups. As shown in Figure 4B, DNA replication, cell cycle, and the TNF signaling pathway were significantly enriched in the KEGG pathway. Within the primary category Environmental Information Processing, Signal transduction, and Signaling molecules and interaction were strongly enriched (Figure 4C).

Figure 3 Identification of DEGs among different frequencies. (AC) Heatmaps of the top 40 DEGs between 0.1 Hz/0.5 Hz/0.7 Hz and static culture, respectively. Red rectangles represent high expression, and blue rectangles represent low expression. (DF) Volcano plot of DEGs between 0.1 Hz/0.5 Hz/0.7 Hz and static culture, respectively. The red plots represent upregulated genes, the blue plots represent downregulated genes, and the black plots represent nonsignificant genes. (G) Venn diagram of upregulated DEGs among 0.1 Hz, 0.5 Hz, and 0.7 Hz. (H) Venn diagram of downregulated DEGs among all the three frequencies.

Figure 4 Functional enrichment analysis of all DEGs. (A) GO enrichment analysis of all DEGs among different frequencies. (B) Top 20 pathways of the KEGG enrichment analysis of all DEGs among different frequencies, with the KEGG pathway annotation (C). The screening criteria for significance were p value < 0.05.

The sequencing data were normalized to the control, and trend analyses of DEGs were identified using STEM. In Figure 5A, within the 20 model profiles, eight mRNA trend profiles were statistically significant. The profile number assigned by STEM was on the top left corner of each profile box, p value was on the bottom left, and the number of the cardinality of each cluster was on the top right corner. As shown in Table 1, among different profiles, the top-ranked KEGG pathways were mainly in the metabolic pathways, PI3K-Akt signaling pathway, cytokine-cytokine receptor interaction, and MAPK signaling pathway. A continuous downregulation pattern was found in profile 0 (Figure 5B), in which the high-ranked BPs, CCs, and MFs in GO enrichment (Figure 5C) were similar to those in Figure 4A, and inflammatory pathways such as arachidonic acid metabolism, peroxisome, and cytokinecytokine receptor interaction were strongly enriched (Figure 5D).

Table 1 Top 10 of KEGG Enrichment Among Different Profiles

Figure 5 Frequency series clustering analysis on expression profiles of mRNAs by STEM. (A) Within the 20 model profiles, eight mRNA trend profiles were statistically significant. The number at the upper-left corner of each profile box was the profile number assigned by STEM, the number on the bottom left was the p value, and the number on the top-right corner was the number of genes within each cluster. (B) Persistently downregulated genes along frequency were clustered in profile 0. (C) GO enrichment of profile 0. (D) Top 10 pathways of the KEGG enrichment of profile 0.

The interactions of 194 DEGs in profile 0 were analyzed using the STRING online database, and the PPI network was obtained using the Cytoscape software (Figure 6A). The MCODE plugin was then used to investigate the key PPI network modules, and one key module with four genes (EYA1, SIX5, SALL1, FRAS1) was identified (Figure 6B). The cytoHubba plugin was then used to analyze hub genes with maximum correlation criterion (MCC)/Degree, and genes with the top 10 scores were respectively identified. The intersection (EYA1, SALL1) of hub genes according to the above three methods were selected for further RTqPCR validation (Figure 6C). The results of RTqPCR (Figure 6D and E) showed that the mRNA expression of EYA1 and SALL1 decreased with increasing frequency from 0.1 Hz to 0.7 Hz, which were highly consistent with our high-throughput sequencing.

Figure 6 Identification and validation of mechanofreqency-sensitive hub genes. (A) The interaction network between proteins coded by the DEGs in profile 0. The nodes represent genes, and the edges represent links between genes. Blue represents downregulated genes. (B) The highest scoring module was extracted by MCODE. (C) The intersection was obtained among modules measured by MCODE, and the top 10 highly connected genes were identified using MCC and Degree in cytoHubba. (D and E) Validation of the expression of the two intersection genes, EYA1 and SALL1, using RTqPCR. *p < 0.05, vs control group.

OTM is based on remodeling processes in the periodontal ligament and the alveolar bone. PDLSCs play an important role in mechano-transduction and in promoting periodontal tissue regeneration in OTM.31,32 It is evident that cyclic tensile force regulates the osteogenic differentiation of PDLSCs.33 A complex network of signaling molecules regulates the osteoblastic differentiation of PDLSCs under cyclic tension.5,17,34 The heterogeneity of mechanical force parameters (duration, magnitude, frequency, and others)9 led to the heterogeneity of the osteogenic phenotype and gene regulation in different studies. This study focused on the effect of tensile frequency on the osteogenic differentiation of PDLSCs and attempted to elucidate a potential mechanism.

In the current study, we successfully isolated and characterized human PDSLCs. The cells at the 4th to 6th passage were used, whose phenotype was generally believed to be maintained.35 Mechanical tension was applied using the Flexcell tension system, which has been widely used in PDLSCs studies.2,20 We observed that the cellar reorientation after force loading was similar to that of the previous study,23 and this could be attributed to the mechano-responsive stress fibersfocal adhesion system.36 The present study showed that cyclic mechanical tension (magnitude: 12% deformation, duration: 10 h) in the range of 0.10.7 Hz promoted osteogenic marker genes including Runx2 and COL-I in PDLSCs, and their protein expressions increased with the increasing tensile frequency. Runx2 is an osteogenic lineage commitment specific transcription factor, which binds to the specific cis-acting elements of osteoblasts to promote the transcription and translation of OCN, osteopontin (OPN), bone sialoprotein (BSP), and COL-I.8 COL-I, which acts as a template onto which minerals are deposited to form bone matrix,37 is the major constituent of extracellular matrix in the periodontal ligament and bone, and is confirmed to be essential for osteogenesis in response to tension during OTM.38 Consistently, upregulation of RUNX2 and COL-I in response to tension was reported in most studies.9 To the best of our knowledge, the present study is the first to reveal the frequency dependence during cyclic tension in enhancing the expression of osteogenic markers within the first 10 h of cyclic tension application, which may inform a new method of accelerating OTM.

Using RNA-seq, we observed that mRNAs in strained PDLSCs were mainly enriched in response to the stimulus process, signal transduction, and relative pathways such as mismatch repair, TNF signaling pathway, and FOXO signaling pathway, which were associated with cell survival and differentiation as well as immune and inflammatory responses.39,40 The STEM platform was also used to investigate how gene expression profiles change with tensile frequency during the osteoblast differentiation of PDLSCs under cyclic tension. Eight trend profiles were noted as significant. Genes in these profiles were mainly enriched in the metabolic pathways, PI3K-Akt signaling pathway, cytokine-cytokine receptor interaction, and MAPK signaling pathway. The PI3K-Akt signaling pathway has been reported to be involved in the mechanical force-induced osteoblast differentiation of PDLSCs.41,42 The MAPK signaling pathway also have been found to participate in the mechano-transduction of PDLSCs.43,44

Genes in profile 0 showed a continuous downward trend from 0.1 Hz to 0.7 Hz, an inverse trend of osteogenic genes, and were mainly enriched for pathways related to an inflammatory response, such as arachidonic acid metabolism and cytokinecytokine receptor interaction. Cyclooxygenase-2 (COX2) and prostaglandin E synthase (PTGES) participate in the arachidonic acid metabolism pathway. TNF Receptor Superfamily Member 14 (TNFRSF14) and Interleukin 9 (IL9) are involved in the cytokinecytokine receptor interaction. PTGES is induced by inflammatory mediators.45 COX2 is involved in the synthesis of prostaglandin E2 (PGE2), which is a potent pro-inflammatory cytokine, and participates in bone resorption.2 The expression of COX2 and PGE2 after mechanical stimulation was previously reported to be correlated with force duration and force magnitude,9 and it showed a negative correlation with tensile frequency in the present study. TNFRSF14 is a membrane-bound receptor leading to the induction of proinflammatory genes by activating the NF-B pathway.46 IL-9 plays a role in regulating inflammatory immunity, and it has demonstrated pro-inflammatory activity in several mouse models of inflammation.47 The response of periodontal ligament to mechanical stress generated by OTM is known as an aseptic transitory inflammatory process, which is regulated by various cytokines and chemokines.48 Proinflammatory cytokines activate matrix metalloproteinases (MMPs), degrade the ECM, and inhibit the expression of COL-I.49 Increased osteogenesis is usually accompanied by lower levels of inflammatory cytokines and chemokines.10,50,51 Accordingly, in the present study, with increasing frequency, the osteogenic commitment increased, and the suppression of pro-inflammatory genes and the relative inflammatory response pathway were observed.

