Category Archives: Minnesota Stem Cells

A senior University of Minnesota scientist said it is "devastating" that a colleague might have doctored images to prop up research, but she defended the authenticity of her groundbreaking work on the origins of Alzheimer's disease.

Dr. Karen Ashe declined to comment about a U investigation into the veracity of studies led by Sylvain Lesn, a neuroscientist she hired and a rising star in the field of Alzheimer's research. However, she criticized an article in Science magazine that raised concerns this week about Lesn, because she said it confused and exaggerated the effect the U's work had on downstream drug development to treat Alzheimer's-related dementia.

"Having worked for decades to understand the cause of Alzheimer disease, so that better treatments can be found for patients, it is devastating to discover that a co-worker may have misled me and the scientific community through the doctoring of images," Ashe said in an e-mail Friday morning. "It is, however, additionally distressing to find that a major scientific journal has flagrantly misrepresented the implications of my work."

Questions have surfaced about as many as 10 papers written by Lesn, and often coauthored by Ashe and other U scientists, and whether they used manipulated or duplicated images to inflate the role of a protein in the onset of Alzheimer's.

The Science article detailed efforts by Dr. Matthew Schrag, an Alzheimer's researcher in Tennessee, who colorized and magnified images from Lesn's studies in ways that revealed questions about whether they were doctored or copied. Expert consultants agreed in the article that some of the images in the U studies appeared manipulated in ways that elevated the importance of a protein called A*56.

Many of the images were of Western blot tests showing that A*56, also called amyloid beta star 56, was more prevalent in mice that were older and showed signs of memory loss.

The U studies have been so influential on the course of Alzheimer's research over the past two decades that any evidence of manipulation or false study results could fundamentally shift thinking on the causes of the disease and dementia. The investigation also implicates two successful researchers on a key measure by which they are judged: their ability to pull in federal grants.

Lesn was a named recipient of $774,000 in National Institutes of Health grants specifically involving A*56 from 2008 through 2012. He subsequently received more than $7 million in additional NIH grants related to the origins of Alzheimer's.

Lesn, who did not reply to an e-mail asking for comment, came to the U in 2002 as a postdoctoral research associate after earning his doctorate at the University of Caen Normandy. He took charge of his own U lab by 2009 and became associate director of graduate studies in the neuroscience program in 2020. He was the first- or last-named author on all of the disputed studies, meaning he either instigated the research or was the senior scientist overseeing the work.

Ashe said there are two classes of A proteins, which she refers to as Abeta, and that her efforts have focused on one while drugmakers have unsuccessfully targeted the other with potential Alzheimer's treatments. As a result, she said it was unfair of the Science article even as it raised concerns about research improprieties to pin an entire industry's lack of progress on the scrutinized U research.

"It is this latter form that drug developers have repeatedly but unsuccessfully targeted," she said. "There have been no clinical trials targeting the type 1 form of Abeta, the form which my research has suggested is more relevant to dementia. [The article] has erroneously conflated the two forms of Abeta."

The scientific journal Nature is reviewing a 2006 study led by Lesn regarding the existence and role of A*56 and urging people to use it cautiously for now. Concerns emerged in part because researchers at other institutions struggled to replicate the results.

Two other 2012 and 2013 papers were corrected earlier this year, with U researchers acknowledging errant images but stating that they didn't affect the overall conclusions. However, Schrag said he has concerns the corrected images also were manipulated.

"I think those corrected images are quite problematic," he said.

Beneath the research controversy is a fundamental search and debate over the causes of Alzheimer's and related dementia. One theory is that certain Abeta proteins result in the development of amyloid plaques, which clog up space between nerve cells in the brain and inhibit memory and cognition. Another is that tau proteins clump inside the brain's thinking cells and disrupt them.

Ashe's research has explored both possibilities. Since 1986, she has been a named recipient of more than $28 million in NIH grants, making her one of the most productive researchers in U history.

Complicated legacy

Despite a remarkable history of life-saving inventions and surgical accomplishments, the U also has a legacy of research stars being implicated in scandals.

The late Dr. S. Charles Schulz stepped down as U psychiatry chair in 2015 amid claims by a grieving family that their son, who died by suicide, was coercively recruited into a schizophrenia drug trial.

Duplicated images and errors forced the correction of a 2002 Nature study, led by Dr. Catherine Verfaillie, claiming that certain adult stem cells possessed flexible abilities to grow and develop other cell types.

The late Dr. John Najarian was a pioneer in organ transplantation who elevated the U's global profile, but he faced federal sanctions in the 1990s related to illicit sales of an experimental anti-rejection medication that improved transplant outcomes.

A U investigation of Lesn's work will follow its standard policy of research misconduct allegations, according to a statement from the medical school.

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University of Minnesota scientist responds to fraud allegations in Alzheimer's research - Star Tribune

Loren and Mike Gordon. Image by Sonya Yruel

A personal investigation into the lifelong implications of his type 1 diabetes (T1D) culminated in a $7 million gift from Mike Gordon, co-founder of Meritech Capital Partners, and his wife, Loren, to help UCSF surmount a key impediment to treating the disease. The funds will support world-class stem cell biologists, immunologists, and bioengineers who are working to overcome significant barriers to beta-cell replacement therapy as an effective treatment for T1D.

Diagnosed when he was just 22 months old, Gordon went through a whole pancreas transplant at UCSF nearly 12 years ago. As a result, he has fewer complications from T1D but must take immunosuppressant drugs and endure the health risks that come with them for the rest of his life.

We wanted to give these researchers freedom to explore bold ideas.