Furthermore, through PPI network screening, we identified two candidate genes, EYA1 and SALL1, which were specifically sensitized to tensile frequency. The result was validated by RTqPCR, which confirmed the decreased expression of EYA1 and SALL1 with increasing frequency of tension stimulation. EYA1 is a conserved critical regulator of organ-specific stem cells.52 SALL1 is also considered a stem cell marker.53 The osteoblastic differentiation of PDLSCs increased with increasing tensile frequency; thus, stemness and related genes correspondingly reduced. In view of the high-throughput sequencing and validation after 6 h of tensile force exposure, further studies over a longer period are needed. Whether overexpression of EYA1 and SALL1 would reverse the frequency-dependent trend of the osteogenic differentiation of PDLSCs also deserves further study.

In the present study, the role of tensile frequency on osteogenic commitment of PDLSCs were identified, and the mRNA transcriptomes of PDLSCs during the osteogenic differentiation under cyclic tension with different frequencies were delineated. Frequency series clustering were defined using STEM, and tensile frequency-sensitive genes were identified. This study extends the knowledge about the role of tensile frequency in cyclic tension induced PDLSCs osteogenesis.

The osteoblastic differentiation of PDLSCs under mechanical tensile force is frequency dependent. EYA1 and SALL1 were identified as potential important tensile frequency-sensitive genes, which may contribute to the cyclic tension-induced osteogenic differentiation of PDLSCs in a frequency-dependent manner.

Raw data of the performed RNA sequencing (RNA-seq) have been recorded in the SRA database with the SRA accession: PRJNA665587 (https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA665587). Other data in this study are available from the corresponding author Xi Chen upon request.

The study was performed in accordance with the principles stated in the Declaration of Helsinki and approved by the Medical Ethics Committee of the First Affiliated Hospital of Medical College of Xi an Jiaotong University (No: XJTU1AF2019LSK-078). Informed consent was obtained from all donors and their legal guardians involved in the study. Written informed consent was also obtained from the donors and their legal guardians to publish this paper.

All authors made a significant contribution to the work reported, whether in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising, or critically reviewing the article; gave final approval of the version to be published; agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

This research was funded by the Key Research and Development project of Shaanxi Province under Grant 2018SF-037.

The authors declare that they have no competing interests.

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Stem cell-based regenerative medicine – PMC

Posted: March 25, 2022 at 2:05 am

Stem Cell Investig. 2019; 6: 19.

1Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran;

2Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran;

3Hematology and Oncology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

1Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran;

2Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran;

3Hematology and Oncology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

Contributions: (I) Conception and design: E Fathi, R Farahzadi; (II) Administrative support: E Fathi, R Farahzadi; (III) Provision of study materials or patients: None; (IV) Collection and assembly of data: R Farahzadi, N Rajabzadeh; (V) Data analysis and interpretation: All authors; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors.

#These authors contributed equally to this work.

Received 2018 Nov 11; Accepted 2019 Mar 17.

Recent developments in the stem cell biology provided new hopes in treatment of diseases and disorders that yet cannot be treated. Stem cells have the potential to differentiate into various cell types in the body during age. These provide new cells for the body as it grows, and replace specialized cells that are damaged. Since mesenchymal stem cells (MSCs) can be easily harvested from the adipose tissue and can also be cultured and expanded in vitro they have become a good target for tissue regeneration. These cells have been widespread used for cell transplantation in animals and also for clinical trials in humans. The purpose of this review is to provide a summary of our current knowledge regarding the important and types of isolated stem cells from different sources of animal models such as horse, pig, goat, dog, rabbit, cat, rat, mice etc. In this regard, due to the widespread use and lot of attention of MSCs, in this review, we will elaborate on use of MSCs in veterinary medicine as well as in regenerative medicine. Based on the studies in this field, MSCs found wide application in treatment of diseases, such as heart failure, wound healing, tooth regeneration etc.

Keywords: Mesenchymal stem cells (MSCs), animal model, cell-based therapy, regenerative medicine

Stem cells are one of the main cells of the human body that have ability to grow more than 200 types of body cells (1). Stem cells, as non-specialized cells, can be transformed into highly specialized cells in the body (2). In the other words, Stem cells are undifferentiated cells with self-renewal potential, differentiation into several types of cells and excessive proliferation (3). In the past, it was believed that stem cells can only differentiate into mature cells of the same organ. Today, there are many evidences to show that stem cells can differentiate into the other types of cell as well as ectoderm, mesoderm and endoderm. The numbers of stem cells are different in the tissues such as bone marrow, liver, heart, kidney, and etc. (3,4). Over the past 20 years, much attention has been paid to stem cell biology. Therefore, there was a profound increase in the understanding of its characteristics and the therapeutic potential for its application (5). Today, the utilization of these cells in experimental research and cell therapy represents in such disorders including hematological, skin regeneration and heart disease in both human and veterinary medicine (6).The history of stem cells dates back to the 1960s, when Friedenstein and colleagues isolated, cultured and differentiated to osteogenic cell lineage of bone marrow-derived cells from guinea pigs (7). This project created a new perspective on stem cell research. In the following, other researchers discovered that the bone marrow contains fibroblast-like cells with congenic potential in vitro, which were capable of forming colonies (CFU-F) (8). For over 60 years, transplantation of hematopoietic stem cells (HSCs) has been the major curative therapy for several genetic and hematological disorders (9). Almost in 1963, Till and McCulloch described a single progenitor cell type in the bone marrow which expand clonally and give rise to all lineages of hematopoietic cells. This research represented the first characterization of the HSCs (10). Also, the identification of mouse embryonic stem cells (ESCs) in 1981 revolutionized the study of developmental biology, and mice are now used extensively as one of the best option to study stem cell biology in mammals (11). Nevertheless, their application a model, have limitations in the regenerative medicine. But this model, relatively inexpensive and can be easily manipulated genetically (12). Failure to obtain a satisfactory result in the selection of many mouse models, to recapitulate particular human disease phenotypes, has forced researchers to investigate other animal species to be more probably predictive of humans (13). For this purpose, to study the genetic diseases, the pig has been currently determined as one the best option of a large animal model (14).

Stem cells, based on their differentiation ability, are classified into different cell types, including totipotent, pluripotent, multipotent, or unipotent. Also, another classification of these cells are based on the evolutionary stages, including embryonic, fetal, infant or umbilical cord blood and adult stem cells (15). shows an overview of stem cells classifications based on differentiation potency.

An overview of the stem cell classification. Totipotency: after fertilization, embryonic stem cells (ESCs) maintain the ability to form all three germ layers as well as extra-embryonic tissues or placental cells and are termed as totipotent. Pluripotency: these more specialized cells of the blastocyst stage maintain the ability to self-renew and differentiate into the three germ layers and down many lineages but do not form extra-embryonic tissues or placental cells. Multipotency: adult or somatic stem cells are undifferentiated cells found in postnatal tissues. These specialized cells are considered to be multipotent; with very limited ability to self-renew and are committed to lineage species.

Toti-potent cells have the potential for development to any type of cell found in the organism. In the other hand, the capacity of these cells to develop into the three primary germ cell layers of the embryo and into extra-embryonic tissues such as the placenta is remarkable (15).

The pluripotent stem cells are kind of stem cells with the potential for development to approximately all cell types. These cells contain ESCs and cells that are isolated from the mesoderm, endoderm and ectoderm germ layers that are organized in the beginning period of ESC differentiation (15).

The multipotent stem cells have less proliferative potential than the previous two groups and have ability to produce a variety of cells which limited to a germinal layer [such as mesenchymal stem cells (MSCs)] or just a specific cell line (such as HSCs). Adult stem cells are also often in this group. In the word, these cells have the ability to differentiate into a closely related family of cells (15).

Despite the increasing interest in totipotent and pluripotent stem cells, unipotent stem cells have not received the most attention in research. A unipotent stem cell is a cell that can create cells with only one lineage differentiation. Muscle stem cells are one of the example of this type of cell (15). The word uni is derivative from the Latin word unus meaning one. In adult tissues in comparison with other types of stem cells, these cells have the lowest differentiation potential. The unipotent stem cells could create one cell type, in the other word, these cells do not have the self-renewal property. Furthermore, despite their limited differentiation potential, these cells are still candidates for treatment of various diseases (16).

ESCs are self-renewing cells that derived from the inner cell mass of a blastocyst and give rise to all cells during human development. It is mentioned that these cells, including human embryonic cells, could be used as suitable, promising source for cell transplantation and regenerative medicine because of their unique ability to give rise to all somatic cell lineages (17). In the other words, ESCs, pluripotent cells that can differentiate to form the specialized of the various cell types of the body (18). Also, ESCs capture the imagination because they are immortal and have an almost unlimited developmental potential. Due to the ethical limitation on embryo sampling and culture, these cells are used less in research (19).

HSCs are multipotent cells that give rise to blood cells through the process of hematopoiesis (20). These cells reside in the bone marrow and replenish all adult hematopoietic lineages throughout the lifetime of the human and animal (21). Also, these cells can replenish missing or damaged components of the hematopoietic and immunologic system and can withstand freezing for many years (22).The mammalian hematopoietic system containing more than ten different mature cell types that HSCs are one of the most important members of this. The ability to self-renew and multi-potency is another specific feature of these cells (23).

Adult stem cells, as undifferentiated cells, are found in numerous tissues of the body after embryonic development. These cells multiple by cell division to regenerate damaged tissues (24). Recent studies have been shown that adult stem cells may have the ability to differentiate into cell types from various germ layers. For example, bone marrow stem cells which is derived from mesoderm, can differentiate into cell lineage derived mesoderm and endoderm such as into lung, liver, GI tract, skin, etc. (25). Another example of adult stem cells is neural stem cells (NSCs), which is derived from ectoderm and can be differentiate into another lineage such as mesoderm and endoderm (26). Therapeutic potential of adult stem cells in cell therapy and regenerative medicine has been proven (27).

For the first time in the late 1990s, CSCs were identified by John Dick in acute myeloid diseases. CSCs are cancerous cells that found within tumors or hematological cancers. Also, these cells have the characteristics of normal stem cells and can also give rise to all cell types found in a particular cancer sample (28). There is an increasing evidence supporting the CSCs hypothesis. Normal stem cells in an adult living creature are responsible for the repair and regeneration of damaged as well as aged tissues (29). Many investigations have reported that the capability of a tumor to propagate and proliferate relies on a small cellular subpopulation characterized by stem-like properties, named CSCs (30).

Embryonic connective tissue contains so-called mesenchymes, from which with very close interactions of endoderm and ectoderm all other connective and hematopoietic tissues originate, Whereas, MSCs do not differentiate into hematopoietic cell (31). In 1924, Alexander A. Maxi mow used comprehensive histological detection to identify a singular type of precursor cell within mesenchyme that develops into various types of blood cells (32). In general, MSCs are type of cells with potential of multi-lineage differentiation and self-renewal, which exist in many different kinds of tissues and organs such as adipose tissue, bone marrow, skin, peripheral blood, fallopian tube, cord blood, liver and lung et al. (4,5). Today, stem cells are used for different applications. In addition to using these cells in human therapy such as cell transplantation, cell engraftment etc. The use of stem cells in veterinary medicine has also been considered. The purpose of this review is to provide a summary of our current knowledge regarding the important and types of isolated stem cells from different sources of animal models such as horse, pig, goat, dog, rabbit, cat, rat, mice etc. In this regard, due to the widespread use and lot of attention of MSCs, in this review, we will elaborate on use of MSCs in veterinary medicine.

The isolation method, maintenance and culture condition of MSCs differs from the different tissues, these methods as well as characterization of MSCs described as (36). MSCs could be isolated from the various tissues such as adipose tissue, bone marrow, umbilical cord, amniotic fluid etc. (37).

Diagram for adipose tissue-derived mesenchymal stem cell isolation (3).

Diagram for bone marrow-derived MSCs isolation (33). MSC, mesenchymal stem cell.

Diagram for umbilical cord-derived MSCs isolation (34). MSC, mesenchymal stem cell.

Diagram for isolation of amniotic fluid stem cells (AFSCs) (35).

Diagram for MSCs characterization (35). MSC, mesenchymal stem cell.

The diversity of stem cell or MSCs sources and a wide aspect of potential applications of these cells cause to challenge for selecting an appropriate cell type for cell therapy (38). Various diseases in animals have been treated by cell-based therapy. However, there are immunity concerns regarding cell therapy using stem cells. Improving animal models and selecting suitable methods for engraftment and transplantation could help address these subjects, facilitating eventual use of stem cells in the clinic. Therefore, for this purpose, in this section of this review, we provide an overview of the current as well as previous studies for future development of animal models to facilitate the utilization of stem cells in regenerative medicine (14). Significant progress has been made in stem cells-based regenerative medicine, which enables researchers to treat those diseases which cannot be cured by conventional medicines. The unlimited self-renewal and multi-lineage differentiation potential to other types of cells causes stem cells to be frontier in regenerative medicine (24). More researches in regenerative medicine have been focused on human cells including embryonic as well as adult stem cells or maybe somatic cells. Today there are versions of embryo-derived stem cells that have been reprogrammed from adult cells under the title of pluripotent cells (39). Stem cell therapy has been developed in the last decade. Nevertheless, obstacles including unwanted side effects due to the migration of transplanted cells as well as poor cell survival have remained unresolved. In order to overcome these problems, cell therapy has been introduced using biocompatible and biodegradable biomaterials to reduce cell loss and long-term in vitro retention of stem cells.

Currently in clinical trials, these biomaterials are widely used in drug and cell-delivery systems, regenerative medicine and tissue engineering in which to prevent the long-term survival of foreign substances in the body the release of cells are controlled (40).

Today, the incidence and prevalence of heart failure in human societies is a major and increasing problem that unfortunately has a poor prognosis. For decades, MSCs have been used for cardiovascular regenerative therapy as one of the potential therapeutic agents (41). Dhein et al. [2006] found that autologous bone marrow-derived mesenchymal stem cells (BMSCs) transplantation improves cardiac function in non-ischemic cardiomyopathy in a rabbit model. In one study, Davies et al. [2010] reported that transplantation of cord blood stem cells in ovine model of heart failure, enhanced the function of heart through improvement of right ventricular mass, both systolic and diastolic right heart function (42). In another study, Nagaya et al. [2005] found that MSCs dilated cardiomyopathy (DCM), possibly by inducing angiogenesis and preventing cardial fibrosis. MSCs have a tremendous beneficial effect in cell transplantation including in differentiating cardiomyocytes, vascular endothelial cells, and providing anti-apoptotic as well angiogenic mediators (43). Roura et al. [2015] shown that umbilical cord blood mesenchymal stem cells (UCBMSCs) are envisioned as attractive therapeutic candidates against human disorders progressing with vascular deficit (44). Ammar et al., [2015] compared BMSCs with adipose tissue-derived MSCs (ADSCs). It was demonstrated that both BMSCs and ADSCs were equally effective in mitigating doxorubicin-induced cardiac dysfunction through decreasing collagen deposition and promoting angiogenesis (45).

There are many advantages of small animal models usage in cardiovascular research compared with large animal models. Small model of animals has a short life span, which allow the researchers to follow the natural history of the disease at an accelerated pace. Some advantages and disadvantages are listed in (46).

Despite of the small animal model, large animal models are suitable models for studies of human diseases. Some advantages and disadvantages of using large animal models in a study protocol planning was elaborated in (47).

Chronic wound is one of the most common problem and causes significant distress to patients (48). Among the types of tissues that stem cells derived it, dental tissuederived MSCs provide good sources of cytokines and growth factors that promote wound healing. The results of previous studies showed that stem cells derived deciduous teeth of the horse might be a novel approach for wound care and might be applied in clinical treatment of non-healing wounds (49). However, the treatment with stem cells derived deciduous teeth needs more research to understand the underlying mechanisms of effective growth factors which contribute to the wound healing processes (50). This preliminary investigation suggests that deciduous teeth-derived stem cells have the potential to promote wound healing in rabbit excisional wound models (49). In the another study, Lin et al. [2013] worked on the mouse animal model and showed that ADSCs present a potentially viable matrix for full-thickness defect wound healing (51).

Many studies have been done on dental reconstruction with MSCs. In one study, Khorsand et al. [2013] reported that dental pulp-derived stem cells (DPSCs) could promote periodontal regeneration in canine model. Also, it was shown that canine DPSCs were successfully isolated and had the rapid proliferation and multi-lineage differentiation capacity (52). Other application of dental-derived stem cells is shown in .

Diagram for application of dental stem cell in dentistry/regenerative medicine (53).