Mike Gordon

Ive suffered a lot from this disease, Gordon said. People say, Its not that bad. Its a chronic condition. But you can fall apart.

Some 1.6 million people in the US have T1D, a disease that is often disabling and can become life-threatening. Diagnoses typically occur in childhood, but not always. The disease develops when the patients immune system attacks its own beta cells, which make insulin in the pancreas. The resulting lack of insulin leaves the body unable to absorb sugar from the bloodstream and convert it into energy, so sugar builds up in the blood. Patients are subject to a lifelong dependence on insulin and are at a higher risk for heart disease, blindness, kidney failure, and other chronic conditions, in addition to shortened average life expectancy.

Beta cell-replacement therapy has shown enormous promise for T1D patients. Beta cells make up 50%-70% of the cells in human pancreatic islets groups of cells in the pancreas that produce blood glucose-regulating hormones and they are the sole producers of insulin in the body. However, replacement beta cells dont live long, and the immune system often rejects the ones that do survive. To prevent the immune system from attacking the replacement cells, immunosuppressants are necessary, but they can be toxic and leave patients vulnerable to malignancies and other infections. UCSF scientists are poised to find solutions to these challenges.

So often the NIH provides funds for low-risk projects where outcomes are more predictable, Gordon said. We didnt want that; we wanted to give these researchers freedom to explore bold ideas.

The research team will use the Gordons investment to help answer two big questions: How can they prolong the survival of replacement beta cells after transplantation? And, can the need for patients to take immunosuppressive drugs be eliminated? The answers to these questions will be a game-changer for patients around the world.

Four interdisciplinary investigators will lead the research:

Julie B. Sneddon, PhDAssistant professor in the UCSF Diabetes Center, the UCSF Broad Center of Regeneration Medicine and Stem Cell Research, and the UCSF Department of Cell and Tissue Biology

Currently, islet-cell replacement relies on obtaining pancreatic tissue from deceased human donors. Thanks to groundbreaking advances during the last decade, beta cells can now be laboratory-generated from pluripotent stem cells, which means supplies, in theory, are unlimited. This creates an opportunity to engineer the stem cell-derived beta cells in ways that support their survival and help them avoid attack by the immune system.

We used to say we were 10 years away from a cure for T1D. We still might be. But if you look at the advances in cell biology and immunology, we have the road map now, Parent said.

Over the past five years, Drs. Parent, Tang, Sneddon, and Desai have co-advised trainees, joined forces on numerous projects, and published papers together. Their labs combine the fresh perspectives and innovation of junior faculty members with the expertise and experience of senior faculty members. The groups collective knowledge, unique understanding, and productive ongoing collaborations position them as an effective group to take on this challenge.

Insulin was first used to successfully treat a patient with type 1 diabetes a century ago, but it wasnt until 1980 that two Minnesota surgeons demonstrated successful intraportal islet transplantation in 10 patients with surgically induced diabetes (in which the patients own islet cells, or autografts, were infused back into their bodies after islet isolation). Ultimately, three of those patients achieved insulin independence for one, nine, and 38 months, respectively. In the last decade, significant strides have been made in groundbreaking technologies such as immunotherapy, metabolomics, and genomics. UCSF has been at the forefront of these advances, especially in immunology, with the Bakar ImmunoX initiative driving collaborative science and the UCSF Helen Diller Family Comprehensive Cancer Center using immunotherapies to find cures for some cancer patients.

All this progress has positively impacted diabetes research. Mark Anderson, MD, PhD, the Robert B. Friend and Michelle M. Friend Professor of Diabetes Research and the new director of the UCSF Diabetes Center, is optimistic about this moment in time and what it means for our patients with T1D and other diseases.

These developments provide unique opportunities for physician-scientists to research the molecular causes of diseases like T1D and potentially replace damaged tissues and repair malfunctioning organs, Anderson said.

Anderson believes that UCSF is one of the few places in the world capable of assembling this type of collaborative team. We are so fortunate to have the Gordons support and shared vision to help us realize the potential of cell transplantation without immune suppression, which could completely change the lives of those affected by T1D.

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Accelerating Transformational Research into Cell Transplantation for Patients with Type 1 Diabetes - UCSF

EDINBURGH, Scotland, May 18, 2022 /PRNewswire/ -- TC Biopharm (Holdings) PLC ("TC Biopharm" or the "Company") (NASDAQ: TCBP) (NASDAQ: TCBPW), a clinical stage biotechnology company developing platform allogeneic gamma-delta T cell therapies for cancer and viral indications, announced today announced the formation of a scientific advisory board (SAB) to advance its gamma-delta T cell therapy, OmnImmune, for the treatment of Acute Myeloid Leukemia (AML).

"We are honored to have these remarkable and accomplished cell therapeutics and scientific leaders join TC BioPharm's Scientific Advisory Board," said Bryan Kobel, CEO of TC BioPharm. "These individuals have made significant contributions and pioneered breakthroughs in cell therapy research and therapeutics, and together, they bring a wealth of knowledge and experience for TC BioPharm, as we work to develop our proprietary therapies to treat blood cancers and develop our platform into other oncological areas. We wil continue to expand our SAB to bring other expertise in cell therapy modalities to reflect our ongoing R&D efforts as well. TCBP looks forward to the input of these outstanding individuals as we advance our platform technology in allogeneic gamma deltas and their contribution to our ongoing research and development efforts in a number of project areas."

Members of the TC BioPharm Scientific Advisory include;

Mark Bonyhadi, Ph D., will lead the SAB. He is a senior advisor to Qiming Venture Partners USA and former Vice President of Research at Juno Therapeautics (acquired by Celgene). Dr. Bonyhadi has more than 30 years of experience in biopharmaceutical leadership roles in the US, specifically in the research and development of commercially viable approaches to take cell and gene therapies, as well as regenerative medicines, from the lab to the clinic and for subsequent commercial development. Prior to his role as vice president of Research at Juno Therapeutics Inc (acquired by Celgene Corporation), he was Director of Global Business Development for Cell Therapy at Invitrogen (which merged to become Life Technologies and was subsequently acquired by Thermo-Fisher) and prior to that, Vice President ofResearch at Xycte Therapies and a Senior Scientist at SyStemix, Inc. He was formerly the chair of the Industry Liaison Committee for the American Society for Gene and Cell Therapy (2015-2016). He is also the inventor on 11 patents and an author on 40 publications. He currently is a member of the scientific advisory board for Akron Biotech and also serves as a Non-executive Director at TCBP and as a Non-executive Director at Integra Therapeutics.

Uma Lakshmipathy, Ph D., has two decades of experience in cell biology, stem cells and translational research. She is currently the Director of R&D in Science and Technology and Head of Patheon Translation Services in Pharma Services Group at Thermo Fisher Scientific. Her work is focused on developing end-to-end, standardized processes and analytics for cell therapy and support translational services destined towards cGMP manufacturing. She has a strong foundation in development of clinical-grade reagents and processes, viral and non-viral methods of cell modification and, analytical platforms for comprehensive cell therapy product characterization. As a junior faculty at the Stem Cell Institute, University of Minnesota, she was involved in ex vivo gene repair of disease mutations in adult stem cells. She has a doctoral degree in Molecular Biophysics from the Center for Cellular and Molecular Biology in India, postdoctoral experience in DNA double strand break repair from University of Minnesota Medical School and has authored several scientific publications, books and patents.

Erin Adams, Ph D., is the Joseph Regenstein Professor of Biochemistry and Molecular Biology at the University of Chicago and an expert in molecular immunology. She explores the molecular cues that the human immune system uses to distinguish between healthy and diseased tissue. Her primary focus is on unconventional, tissue resident effector cells in the human immune system including T cells, MR1-restricted and CD1-restricted T cells. Her laboratory research seeks to understand the role of these cells types in the immune response process and what signals regulate their activity in tissue homeostasis and disease. She has received multiple honors, including being named a Searle Scholar, a Kavli Fellow and awarded a Cancer Research Foundation Junior Investigator Award.

About TC BioPharm (Holdings) PLCTC BioPharm is a clinical-stage biopharmaceutical company focused on the discovery, development and commercialization of gamma-delta T cell therapies for the treatment of cancer and viral infections with human efficacy data in acute myeloid leukemia. Gamma-delta T cells are naturally occurring immune cells that embody properties of both the innate and adaptive immune systems and can intrinsically differentiate between healthy and diseased tissue. TC BioPharm uses an allogeneic approach in both unmodified and CAR modified gamma delta t-cells to effectively identify, target and eradicate both liquid and solid tumors in cancer.

TC BioPharm is the leader in developing gamma-delta T cell therapies, and the first company to conduct phase II/pivotal clinical studies in oncology. The Company is conducting two investigator-initiated clinical trials for its unmodified gamma-delta T cell product line - Phase 2b/3 pivotal trial for OmnImmune in treatment of acute myeloid leukemia and Phase I trial for ImmuniStim in treatment of Covid patients using the Company's proprietary allogenic CryoTC technology to provide frozen product to clinics worldwide. TC BioPharm also maintains a robust pipeline for future indications in solid tumors and other aggressive viral infections as well as a significant IP/patent portfolio in the use of CARs with gamma delta t-cells and owns our manufacturing facility to maintain cost and product quality controls.

Forward Looking StatementsThis press release may contain statements of a forward-looking nature relating to future events. These forward-looking statements are subject to the inherent uncertainties in predicting future results and conditions. These statements reflect our current beliefs, and a number of important factors could cause actual results to differ materially from those expressed in this press release. We undertake no obligation to revise or update any forward-looking statements, whether as a result of new information, future events or otherwise. The reference to the website of TC BioPharm has been provided as a convenience, and the information contained on such website is not incorporated by reference into this press release.

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TC BioPharm Announces Formation of Scientific Advisory Board with Renowned Cell Therapy Experts - GuruFocus.com

When La Jollan Todd Witt was diagnosed with ALS last summer, he and his wife, Betsy, reacted with shock and sadness.

ALS, also known as Lou Gehrigs disease, is amyotrophic lateral sclerosis, a progressive neurodegenerative disorder that affects nerve cells in the brain and spinal cord and over time takes away the brains ability to initiate and control muscle movement. Patients may lose the ability to speak, eat, move and breathe. The mean survival time is two to five years, though some people may live five to 10 years or even longer, according to the ALS Association. There is no cure.

After their initial reaction, the Witts galvanized their grief and, with the help of their family which includes several La Jollans started a campaign to raise awareness about the disease and money for research to fight it.

Were going to find a cure and someday somebodys going to beat it, Todd said. It might as well be me, and it might as well be now.

To further the effort, sister-in-law Lisa Witt has organized a workout fundraising event with coach Travis Parkyn of Orangetheory Fitness La Jolla beginning at 9:30 a.m. Saturday, July 16, at 7734 Girard Ave.

The event will start with a one-hour workout with Parkyn and will include speakers, refreshments and raffle prizes from local businesses.