As noted above, stem cells have different therapeutic applications and self-renewal capability. These cells can also differentiate into the different cell types. There is now a great hope that stem cells can be used to treat diseases such as Alzheimer, Parkinson and other serious diseases. In stem cell-based therapy, ESCs are essentially targeted to differentiate into functional neural cells. Today, a specific category of stem cells called induced pluripotent stem (iPS) cells are being used and tested to generate functional dopamine neurons for treating Parkinson's disease of a rat animal model. In addition, NSC as well as MSCs are being used in neurodegenerative disorder therapies for Alzheimers disease, Parkinsons disease, and stroke (54). Previous studies have shown that BMSCs could reduce brain amyloid deposition and accelerate the activation of microglia in an acutely induced Alzheimers disease in mouse animal model. Lee et al. [2009] reported that BMSCs can increase the number of activated microglia, which effective therapeutic vehicle to reduce A deposits in AD patients (55). In confirmation of previous study, Liu et al. [2015] showed that transplantation of BMSCs in brain of mouse model of Alzheimers disease cause to decrease in amyloid beta deposition, increase in brain-derived neurotrophic factor (BDNF) levels and improvements in social recognition (56). In addition of BMSCs, NSCs have been proposed as tools for treating neurodegeneration disease because of their capability to create an appropriate cell types which transplanted. kerud et al. [2001] demonstrated that NSCs efficiently express high level of glial cell line-derived neurotrophic factor (GDNF) in vivo, suggesting a use of these cells in the treatment of neurodegenerative disorders, including Parkinsons disease (57). In the following, Venkataramana et al. [2010] transplanted BMSCs into the sub lateral ventricular zones of seven Parkinsons disease patients and reported encouraging results (58).

The human body is fortified with specialized cells named MSCs, which has the ability to self-renew and differentiate into various cell types including, adipocyte, osteocyte, chondrocyte, neurons etc. In addition to mentioned properties, these cells can be easily isolated, safely transplanted to injured sites and have the immune regulatory properties. Numerous in vitro and in vivo studies in animal models have successfully demonstrated the potential of MSCs for various diseases; however, the clinical outcomes are not very encouraging. Based on the studies in the field of stem cells, MSCs find wide application in treatment of diseases, such as heart failure, wound healing, tooth regeneration and etc. In addition, these cells are particularly important in the treatment of the sub-branch neurodegenerative diseases like Alzheimer and Parkinson.

The authors wish to thank staff of the Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.

Funding: The project described was supported by Grant Number IR.TBZMED.REC.1396.1218 from the Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Conflicts of Interest: The authors have no conflicts of interest to declare.

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Stem cell-based regenerative medicine - PMC

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Stem-cell therapy – Wikipedia

Posted: December 24, 2021 at 2:06 am

This article is about the medical therapy. For the cell type, see Stem cell.

Use of stem cells to treat or prevent a disease or condition

Stem-cell therapy is the use of stem cells to treat or prevent a disease or condition.[1] As of 2016[update], the only established therapy using stem cells is hematopoietic stem cell transplantation.[2] This usually takes the form of a bone-marrow transplantation, but the cells can also be derived from umbilical cord blood. Research is underway to develop various sources for stem cells as well as to apply stem-cell treatments for neurodegenerative diseases[3] and conditions such as diabetes and heart disease.

Stem-cell therapy has become controversial following developments such as the ability of scientists to isolate and culture embryonic stem cells, to create stem cells using somatic cell nuclear transfer and their use of techniques to create induced pluripotent stem cells. This controversy is often related to abortion politics and to human cloning. Additionally, efforts to market treatments based on transplant of stored umbilical cord blood have been controversial.

For over 30 years, hematopoietic stem cell transplantation (HSCT) has been used to treat people with conditions such as leukaemia and lymphoma; this is the only widely practiced form of stem-cell therapy.[4][5][6] During chemotherapy, most growing cells are killed by the cytotoxic agents. These agents, however, cannot discriminate between the leukaemia or neoplastic cells, and the hematopoietic stem cells within the bone marrow. This is the side effect of conventional chemotherapy strategies that the stem-cell transplant attempts to reverse; a donor's healthy bone marrow reintroduces functional stem cells to replace the cells lost in the host's body during treatment. The transplanted cells also generate an immune response that helps to kill off the cancer cells; this process can go too far, however, leading to graft vs host disease, the most serious side effect of this treatment.[7]

Another stem-cell therapy, called Prochymal, was conditionally approved in Canada in 2012 for the management of acute graft-vs-host disease in children who are unresponsive to steroids.[8] It is an allogenic stem therapy based on mesenchymal stem cells (MSCs) derived from the bone marrow of adult donors. MSCs are purified from the marrow, cultured and packaged, with up to 10,000 doses derived from a single donor. The doses are stored frozen until needed.[9]

The FDA has approved five hematopoietic stem-cell products derived from umbilical-cord blood, for the treatment of blood and immunological diseases.[10]

In 2014, the European Medicines Agency recommended approval of limbal stem cells for people with severe limbal stem cell deficiency due to burns in the eye.[11]

Stem cells are being studied for a number of reasons. The molecules and exosomes released from stem cells are also being studied in an effort to make medications.[12] In addition to the functions of the cells themselves, paracrine soluble factors produced by stem cells, known as the stem cell secretome, have been found to be another mechanism by which stem cell-based therapies mediate their effects in degenerative, autoimmune, and inflammatory diseases.[13]

To be used for research or treatment applications, large numbers of high-quality stem cells are needed. Thus, it is necessary to develop culture systems which produce pure populations of tissue-specific stem-cells in vitro without the loss of stem-cell potential. Two main approaches are taken for this purpose: two-dimensional and three-dimensional cell culture.[14]

Cell culture in two dimensions has been routinely performed in thousands of laboratories worldwide for the past four decades. In two-dimensional platforms, cells are typically exposed to a solid, rigid flat surface on the basal side and to liquid at the apicalsurface. Inhabiting such a two-dimensional rigid substrate requires a dramatic adaption for the surviving cells because they lack the extracellular matrix that is unique to each cell type and which may alter cell metabolism and reduce its functionality.[14]

Three-dimensional cell culture systems may create a biomimicking microenvironment for stem cells, resembling their native three-dimensional extracellular matrix (ECM). Advanced biomaterials have significantly contributed to three-dimensional cell culture systems in recent decades, and more unique and complex biomaterials have been proposed for improving stem-cell proliferation and controlled differentiation. Among them, nanostructured biomaterials are of particular interest because they have the advantage of a high surface-to-volume ratio, and they mimic the physical and biological features of natural ECM at the nanoscale.[14]

Research has been conducted on the effects of stem cells on animal models of brain degeneration, such as in Parkinson's disease, Amyotrophic lateral sclerosis, and Alzheimer's disease.[15][16][17] Preliminary studies related to multiple sclerosis have been conducted,[18][19][20] and a 2020 phase 2 trial found significantly improved outcomes for mesenchymal stem cell treated patients compared to those receiving a sham treatment.[21] In January 2021 the FDA approved the first clinical trial for an investigational stem cell therapy to restore lost brain cells in people with advanced Parkinsons disease.[22]

Healthy adult brains contain neural stem cells, which divide to maintain general stem-cell numbers, or become progenitor cells. In healthy adult laboratory animals, progenitor cells migrate within the brain and function primarily to maintain neuron populations for olfaction (the sense of smell). Pharmacological activation of endogenous neural stem cells has been reported to induce neuroprotection and behavioral recovery in adult rat models of neurological disorder.[23][24][25]

Stroke and traumatic brain injury lead to cell death, characterized by a loss of neurons and oligodendrocytes within the brain. Clinical and animal studies have been conducted into the use of stem cells in cases of spinal cord injury.[26][27][28][20]

A small-scale study on individuals 60 year or older with aging frailty showed, after intravenous treatment with MSCs from healthy young donors, showed significant improvements in physical performance measures.[29]

Stem cells are studied in people with severe heart disease.[30] The work by Bodo-Eckehard Strauer[31] was discredited by identifying hundreds of factual contradictions.[32] Among several clinical trials reporting that adult stem cell therapy is safe and effective, actual evidence of benefit has been reported from only a few studies.[33] Some preliminary clinical trials achieved only modest improvements in heart function following use of bone marrow stem cell therapy.[34][35]

Stem-cell therapy for treatment of myocardial infarction usually makes use of autologous bone-marrow stem cells, but other types of adult stem cells may be used, such as adipose-derived stem cells.[36]

Possible mechanisms of recovery include:[15]

In 2013, studies of autologous bone-marrow stem cells on ventricular function were found to contain "hundreds" of discrepancies.[37] Critics report that of 48 reports, just five underlying trials seemed to be used, and that in many cases whether they were randomized or merely observational accepter-versus-rejecter, was contradictory between reports of the same trial. One pair of reports of identical baseline characteristics and final results, was presented in two publications as, respectively, a 578-patient randomized trial and as a 391-subject observational study. Other reports required (impossible) negative standard deviations in subsets of people, or contained fractional subjects, negative NYHA classes. Overall, many more people were reported as having receiving stem cells in trials, than the number of stem cells processed in the hospital's laboratory during that time. A university investigation, closed in 2012 without reporting, was reopened in July 2013.[38]