Lisa is seeking $25 donations to the Witt team for the ALS Associations Greater San Diego chapter and hopes to raise $5,000 from the event.

To register, visit web.alsa.org/goto/LisaWitt_OTF.

Members of the Witt family have rallied behind Todd and Betsys efforts to fund ALS research, naming themselves the Witt Wolfpack.

Were going to find a cure and someday somebodys going to beat it. It might as well be me, and it might as well be now.

Todd Witt

Todd Witt, pictured with his family Betsy and Megan, is battling ALS, or amyotrophic lateral sclerosis.

(Taylor Dunfee)

Awareness is crucial, Lisa said. If you know more about it and you see the signs in someone, maybe you can help them find information faster. I know people who spent years trying to figure out what they had.

A lack of knowledge is very frustrating, Betsy said. Weve really had to do so much of our own research, contacting our own advocates out there to figure out what the next steps are for Todd to beat this thing.

When Todd was diagnosed, she said, doctors told him: Its ALS; youve got two to five years. Go put your affairs in order. Theres nothing out there; no treatment, no cure.

We were truly shattered, Betsy said.

Weeks later, they signed up for the ALS Association Greater San Diego chapters annual Walk to Defeat ALS in October, wanting to get involved in ALS research and awareness.

We know this is sad, but were also not going to let it knock us down, Betsy said. Were going to do everything we can to beat it.

The Witts collective fundraising netted $97,000, making the Witt Wolfpack the second-ranking fundraising team in the nation for the ALS Association.

Doing the walk, Betsy added, just really lifted our spirits.

She hopes to raise $100,000 for the San Diego ALS Association chapter this year. So far, the years total is $22,700.

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The local chapter works to provide equipment and other support to about 220 patients throughout San Diego, Imperial Valley, Temecula and Lake Elsinore.

Lisa said local businesses donated more than what was asked for the July 16 event at Orangetheory Fitness and asked to be educated about ALS.

La Jolla is such an amazing community, she said. It goes to show how much people want to support.

For now, Todd is in physical therapy, acupuncture and other appointments several times a week to help keep his symptoms at bay and is looking forward to a stem cell trial hes participating in later this month at the Mayo Clinic in Minnesota.

We just have this great pack of people and family and friends. Were just so grateful and humbled, Betsy said.

For more information on the Witt Wolfpacks fundraising efforts, visit web.alsa.org/goto/wittwolfpack.

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Witt Wolfpack: Family works out fundraising and awareness after La Jollan's ALS diagnosis - La Jolla Light

Abstract

Antibody-dependent cellular cytotoxicity (ADCC) is a key effector mechanism of natural killer (NK) cells that is mediated by therapeutic monoclonal antibodies (mAbs). This process is facilitated by the Fc receptor CD16a on human NK cells. CD16a appears to be the only activating receptor on NK cells that is cleaved by the metalloprotease a disintegrin and metalloproteinase-17 upon stimulation. We previously demonstrated that a point mutation of CD16a prevents this activation-induced surface cleavage. This noncleavable CD16a variant is now further modified to include the high-affinity noncleavable variant of CD16a (hnCD16) and was engineered into human induced pluripotent stem cells (iPSCs) to create a renewable source for human induced pluripotent stem cellderived NK (hnCD16-iNK) cells. Compared with unmodified iNK cells and peripheral bloodderived NK (PB-NK) cells, hnCD16-iNK cells proved to be highly resistant to activation-induced cleavage of CD16a. We found that hnCD16-iNK cells were functionally mature and exhibited enhanced ADCC against multiple tumor targets. In vivo xenograft studies using a human B-cell lymphoma demonstrated that treatment with hnCD16-iNK cells and anti-CD20 mAb led to significantly improved regression of B-cell lymphoma compared with treatment utilizing anti-CD20 mAb with PB-NK cells or unmodified iNK cells. hnCD16-iNK cells, combined with anti-HER2 mAb, also mediated improved survival in an ovarian cancer xenograft model. Together, these findings show that hnCD16-iNK cells combined with mAbs are highly effective against hematologic malignancies and solid tumors that are typically resistant to NK cellmediated killing, demonstrating the feasibility of producing a standardized off-the-shelf engineered NK cell therapy with improved ADCC properties to treat malignancies that are otherwise refractory.

Cell-based anticancer immunotherapies have experienced great advances in the past few years.1 Although chimeric antigen receptor (CAR)expressing T cells have garnered the most attention, clinical trials using natural killer (NK) cells have demonstrated that they are safe and effective.2-5 In recent clinical trials, NK cells have been shown to possess potent antiacute myeloid leukemia effects without eliciting serious adverse effects, such as graft-versus-host disease, neurotoxicity, and cytokine release syndrome.4,6,7 However, the adoptive transfer of NK cells to patients with B-cell lymphoma, ovarian carcinoma, or renal cell carcinoma has demonstrated low efficacy and has lacked specific tumor-targeting receptors8-10.

NK cellbased clinical trials have used a variety of cell sources, including peripheral bloodderived NK (PB-NK) cells, umbilical cord bloodisolated NK (UCB-NK) cells, umbilical cord blood CD34+ cellderived NK cells, and the NK cell line NK-92.7,11-14 Although these trials have demonstrated clinical safety, each cell source is confined by limitations.11,12,15 The NK cell yields and subsets from PB-NK cells and UCB-NK cells are extremely donor dependent and are not derived from a single renewable source, making product standardization and multiple-dosing strategies difficult.16,17 Additionally, genetic modification of primary NK cells is challenging and highly variable, making it difficult to develop consistent and reproducible engineered NK cell therapies.18 Lastly, although NK-92 cells are from a single source, they lack many conventional NK cell markers and, as a transformed cell, must be mitotically inactivated before infusion to prevent uncontrolled proliferation.13 This eliminates the ability of NK-92 cell treatment to expand upon infusion, a critical factor for NK cell antitumor activity.2,4,7,19 In contrast, human induced pluripotent stem cell (iPSC)derived NK (iNK) cells can be produced in a homogenous and clinically scalable manner, are capable of being genetically edited at the iPSC stage, and have demonstrated in vivo proliferative capacity.20-23 Therefore, iNK cells are an important source of standardized off-the-shelf NK cell therapy to treat refractory malignancies.24

NK cellmediated antitumor activity is regulated through a repertoire of activating and inhibitory cell surface receptors, including natural cytotoxicity receptors, killer immunoglobulin receptors, and immunoglobulin G (IgG) Fc receptor FcRIIIa (CD16a).4,5,25 CD16a binds the Fc portion of IgG when attached to a target cell to mediate antibody-dependent cell-mediated cytotoxicity (ADCC), a key effector and tumor antigen-targeting mechanism of NK cells.26 The binding affinity of CD16a to IgG varies between its allelic variants. Specifically, CD16a with valine at position 158 (158V) has a higher affinity for IgG than does CD16a with phenylalanine at the same position.27,28 In addition to the clinical observation that NK cells enhance the efficacy of therapeutic monoclonal antibodies (mAbs),29 CD16a has been shown to play an important role in the clinical setting, because patients with high-affinity CD16a with 158V have had greater objective responses and progression-free survival when treated with cetuximab, trastuzumab, or rituximab.30-32 Notably, the CD16a molecule is cleaved from the surface of activated NK cells by a disintegrin and metalloproteinase-17 (ADAM17), which is constitutively expressed on the surface of NK cells,33-36 leading to NK cell dysfunction and reduced ADCC capacity.35 Our group previously identified the ADAM17 cleavage site of CD16 and created a high-affinity noncleavable version of CD16a (hnCD16) by mutating the cleavage site in the 158V variant.33

We hypothesized that engineering iNK cells with hnCD16 would overcome the challenges faced with NK cell therapies. Specifically, we demonstrate that iNK cells uniformly expressing hnCD16 (hnCD16-iNK cells) exhibit potent ADCC against hematological malignancies and solid tumors. Notably, a multidose regimen of hnCD16-iNK cells derived from an engineered clonal human iPSC line administered with anti-CD20 mAb treatment mediated potent activity and improved long-term survival in a mouse xenograft lymphoma model. Therefore, standardized off-the-shelf hnCD16-iNK cells with enhanced ADCC effector function, in combination with readily available anti-tumor mAbs, provide a novel clinical strategy to treat cancer.

The derivation of NK cells from human iPSCs has been described previously.20,37 iNK cells were expanded using irradiated K562IL214-1BBL cells (details can be found in supplemental Methods, available on the Blood Web site).

CD107a expression was assessed and interferon- (IFN-) staining was performed as previously described.23 NK cells were cocultured with tumor targets at a 1:1 effector-to-target ratio. Details about the staining procedure are available in supplemental Methods.

NOD/SCID/c/ (NSG) mice (The Jackson Laboratory; n = 5 per group) were used for in vivo experiments. Mice were sublethally irradiated (225 cGy) 1 day prior to tumor engraftment. Mice were given 1 105 to 2.5 105 Luc-expressing tumor cells IV or via intraperitoneal injection. For the intraperitoneal injection tumor models, NK cells (107 cells per mouse) were injected intraperitoneally 4 days after tumor transplant. One day prior to NK cell injection, mice were assayed for tumor burden using bioluminescent imaging (BLI) and then placed into equivalent BLI-expressing groups. For the IV models, NK cells (107 cells per mouse) were injected IV 1 day after tumor cell infusion. NK cells were supported by the injection of interleukin-2 (IL-2) and/or IL-15, as reported previously.22 Tumor burden was determined by BLI using a Xenogen IVIS Imaging system. Mice were euthanized when they lost the ability to ambulate. All mice were housed, treated, and handled in accordance with the guidelines set forth by the University of California, San Diego Institutional Animal Care and Use Committee and the National Institutes of Healths Guide for the Care and Use of Laboratory Animals.

All antibodies used are listed in supplemental Methods. Flow cytometry was performed on a BD FACSCalibur, a BD LSR II, or a NovoCyte 3000, and data were analyzed using FlowJo or NovoExpress software.

Data are presented as the mean standard error of the mean (SEM). In vitro data are from 3 independent experiments. Differences between groups were evaluated using 1-way analysis of variance. For the quantification of in vivo image, data are presented as the mean SEM, and differences between groups were analyzed using a 2-tailed Student t test. The survival curve was analyzed using the log-rank (Mantel-Cox) test. Statistical analyses were performed using GraphPad Prism. All tests were considered significant at P < .05.