In 2014, a meta-analysis on stem cell therapy using bone-marrow stem cells for heart disease revealed discrepancies in published clinical trial reports, whereby studies with a higher number of discrepancies showed an increase in effect sizes.[39] Another meta-analysis based on the intra-subject data of 12 randomized trials was unable to find any significant benefits of stem cell therapy on primary endpoints, such as major adverse events or increase in heart function measures, concluding there was no benefit.[40]

The TIME trial, which used a randomized, double-blind, placebo-controlled trial design, concluded that "bone marrow mononuclear cells administration did not improve recovery of LV function over 2 years" in people who had a myocardial infarction.[41] Accordingly, the BOOST-2 trial conducted in 10 medical centers in Germany and Norway reported that the trial result "does not support the use of nucleated BMCs in patients with STEMI and moderately reduced LVEF".[42] Furthermore, the trial also did not meet any other secondary MRI endpoints,[43] leading to a conclusion that intracoronary bone marrow stem cell therapy does not offer a functional or clinical benefit.[44]

The specificity of the human immune-cell repertoire is what allows the human body to defend itself from rapidly adapting antigens. However, the immune system is vulnerable to degradation upon the pathogenesis of disease, and because of the critical role that it plays in overall defense, its degradation is often fatal to the organism as a whole. Diseases of hematopoietic cells are diagnosed and classified via a subspecialty of pathology known as hematopathology. The specificity of the immune cells is what allows recognition of foreign antigens, causing further challenges in the treatment of immune disease. Identical matches between donor and recipient must be made for successful transplantation treatments, but matches are uncommon, even between first-degree relatives. Research using both hematopoietic adult stem cells and embryonic stem cells has provided insight into the possible mechanisms and methods of treatment for many of these ailments.[45]

Fully mature human red blood cells may be generated ex vivo by hematopoietic stem cells (HSCs), which are precursors of red blood cells. In this process, HSCs are grown together with stromal cells, creating an environment that mimics the conditions of bone marrow, the natural site of red-blood-cell growth. Erythropoietin, a growth factor, is added, coaxing the stem cells to complete terminal differentiation into red blood cells.[46] Further research into this technique should have potential benefits to gene therapy, blood transfusion, and topical medicine.

In 2004, scientists at King's College London discovered a way to cultivate a complete tooth in mice[47] and were able to grow bioengineered teeth stand-alone in the laboratory. Researchers are confident that the tooth regeneration technology can be used to grow live teeth in people.

In theory, stem cells taken from the patient could be coaxed in the lab turning into a tooth bud which, when implanted in the gums, will give rise to a new tooth, and would be expected to be grown in a time over three weeks.[48] It will fuse with the jawbone and release chemicals that encourage nerves and blood vessels to connect with it. The process is similar to what happens when humans grow their original adult teeth. Many challenges remain, however, before stem cells could be a choice for the replacement of missing teeth in the future.[49][50]

Heller has reported success in re-growing cochlea hair cells with the use of embryonic stem cells.[51]

In a 2019 review that looked at hearing regeneration and regenerative medicine, stem cell-derived otic progenitors have the potential to greatly improve hearing.[52]

Since 2003, researchers have successfully transplanted corneal stem cells into damaged eyes to restore vision. "Sheets of retinal cells used by the team are harvested from aborted fetuses, which some people find objectionable." When these sheets are transplanted over the damaged cornea, the stem cells stimulate renewed repair, eventually restore vision.[53] The latest such development was in June 2005, when researchers at the Queen Victoria Hospital of Sussex, England were able to restore the sight of forty people using the same technique. The group, led by Sheraz Daya, was able to successfully use adult stem cells obtained from the patient, a relative, or even a cadaver. Further rounds of trials are ongoing.[54]

People with Type 1 diabetes lose the function of insulin-producing beta cells within the pancreas.[55] In recent experiments, scientists have been able to coax embryonic stem cell to turn into beta cells in the lab. In theory if the beta cell is transplanted successfully, they will be able to replace malfunctioning ones in a diabetic patient.[56]

Use of mesenchymal stem cells (MSCs) derived from adult stem cells is under preliminary research for potential orthopedic applications in bone and muscle trauma, cartilage repair, osteoarthritis, intervertebral disc surgery, rotator cuff surgery, and musculoskeletal disorders, among others.[57] Other areas of orthopedic research for uses of MSCs include tissue engineering and regenerative medicine.[57]

Stem cells can also be used to stimulate the growth of human tissues. In an adult, wounded tissue is most often replaced by scar tissue, which is characterized in the skin by disorganized collagen structure, loss of hair follicles and irregular vascular structure. In the case of wounded fetal tissue, however, wounded tissue is replaced with normal tissue through the activity of stem cells.[58] A possible method for tissue regeneration in adults is to place adult stem cell "seeds" inside a tissue bed "soil" in a wound bed and allow the stem cells to stimulate differentiation in the tissue bed cells. This method elicits a regenerative response more similar to fetal wound-healing than adult scar tissue formation.[58] Researchers are still investigating different aspects of the "soil" tissue that are conducive to regeneration.[58] Because of the general healing capabilities of stem cells, they have gained interest for the treatment of cutaneous wounds, such as in skin cancer.[59]

Destruction of the immune system by the HIV is driven by the loss of CD4+ T cells in the peripheral blood and lymphoid tissues. Viral entry into CD4+ cells is mediated by the interaction with a cellular chemokine receptor, the most common of which are CCR5 and CXCR4. Because subsequent viral replication requires cellular gene expression processes, activated CD4+ cells are the primary targets of productive HIV infection.[60] Recently scientists have been investigating an alternative approach to treating HIV-1/AIDS, based on the creation of a disease-resistant immune system through transplantation of autologous, gene-modified (HIV-1-resistant) hematopoietic stem and progenitor cells (GM-HSPC).[61]

Stem cells are thought to mediate repair via five primary mechanisms: 1) providing an anti-inflammatory effect, 2) homing to damaged tissues and recruiting other cells, such as endothelial progenitor cells, that are necessary for tissue growth, 3) supporting tissue remodeling over scar formation, 4) inhibiting apoptosis, and 5) differentiating into bone, cartilage, tendon, and ligament tissue.[62][63]

To further enrich blood supply to the damaged areas, and consequently promote tissue regeneration, platelet-rich plasma could be used in conjunction with stem cell transplantation.[64][65] The efficacy of some stem cell populations may also be affected by the method of delivery; for instance, to regenerate bone, stem cells are often introduced in a scaffold where they produce the minerals necessary for generation of functional bone.[64][65][66][67]

Stem cells have also been shown to have a low immunogenicity due to the relatively low number of MHC molecules found on their surface. In addition, they have been found to secrete chemokines that alter the immune response and promote tolerance of the new tissue. This allows for allogeneic treatments to be performed without a high rejection risk.[68]

The ability to grow up functional adult tissues indefinitely in culture through Directed differentiation creates new opportunities for drug research. Researchers are able to grow up differentiated cell lines and then test new drugs on each cell type to examine possible interactions in vitro before performing in vivo studies. This is critical in the development of drugs for use in veterinary research because of the possibilities of species-specific interactions. The hope is that having these cell lines available for research use will reduce the need for research animals used because effects on human tissue in vitro will provide insight not normally known before the animal testing phase.[69]

Stem cells are being explored for use in conservation efforts. Spermatogonial stem cells have been harvested from a rat and placed into a mouse host and fully mature sperm were produced with the ability to produce viable offspring. Currently research is underway to find suitable hosts for the introduction of donor spermatogonial stem cells. If this becomes a viable option for conservationists, sperm can be produced from high genetic quality individuals who die before reaching sexual maturity, preserving a line that would otherwise be lost.[70]

Most stem cells intended for regenerative therapy are generally isolated either from the patient's bone marrow or from adipose tissue.[65][67] Mesenchymal stem cells can differentiate into the cells that make up bone, cartilage, tendons, and ligaments, as well as muscle, neural and other progenitor tissues. They have been the main type of stem cells studied in the treatment of diseases affecting these tissues.[71][72] The number of stem cells transplanted into damaged tissue may alter the efficacy of treatment. Accordingly, stem cells derived from bone marrow aspirates, for instance, are cultured in specialized laboratories for expansion to millions of cells.[65][67] Although adipose-derived tissue also requires processing prior to use, the culturing methodology for adipose-derived stem cells is not as extensive as that for bone marrow-derived cells.[73] While it is thought that bone-marrow-derived stem cells are preferred for bone, cartilage, ligament, and tendon repair, others believe that the less challenging collection techniques and the multi-cellular microenvironment already present in adipose-derived stem cell fractions make the latter the preferred source for autologous transplantation.[64]

New sources of mesenchymal stem cells are being researched, including stem cells present in the skin and dermis which are of interest because of the ease at which they can be harvested with minimal risk to the animal.[74] Hematopoietic stem cells have also been discovered to be travelling in the blood stream and possess equal differentiating ability as other mesenchymal stem cells, again with a very non-invasive harvesting technique.[75]

There has been more recent interest in the use of extra embryonic mesenchymal stem cells. Research is underway to examine the differentiating capabilities of stem cells found in the umbilical cord, yolk sac and placenta of different animals. These stem cells are thought to have more differentiating ability than their adult counterparts, including the ability to more readily form tissues of endodermal and ectodermal origin.[68]

There is widespread controversy over the use of human embryonic stem cells. This controversy primarily targets the techniques used to derive new embryonic stem cell lines, which often requires the destruction of the blastocyst. Opposition to the use of human embryonic stem cells in research is often based on philosophical, moral, or religious objections.[76] There is other stem cell research that does not involve the destruction of a human embryo, and such research involves adult stem cells, amniotic stem cells, and induced pluripotent stem cells.