To maintain high levels of stable CD16a expression on mature NK cells, we engineered human iPSCs with a non-ADAM17 cleavable version of the high-affinity CD16a 158V variant (supplemental Figure 1A-B).33 From the transduced hnCD16 pool, clonal iPSC lines were generated and screened to ensure a homogenous population of starting material (supplemental Figure 1C).38 The selected hnCD16-engineered iPSC clonal cell line stably expressed homogenous levels of hnCD16 (>99% CD16+; supplemental Figure 1D). Overexpression of hnCD16 had no significant effect on the morphology or growth rate of hnCD16-engineered iPSCs, which maintained homogenous expression of pluripotency master regulators NANOG and OCT4, as determined by flow cytometry (supplemental Figure 1E), as well as hiPSC surface markers (SSEA4, TRA-1-81, and CD30; supplemental Figure 1F). Moreover, integration site analysis showed that the hnCD16-iPSC clonal line that we selected has 3 copies of hnCD16 inserted. The hnCD16 transgene was inserted into intron regions or an intergenic region that would not lead to any changes in NK cell growth or activity (supplemental Table 1).

We then generated NK cells from hnCD16-engineered iPSCs using an in vitro differentiation method previously reported by our group (supplemental Figure 1B).20,39 Similar to PB-NK cells, unmodified iNK cells and hnCD16-iNK cells consist of a homogeneous population of CD56+ NK cells that also coexpress typical NK cell surface antigens: NKp44, NKp46, NKG2D, TRAIL, and Fas ligand (). They also expressed NK cell maturation markers (eg, CD94, CD2, NKG2C, and CD57) and homing receptors (eg, CXCR4 and CCR6) (). Expression of CD62L, another receptor that can be cleaved by ADAM17,40,41 was very low in iNK cells compared with PB-NK cells (supplemental Figure 2A). Additional characterization studies demonstrate that iNK cells have a longer telomere length than do PB-NK cells (supplemental Figure 1F).

hnCD16-iPSCderived NK cells are functionally mature and do not downregulate CD16 expression upon activation. (A) Unmodified iNK cells, hnCD16-iNK cells, and adult PB-NK cells were stained and analyzed by flow cytometry for CD56 and CD16 and the indicated NK cell surface receptors. In each panel, red line: isotype control; blue line: stained sample. Data were repeated independently in 3 separate experiments. (B) hnCD16-iNK cells, unmodified iNK cells, or PB-NK cells were stimulated as indicated for 4 hours, and CD16 expression was determined by flow cytometry (n = 4-6 per group). (C) Representative flow cytometric analysis of CD16 and TNF- expression on unmodified iNK cells, hnCD16-iNK cells, and PB-NK cells that were left unstimulated or stimulated with K562 cells or PMA/ionomycin. (D) Representative flow cytometric analysis of intracellular TNF- and IFN- production after a 4-hour incubation with culture media only (unstimulated), with P815 cells, or with P815 cells + anti-CD16 antibody. Data in panels C-D were repeated in 3 separate experiments.

hnCD16-iNK cells were expanded using artificial antigen presenting cells (aAPCs), as previously reported.22,42 Under these culture and expansion conditions, nearly all hnCD16-iNK cells expressed CD16 (99% CD16+; ), whereas the endogenous expression of CD16 on unmodified iNK cells and PB-NK cells under the same culture conditions is typically between 20% and 60% (). These results are consistent with previous studies demonstrating that endogenous CD16 is cleaved from the surface of NK cells by ADAM17 upon activation by stimuli, such as cytokines and target cells.33,36 To assess the stability of CD16 expression by hnCD16-iNK cells, we activated unmodified iNK cells, hnCD16-iNK cells, and PB-NK cells with different stimuli, including PMA/ionomycin, K562 cells, the CD20+ Burkitt lymphoma cell line Raji, and Raji cells in the presence of the anti-CD20 mAb rituximab (). For example, after stimulation with K562 target cells or PMA/ionomycin, PB-NK cells and unmodified iNK cells lost the majority of their CD16 expression, whereas the majority of hnCD16-iNK cells maintained high levels of CD16 expression (). It is also important to note that unmodified iNK cells are homozygous for low-affinity CD16 (158F), whereas PB-NK cells are heterozygous (supplemental Figure 1G), indicating that low-affinity and high-affinity CD16 can be downregulated on activated NK cells. Importantly, stable CD16 surface expression on hnCD16-iNK cells led to enhanced cytokine production when CD16 was activated directly (). In support of this, the production of tumor necrosis factor- (TNF-) and IFN- in response to CD16a stimulation was directly correlated with the level of surface CD16 expression ().

Because CD16 is a key activating receptor on NK cells that mediates ADCC, we examined the ability of hnCD16-iNK cells to mediate ADCC against multiple cancer cell lines, including Raji cells, A549 (lung adenocarcinoma) cells, SKOV-3 (ovarian adenocarcinoma) cells, and Cal27 (squamous cell carcinoma) cells. The antibodies that recognize antigens on these tumor cell lines (CD20 on Raji; EGFR on A549, SKOV-3, and Cal27; HER2 on SKOV-3) are all routinely used clinically with proven efficacy.43-45

We first used degranulation (indicated by the cell surface expression of CD107a) and IFN-/TNF- expression as parameters for NK cell activity to evaluate antibody-dependent effector function against different cancer cell populations.21,23 When Raji, SKOV-3, and Cal27 cells were cocultured with unmodified iNK cells or with hnCD16-iNK cells alone, minimal expression of CD107a, IFN- (), and TNF- (supplemental Figure 2B) was detected. However, when target cells were pretreated with their respective antibodies (anti-CD20: rituximab; anti-HER2: trastuzumab; and anti-EGFR: cetuximab), CD107a+ hnCD16-iNK cells were increased 3.9-, 8-, and 4.5-fold for Raji cells + rituximab, SKOV-3 cells + trastuzumab, and Cal27 cells + cetuximab, respectively (), and IFN+ hnCD16-iNK cells were increased 5.1-, 12-, and 6.5-fold () for Raji cells + rituximab, SKOV-3 cells + trastuzumab, and Cal27 cells + cetuximab, respectively. Production of IFN- correlates closely with production of TNF- in NK cells (supplemental Figure 2). In contrast, stimulation of unmodified iNK cells with target cells and antibodies did not mediate increased CD107a, IFN-, () or TNF- (supplemental Figure 2) expression, suggesting that hnCD16 improved the antibody-dependent cytokine response against these tumor cells. In addition, we systemically evaluated cytokine production by comparing TNF- and IFN- production and CD107a surface expression of unmodified iNK cells, hnCD16-iNK cells, UCB-NK cells, and PB-NK cells upon stimulation with P815 cells (mouse lymphoblast-like mastocytoma cell line),46 P815 cells + anti-CD16 mAb, Raji cells, and Raji cells + anti-CD20 mAb. Similarly, hnCD16-iNK cells showed the greatest TNF- and IFN- production and CD107a surface expression with P815 + anti-CD16 mAb or Raji + anti-CD20 mAb stimulation, suggesting the strongest antibody-mediated response against these tumor targets ().

hnCD16-iNK cells demonstrate improved in vitro ADCC against multiple tumor types. (A) hnCD16-iNK cells and unmodified iNK cells produce CD107 and IFN- in response to Raji cells with or without anti-CD20 antibody, in response to SKOV-3 cells with or without anti-HER2, and in response to Cal27 cells with or without anti-EGFR. hnCD16-iNK cells or unmodified iNK cells were left unstimulated or were stimulated with a 1:1 ratio of target cells with or without antibody and stained for CD107a and IFN- 4 hours later. (B) Quantification of CD107a (left panel) and IFN- (right panel) expression by cells in panel A. An increase in CD107a+ or IFN+ positive cells in the antibody group was normalized to the without-antibody group (fold increase: antibody/without antibody). Studies were repeated independently 3 times, and data are mean standard deviation. (C) Quantification of flow cytometric analysis of TNF- and IFN- production and CD107a surface expression after a 4-hour incubation with culture media only (unstimulated) or with the indicated stimuli. Heat maps quantify the frequency of NK cells that are positive for IFN-, TNF-, or CD107a and are scaled from 0% (black) to 30% (yellow), with background expression subtracted such that unstimulated = 0. (D) ADCC against Raji cells was analyzed using a caspase-3/7 green flow cytometry assay. Raji cells were incubated with NK cells, with or without anti-CD20 antibody, for 4 hours. (E) ADCC against Raji cells was analyzed over a 24-hour period using an IncuCyte real-time imaging system. Anti-CD20 was titrated from 0.001 g/mL to 20 g/mL. (F-G) Long-term (66-hour) ADCC assays using the IncuCyte real-time imaging system. ADCC against the lung cancer cell line A549 with and without anti-EGFR mAb (F) and against the ovarian cancer cell line SKOV-3 with and without anti-HER2 mAb (G). Data in panels F-G are presented as the normalized frequency of target cells remaining, where target cells without NK effectors = 100%. Data in panels D-G were repeated independently in 3 separate experiments. ***P < .001, 2-tailed Student t test.

We then assessed these different NK cell populations for ADCC. Similar to the results for NK cell degranulation and IFN- expression, unmodified iNK cells demonstrated relatively limited killing of Raji cells, even with the addition of an anti-CD20 mAb (). In contrast, the addition of an anti-CD20 mAb to hnCD16-iNK cells cocultured with Raji cells led to a marked increase in cell killing (). A prolonged time course analysis using various doses of anti-CD20 mAb further demonstrated the potent ADCC activity of hnCD16-iNK cells, even at antibody concentrations as low as 0.1 g/mL, which may improve the efficacy of mAb treatment41 (). Studies comparing hnCD16-iNK cells with PB-NK cells using this longer (>60 hours) cytotoxicity assay against SKOV-3 cells (with or without trastuzumab; ) and A549 cells (with or without cetuximab; ) also demonstrated that hnCD16-iNK cells mediated improved ADCC compared with PB-NK cells. To further investigate the contribution of the noncleavable CD16 variant to the improved ADCC, we compared hnCD16-iNK cells and iNK cells engineered with natural (cleavable) high-affinity CD16 (WTCD16-iNK cells).33 hnCD16-iNK cells and WTCD16-iNK cells exhibit similar expression levels of CD16 (supplemental Figure 3A-B). In line with our previous findings (), CD16 expression in WTCD16-iNK cells was downregulated when cells were activated with PMA/ionomycin, Raji cells + anti-CD20 mAb rituximab, or Cal-27 cells + anti-EGFR mAb cetuximab for 4 hours (supplemental Figure 3C). In contrast, hnCD16-iNK cells maintained uniformly high levels of CD16 expression (supplemental Figure 3C). Notably, hnCD16-iNK cells mediated ADCC better than did WTCD16-iNK cells (supplemental Figure 3D-E). These results demonstrate that noncleavable CD16 contributes to the improved ADCC in hnCD16-iNK cells.