On 23 January 2009, the US Food and Drug Administration gave clearance to Geron Corporation for the initiation of the first clinical trial of an embryonic stem-cell-based therapy on humans. The trial aimed to evaluate the drug GRNOPC1, embryonic stem cell-derived oligodendrocyte progenitor cells, on people with acute spinal cord injury. The trial was discontinued in November 2011 so that the company could focus on therapies in the "current environment of capital scarcity and uncertain economic conditions".[77] In 2013 biotechnology and regenerative medicine company BioTime (AMEX:BTX) acquired Geron's stem cell assets in a stock transaction, with the aim of restarting the clinical trial.[78]

Scientists have reported that MSCs when transfused immediately within few hours post thawing may show reduced function or show decreased efficacy in treating diseases as compared to those MSCs which are in log phase of cell growth (fresh), so cryopreserved MSCs should be brought back into log phase of cell growth in invitro culture before administration. Re-culturing of MSCs will help in recovering from the shock the cells get during freezing and thawing. Various MSC clinical trials which used cryopreserved product immediately post thaw have failed as compared to those clinical trials which used fresh MSCs.[79]

Research has been conducted on horses, dogs, and cats can benefit the development of stem cell treatments in veterinary medicine and can target a wide range of injuries and diseases such as myocardial infarction, stroke, tendon and ligament damage, osteoarthritis, osteochondrosis and muscular dystrophy both in large animals, as well as humans.[80][81][82][83] While investigation of cell-based therapeutics generally reflects human medical needs, the high degree of frequency and severity of certain injuries in racehorses has put veterinary medicine at the forefront of this novel regenerative approach.[84] Companion animals can serve as clinically relevant models that closely mimic human disease.[85][86]

Veterinary applications of stem cell therapy as a means of tissue regeneration have been largely shaped by research that began with the use of adult-derived mesenchymal stem cells to treat animals with injuries or defects affecting bone, cartilage, ligaments and/or tendons.[87][71][88] There are two main categories of stem cells used for treatments: allogeneic stem cells derived from a genetically different donor within the same species[67][89] and autologous mesenchymal stem cells, derived from the patient prior to use in various treatments.[64] A third category, xenogenic stem cells, or stem cells derived from different species, are used primarily for research purposes, especially for human treatments.[69]

Bone has a unique and well documented natural healing process that normally is sufficient to repair fractures and other common injuries. Misaligned breaks due to severe trauma, as well as treatments like tumor resections of bone cancer, are prone to improper healing if left to the natural process alone. Scaffolds composed of natural and artificial components are seeded with mesenchymal stem cells and placed in the defect. Within four weeks of placing the scaffold, newly formed bone begins to integrate with the old bone and within 32 weeks, full union is achieved.[90] Further studies are necessary to fully characterize the use of cell-based therapeutics for treatment of bone fractures.

Stem cells have been used to treat degenerative bone diseases. The normally recommended treatment for dogs that have LeggCalvePerthes disease is to remove the head of the femur after the degeneration has progressed. Recently, mesenchymal stem cells have been injected directly in to the head of the femur, with success not only in bone regeneration, but also in pain reduction.[90]

Autologous stem cell-based treatments for ligament injury, tendon injury, osteoarthritis, osteochondrosis, and sub-chondral bone cysts have been commercially available to practicing veterinarians to treat horses since 2003 in the United States and since 2006 in the United Kingdom. Autologous stem cell based treatments for tendon injury, ligament injury, and osteoarthritis in dogs have been available to veterinarians in the United States since 2005. Over 3000 privately owned horses and dogs have been treated with autologous adipose-derived stem cells. The efficacy of these treatments has been shown in double-blind clinical trials for dogs with osteoarthritis of the hip and elbow and horses with tendon damage.[91][92]

Race horses are especially prone to injuries of the tendon and ligaments. Conventional therapies are very unsuccessful in returning the horse to full functioning potential. Natural healing, guided by the conventional treatments, leads to the formation of fibrous scar tissue that reduces flexibility and full joint movement. Traditional treatments prevented a large number of horses from returning to full activity and also have a high incidence of re-injury due to the stiff nature of the scarred tendon. Introduction of both bone marrow and adipose derived stem cells, along with natural mechanical stimulus promoted the regeneration of tendon tissue. The natural movement promoted the alignment of the new fibers and tendocytes with the natural alignment found in uninjured tendons. Stem cell treatment not only allowed more horses to return to full duty and also greatly reduced the re-injury rate over a three-year period.[68]

The use of embryonic stem cells has also been applied to tendon repair. The embryonic stem cells were shown to have a better survival rate in the tendon as well as better migrating capabilities to reach all areas of damaged tendon. The overall repair quality was also higher, with better tendon architecture and collagen formed. There was also no tumor formation seen during the three-month experimental period. Long-term studies need to be carried out to examine the long-term efficacy and risks associated with the use of embryonic stem cells.[68] Similar results have been found in small animals.[68]

Osteoarthritis is the main cause of joint pain both in animals and humans. Horses and dogs are most frequently affected by arthritis. Natural cartilage regeneration is very limited. Different types of mesenchymal stem cells and other additives are still being researched to find the best type of cell and method for long-term treatment.[68]

Adipose-derived mesenchymal cells are currently the most often used for stem cell treatment of osteoarthritis because of the non-invasive harvesting. This is a recently developed, non-invasive technique developed for easier clinical use. Dogs receiving this treatment showed greater flexibility in their joints and less pain.[93]

Stem cells have successfully been used to ameliorate healing in the heart after myocardial infarction in dogs. Adipose and bone marrow derived stem cells were removed and induced to a cardiac cell fate before being injected into the heart. The heart was found to have improved contractility and a reduction in the damaged area four weeks after the stem cells were applied.[94]

A different trial is underway for a patch made of a porous substance onto which the stem cells are "seeded" in order to induce tissue regeneration in heart defects. Tissue was regenerated and the patch was well incorporated into the heart tissue. This is thought to be due, in part, to improved angiogenesis and reduction of inflammation. Although cardiomyocytes were produced from the mesenchymal stem cells, they did not appear to be contractile. Other treatments that induced a cardiac fate in the cells before transplanting had greater success at creating contractile heart tissue.[95]

Recent research, such as the European nTRACK research project, aims to demonstrate that multimodal nanoparticles can structurally and functionally track stem cell in muscle regeneration therapy. The idea is to label stem cells with gold nano-particles that are fully characterised for uptake, functionality, and safety. The labelled stem cells will be injected into an injured muscle and tracked using imaging systems.[96] However, the system still needs to be demonstrated at lab scale.

Spinal cord injuries are one of the most common traumas brought into veterinary hospitals.[90] Spinal injuries occur in two ways after the trauma: the primary mechanical damage, and in secondary processes, like inflammation and scar formation, in the days following the trauma. These cells involved in the secondary damage response secrete factors that promote scar formation and inhibit cellular regeneration. Mesenchymal stem cells that are induced to a neural cell fate are loaded onto a porous scaffold and are then implanted at the site of injury. The cells and scaffold secrete factors that counteract those secreted by scar forming cells and promote neural regeneration. Eight weeks later, dogs treated with stem cells showed immense improvement over those treated with conventional therapies. Dogs treated with stem cells were able to occasionally support their own weight, which has not been seen in dogs undergoing conventional therapies.[97][98][99]

In a study to evaluate the treatment of experimentally induced MS in dogs using laser activated non-expanded adipose derived stem cells. The results showed amelioration of the clinical signs over time confirmed by the resolution of the previous lesions on MRI. Positive migration of the injected cells to the site of lesion, increased remyelination detected by Myelin Basic Proteins, positive differentiation into Olig2 positive oligodendrocytes, prevented the glial scar formation and restored axonal architecture.[20]

Treatments are also in clinical trials to repair and regenerate peripheral nerves. Peripheral nerves are more likely to be damaged, but the effects of the damage are not as widespread as seen in injuries to the spinal cord. Treatments are currently in clinical trials to repair severed nerves, with early success. Stem cells induced to a neural fate injected in to a severed nerve. Within four weeks, regeneration of previously damaged stem cells and completely formed nerve bundles were observed.[74]

Stem cells are also in clinical phases for treatment in ophthalmology. Hematopoietic stem cells have been used to treat corneal ulcers of different origin of several horses. These ulcers were resistant to conventional treatments available, but quickly responded positively to the stem cell treatment. Stem cells were also able to restore sight in one eye of a horse with retinal detachment, allowing the horse to return to daily activities.[75]

In the late 1990s and early 2000s, there was an initial wave of companies and clinics offering stem cell therapy, while not substantiating health claims or having regulatory approval.[100] By 2012, a second wave of companies and clinics had emerged, usually located in developing countries where medicine is less regulated and offering stem cell therapies on a medical tourism model.[101][102] Like the first wave companies and clinics, they made similar strong, but unsubstantiated, claims, mainly by clinics in the United States, Mexico, Thailand, India, and South Africa.[101][102] By 2016, research indicated that there were more than 550 stem cell clinics in the US alone selling generally unproven therapies for a wide array of medical conditions in almost every state in the country,[103] altering the dynamic of stem cell tourism. In 2018, the FDA sent a warning letter to StemGenex Biologic Laboratories in San Diego, which marketed a service in which it took body fat from people, processed it into mixtures it said contained various forms of stem cells, and administered it back to the person by inhalation, intravenously, or infusion into their spinal cords; the company said the treatment was useful for many chronic and life-threatening conditions.[104]

Costs of stem cell therapies range widely by clinic, condition, and cell type, but most commonly range between $10,000-$20,000.[105] Insurance does not cover stem cell injections at clinics so patients often use on-line fundraising.[106] In 2018, the US Federal Trade Commission found health centers and an individual physician making unsubstantiated claims for stem cell therapies, and forced refunds of some $500,000.[107] The FDA filed suit against two stem cell clinic firms around the same time, seeking permanent injunctions against their marketing and use of unapproved adipose stem cell products.[108]

Although according to the NIH no stem cell treatments have been approved for COVID-19 and the agency recommends against the use of MSCs for the disease,[109] some stem cell clinics began marketing both unproven and non-FDA-approved stem cells and exosomes for COVID-19 in 2020.[110] The FDA took prompt action by sending letters to the firms in question.[111][112] The FTC also warned a stem cell firm for COVID-19-related marketing.[113][114]

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Stem-cell therapy - Wikipedia

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New Mexico Stem Cells | Stem Cell TV

Posted: September 12, 2019 at 1:42 pm

Stem cell therapy can be described as a means or process by which stem cells are used for the prevention, treatment or the cure of diseases. Stem cells are a special kind of cells that have features other types of cells dont have. As an illustration, stem cells are capable of proliferation. This implies that they can develop into any type of cell, and grow to start performing the functions of the tissue. In addition, they can regenerate. This means they can multiply themselves. This is most important when a new tissue has to be formed. Also, they modulate immune reactions. This has made them useful for the treatment of autoimmune diseases, especially those that affect the musculoskeletal system such as rheumatoid arthritis, systemic lupus erythematosus and so on. Stem cells can be derrived from different sources. They can be extracted from the body, and in some specific parts of the body. This includes the blood, bone marrow, umbilical cord in newborns, adipose tissue, and from embryos. There are 2 main types of stem cell transplant. These are autologous stem cell transplant, and allogeneic stem cell transplant. The autologous stem cell transplant means that stem cells are extracted from the patient, processed, and then transplanted back to the patient, for therapeutic purposes. On the other hand, allogeneic stem cell transplant means the transplant of stem cells or from another individual, known as the donor, to another person, or recipient. Some treatments must be given to the receiver to prevent any cases of rejections, and other complications. The autologous is usually the most preferred type of transplant because of its almost zero side effects. Below are some of the stem cell treatments. Our goal is to provide education, research and an opportunity to connect with Stem Cell Doctors, as well as provide stem cell reviews

Adipose Stem Cell TreatmentsAdipose stem cell treatment is one of the most commonly used. This is because large quantities of stem cells can be derrived from them. According to statistics, the number of stem cells in adipose tissue are usually hundreds of times higher than what can be obtained from other sources, such as the bone marrow stem cells. Adipose stem cells have taken the center stage in the world of stem cell therapy. Apart from the ease that comes with the harvesting of these cells from the adipose tissue, they also have some special features, that separates them from other types of cells. Adipose stem cells are capable of regulating and modulating the immune system. This includes immune suppression, which is important for the treatment of autoimmune diseases. In addition, adipose stem cells can differentiate to form other types of cells. Some of them include the bone forming cells, cardiomyocytes, and cells of the nervous system.

This process can be divided into four parts. These are

Stem cell joint injection is fast becoming the new treatment of joint diseases. Stem cells derived from bone marrow, adipose and mesenchymal stem cells are the most commonly used. The stem cells are injected into the joints, and they proceed to repair and replace the damaged tissues. The cells also modulate the inflammatory process going on. Overall, stem cell joint injections significantly reduce the recovery time of patients and also eliminates pain and risks associated with surgery. Examples of diseases where this treatment is used include osteoarthritis, rheumatoid arthritis, and so on. Researchers and physicians have rated this procedure to be the future of joint therapy.

Losing a tooth as a kid isnt news because youd eventually grow them back, but losing one as an adult isnt a pleasant experience. Youd have to go through the pains of getting a replacement from your dentist. Apart from the cost of these procedures, the pain and number of days youd have to stay at home nursing the pain is also a problem. Nevertheless, there are great teeth replacement therapies available for all kinds of dental problems. Although there are already good dental treatment methods, stem cell therapy might soon become the future of dental procedures. Currently, a lot of research is being done on how stem cells can be used to develop teeth naturally, especially in patients with dental problems. The aim of the project is to develop a method whereby peoples stem cells are used in regenerating their own teeth and within the shortest time possible. Some of the benefits of the stem cell tooth would be:

The quality of life of those that underwent serious procedures, especially those that had an allogeneic hematopoietic stem cell transplantation done was studied. It was discovered that this set of people had to cope with some psychological problems, even years after the procedure. In addition, allogeneic stem cell transplantation often comes with some side effects. However, this a small price to pay, considering that the adverse effects are not usually life-threatening. Also theses types of procedures are used for severe disorders or even terminal diseases. On the other hand, autologous stem cell transplantation bears the minimum to no side effects. Patients do have a great quality of life, both in the short term and in the long term.

This is one of the many uses of stem cells. The stem cell gun is a device that is used in treating people with wounds or burns. This is done by simply triggering it, and it sprays stem cells on the affected part. This kind of treatment is crucial for victims of a severe burn. Usually, people affected by severe burns would have to endure excruciating pain. The process of recovery is usually long, which might vary from weeks to months, depending on the severity of the burn. Even after treatment, most patients are left with scars forever. However, the stem cell gun eliminates these problems, the skin can be grown back in just a matter of days. The new skin also grows evenly and blends perfectly with the other part of the body. This process is also without the scars that are usually associated with the traditional burns therapy. The stem cell gun is without any side effects.

There is one company that focuses on the production of stem cell supplements. These stem cells are usually natural ingredients that increase the development of stem cells, and also keeps them healthy. The purpose of the stem cell supplements is to help reduce the aging process and make people look younger. These supplements work by replacing the dead or repairing the damaged tissues of the body. There have been a lot of testimonials to the efficacy of these supplements.

It is the goal of researchers to make stem cell therapy a good alternative for the millions of patients suffering from cardiac-related diseases. According to some experiments carried out in animals, stem cells were injected into the ones affected by heart diseases. A large percentage of them showed great improvement, even within just a few weeks. However, when the trial was carried out in humans, some stem cells went ahead to develop into heart muscles, but overall, the heart function was generally improved. The reason for the improvement has been attributed to the formation of new vessels in the heart. The topic that has generated a lot of arguments have been what type of cells should be used in the treatment of heart disorders. Stem cells extracted from the bone marrow, embryo have been in use, although bone marrow stem cells are the most commonly used. Stem cells extracted from bone marrow can differentiate into cardiac cells, while studies have shown that other stem cells cannot do the same. Even though the stem cell therapy has a lot of potential in the future, more research and studies have to be done to make that a reality.

The use of stem cells for the treatment of hair loss has increased significantly. This can be attributed to the discovery of stem cells in bone marrow, adipose cells, umbilical cord, and so on. Stem cells are extracted from the patient, through any of the sources listed above. Adipose tissue stem cells are usually the most convenient in this scenario, as they do not require any special extraction procedure. Adipose tissue is harvested from the abdominal area. The stem cells are then isolated from the other cells through a process known as centrifugation. The stem cells are then activated and are now ready for use. The isolated stem cells are then introduced into the scalp, under local anesthesia. The entire process takes about three hours. Patients are free to go home, after the procedure. Patients would begin to see improvements in just a few months, however, this depends largely on the patients ability to heal. Every patient has a different outcome.

Human umbilical stem cells are cells extracted from the umbilical cord of a healthy baby, shortly after birth. Umbilical cord tissue is abundant in stem cells, and the stem cells can differentiate into many types of cells such as red blood cells, white blood cells, and platelets. They are also capable of differentiating into non-blood cells such as muscle cells, cartilage cells and so on. These cells are usually preferred because its' extraction is minimally non invasive. It also is nearly painless. It also has zero risks of rejecting, as it does not require any form of matching or typing.Human umbilical stem cell injections are used for the treatment of spinal cord injuries. A trial was done on twenty-five patients that had late-stage spinal cord injuries. They were placed on human umbilical stem cell therapy, while another set of 25 patients were simultaneously placed on the usual rehabilitation therapy. The two groups were studied for the next twelve months. The results of the trial showed that those people placed on stem cell therapy by administering the human umbilical cell tissue injections had a significant recovery, as compared to the other group that underwent the traditional rehabilitation therapy. It was concluded that human umbilical tissue injections applied close to the injured part gives the best outcomes.

Stem cell therapy has been used for the treatment of many types diseases. This ranges from terminal illnesses such as cancer, joint diseases such as arthritis, and also autoimmune diseases. Stem cell therapy is often a better alternative to most traditional therapy today. This is because stem cell procedure is minimally invasive when compared to chemotherapy and so on. It harnesses the bodys own ability to heal. The stem cells are extracted from other parts of the body and then transplanted to other parts of the body, where they would repair and maintain the tissues. They also perform the function of modulating the immune system, which makes them important for the treatment of autoimmune diseases. Below are some of the diseases that stem cell therapies have been used successfully:

A stem cell bank can be described as a facility where stem cells are stored for future purposes. These are mostly amniotic stem cells, which are derived from the amnion fluid. Umbilical cord stem cells are also equally important as it is rich in stem cells and can be used for the treatment of many diseases. Examples of these diseases include cancer, blood disorders, autoimmune diseases, musculoskeletal diseases and so on. According to statistics, umbilical stem cells can be used for the treatment of over eighty diseases. Storing your stem cells should be seen as an investment in your health for future sake. Parents do have the option of either throwing away their babys umbilical cord or donating it to stem cell banks.

The adipose tissue contains a lot of stem cells, that has the ability to transform into other cells such as muscle, cartilage, neural cells. They are also important for the treatment of some cardiovascular diseases. This is what makes it important for people to want to store their stem cells. The future health benefit is huge. The only way adults can store their stem cells in sufficient amounts is to extract the stem cells from their fat tissues. This process is usually painless and fast. Although, the extraction might have to be done between 3 to 5 times before the needed quantity is gotten. People that missed the opportunity to store their stem cells, using their cord cells, can now store it using their own adipose tissues. This can be used at any point in time.

Side effects often accompany every kind of treatment. However, this depends largely on the individual. While patients might present with side effects, some other people wouldnt. Whether a patient will present with adverse effects, depends on the following factors;

Some of the common side effects of stem cell transplant are;

Stem cell treatment has been largely successful so far, however, more studies and research needs to be done. Stem cell therapy could be the future.

Stem cells are unique cells that have some special features such as self-regeneration, tissue repair, and modulation of the immune system. These are the features that are employed in the treatment of diseases.

Our doctors are certified by iSTEMCELL but operate as part of a medical group or as independent business owners and as such are free to charge what the feel to be the right fit for their practice and clients. We have seen Stem Cell Treatment costs range from $3500 upwards of $30,000 depending on the condition and protocol required for intended results. Find the Best Stem Cell Doctor Near me If you are interested in saving money, try our STEM CELL COUPON!

Travel Medcations are becoming very popular around the globe for several reasons but not for what one might think. It is not about traveling to Mexico to save money, but to get procedures or protocols that are not yet available in your home country. Many procedures are started in your home country, then the tissue is set to the tissue lab where it is then grown in a process to maximize live cells, then sent to a hospital in Mexico designed to treat or provide different therapies for different conditions. If you're ready to take a medical vacation call 972-800-6670 for our"WHITE GLOVE" service.

Chen, C. and Hou, J. (2016). Mesenchymal stem cell-based therapy in kidney transplantation. Stem Cell Research & Therapy, 7(1).

Donnelly, A., Johar, S., OBrien, T. and Tuan, R. (2010). Welcome to Stem Cell Research & Therapy. Stem Cell Research & Therapy, 1(1), p.1.

Groothuis, S. (2015). Changes in Stem Cell Research. Stem Cell Research, 14(1), p.130.

Rao, M. (2012). Stem cells and regenerative medicine. Stem Cell Research & Therapy, 3(4), p.27.

Vunjak-Novakovic, G. (2013). Physical influences on stem cells. Stem Cell Research & Therapy, 4(6), p.153.

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This process can be divided into four parts. These are

Some of the common side effects of stem cell transplant are;

Stem cell treatment has been largely successful so far, however, more studies and research needs to be done. Stem cell therapy could be the future.

Chen, C. and Hou, J. (2016). Mesenchymal stem cell-based therapy in kidney transplantation. Stem Cell Research & Therapy, 7(1).

Donnelly, A., Johar, S., OBrien, T. and Tuan, R. (2010). Welcome to Stem Cell Research & Therapy. Stem Cell Research & Therapy, 1(1), p.1.

Groothuis, S. (2015). Changes in Stem Cell Research. Stem Cell Research, 14(1), p.130.

Rao, M. (2012). Stem cells and regenerative medicine. Stem Cell Research & Therapy, 3(4), p.27.

Vunjak-Novakovic, G. (2013). Physical influences on stem cells. Stem Cell Research & Therapy, 4(6), p.153.

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Human urine used as source of stem cells to grow teeth

Posted: July 31, 2013 at 2:46 am

GUANGZHOU, China, July 30 (UPI) -- Scientists in China report they've grown rudimentary teeth from stem cells taken from an unexpected source -- human urine.

The technique of using stem cells that could be grown into tiny tooth-like structures could be developed into a way of replacing lost teeth, they said.

Researchers in many countries looking for ways of growing new teeth to replace those lost with age and poor dental hygiene are increasingly focusing on stem cells.

Scientists at the Guangzhou Institutes of Biomedicine and Health used urine as the starting point, harvesting cells passed from the body and modifying them into stem cells.

The cells, when implanted into animals, began to resemble a tooth, the researchers said.

"The tooth-like structure contained dental pulp, dentin, enamel space and enamel organ," they reported in the Cell Regeneration Journal.

Some stem cell scientists have expressed doubts about using urine as a cell source.

"It is probably one of the worst sources; there are very few cells in the first place and the efficiency of turning them into stem cells is very low," stem cell scientist Chris Mason of University College London told the BBC. "You just wouldn't do it in this way."

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Stem cells from urine can be used to create teeth, study shows

Posted: July 31, 2013 at 2:46 am

Scientists have discovered a way to grow teeth from stem cells taken from human urine, Medical Daily reported.

Previous research has demonstrated the possibility for generating induced pluripotent stem cells (iPSCs) from urine samples. Now, in a study published in Cell Regeneration Journal, researchers have detailed how they were able to use these cells to grow structures resembling human teeth in mice models.

The tooth-like structure contained dental pulp, dentin, enamel space and enamel organ," the researchers wrote. "In particular, these regenerative teeth contain enamel with ameloblast-like cells of human origin and possess physical properties found in the regular human tooth. Thus, human [stem cells] could be a candidate source of seed cells on human tooth tissue-engineering for further drug screening or regenerative therapies."

The researchers, from the Guangzhou Institutes of Biomedicine and Health in China, expressed enthusiasm at the possibility of using stem cells from urine in other areas of regenerative medicine as well, Medical Daily reported. However, other experts remain skeptical because of the high-contamination risk for stem cells derived from urine among other reasons.

"It is probably one of the worst sources; there are very few cells in the first place, and the efficiency of turning them into stem cells is very low," Chris Mason, a researcher at University College, London, told reporters. "You just wouldn't do it in this way."

Despite this, the studys authors maintain that their discovery is a strong preliminary step towards the idea that stem cells could eventually be used to address patient-specific dental issues.

Click for more from Medical Daily.

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