Next, we investigated downstream signaling mediated by CD16 activation. Upon cross-linking by anti-CD16 antibody, all 3 cell populations mediate efficient downstream signaling activation, as shown by phospho-flow staining of CD3, ZAP70, and SLP76, which are downstream targets of CD16 activation47 (supplemental Figure 4A). This was supported by immunoblot analysis that also demonstrates ERK phosphorylation (supplemental Figure 4B). Furthermore, we studied the detachment of NK cells after killing target cells using an IncuCyte real-time imaging system. Again, hnCD16-iNK cells show significantly better killing against Cal27 cells with the addition of cetuximab (supplemental Figure 5A-D). Notably, the number of target cells killed per single hnCD16-iNK cell was significantly higher than that seen with unmodified iNK cells and PB-NK cells (supplemental Figure 5D). The percentage of NK cells detached from targets and the detachment time after killing targets are also similar among hnCD16-iNK cells, unmodified iNK cells, and PB-NK cells (supplemental Figure 5E-F), demonstrating that hnCD16 does not inhibit the detachment of NK cells from targets (see also supplemental Videos 1-3).

To evaluate the in vivo ADCC activity of the hnCD16-iNK cells, we used a Raji-Luc xenograft mouse model () to compare 8 treatment groups: tumor alone (untreated), anti-CD20 mAb alone, and PB-NK cells, unmodified iNK cells, or hnCD16-iNK cells alone or in combination with anti-CD20 mAb (). As previously seen with NK cells in this in vivo xenograft model,48 treatment with PB-NK cells, unmodified iNK cells, or hnCD16-iNK cells alone did not inhibit tumor growth, and mice in these 3 groups show tumor burden similar to that seen in the untreated tumor group at all time points (). In comparison with anti-CD20 mAb treatment alone, combination treatment with hnCD16-iNK and anti-CD20 mAb was the only one to result in a significant decrease in tumor burden (at day 10 posttreatment, **P < .001) (). Specifically, a single dose of anti-CD20 mAb improved median survival from 27 to 38 days, whereas the combination of PB-NK cells or unmodified iNK cells with anti-CD20 mAb further inhibited tumor progression and resulted in significantly better median survival (43 and 44 days, respectively; ). Combination treatment with hnCD16-iNK cells and anti-CD20 mAb led to an additional increase in median survival to 52 days (; anti-CD20 vs hnCD16-iNK cells + anti-CD20; P = .0027). However, survival in the hnCD16-iNK cells + anti-CD20 mAb group was not statistically better than in the PB-NK cells + anti-CD20 mAb group or the unmodified iNK cells + anti-CD20 mAb group (). Therefore, in this single-dose model, any of the 3 NK cell populations combined with anti-CD20 treatment mediated effective antitumor activity in vivo ().

A single dose of hnCD16-iNK cells effectively mediates in vivo ADCC against human B-cell lymphoma. (A) Schema of single-dose NK infusion in vivo study. NSG mice were inoculated intraperitoneally with 2 105 Luc-expressing Raji cells, and tumor engraftment was assessed by IVIS imaging 3 days later for a baseline pretreatment reading. On day 4 after transplant, mice were left untreated or were treated with 1 107 PB-NK cells, unmodified iNK cells, or hnCD16-iNK cells, alone or in combination with 300 g of anti-CD20 antibody. Mice were treated with IL-15 for the first week and with IL-2 for 3 weeks, and IVIS imaging was performed to track tumor progression. (B) Tumor burden was determined by BLI. (C) Quantification of IVIS imaging time course. Data are mean SEM for the mice in panel B. Data were not significant for anti-CD20 alone vs unmodified iNK cells + anti-CD20 or for PB-NK cells + anti-CD20 at all time points. (D) Kaplan-Meier curve demonstrating survival of the experimental groups. The median survival for the untreated group and the groups treated with anti-CD20, PB-NK cells + anti-CD20, unmodified iNK cells + anti-CD20, and hnCD16-iNK cells + anti-CD20 was 27, 38, 43, 44, and 52 days, respectively. Anti-CD20 vs PB-NK+anti-CD20, P = .0047; anti-CD20 vs unmodified-iNK+anti-CD20, P = .0067; anti-CD20 vs hnCD16-iNK+anti-CD20, P = .0027; PB-NK+anti-CD20 vs hnCD16-iNK+anti-CD20, P = .1098; unmodified-iNK+anti-CD20 vs hnCD16-iNK+anti-CD20, P = .3127; 2-tailed log-rank test. *P < .05, **P < .01, 2-tailed Student t test, anti-CD20 alone vs hnCD16-iNK cells + anti-CD20.

In the single-dose study, we identified a decrease in tumor burden at day 10 (); however, relapse was observed in most of the treated mice, which may be due to human NK cells having a limited life span after adoptive transfer.2,22,23 We next examined whether multiple doses of NK cells + anti-CD20 mAb would further augment in vivo ADCC. We performed a 1-month dosing study consisting of 4 weekly doses of NK cells, with or without anti-CD20 mAb (). Because unmodified iNK cells and PB-NK cells, in combination with anti-CD20, show similar tumor suppression in the single-dose study, in this study we focused our comparison on hnCD16-iNK cells and PB-NK cells. Similar to the single-dose study, all of the mice in the untreated groups died between day 23 and day 26 (median survival, 25 days; ). Again, 4 weekly doses of PB-NK cells or hnCD16-iNK cells alone had no effect on tumor progression (). As expected, 4 weekly doses of anti-CD20 mAb alone induced tumor regression () and prolonged the median survival to 47 days (). Notably, median survival was longer with multiple NK cell doses compared with the single-dose study (hnCD16-iNK cells + anti-CD20 mAb), demonstrating significant improvement in antitumor activity, with a mean survival of 76 days (; anti-CD20 vs hnCD16-iNK cells + anti-CD20; P = .0065).

Multiple doses of hnCD16-iNK cells effectively mediate improved ADCC in vivo against B-cell lymphoma. (A) Schema of multiple NK cell dosing study. NSG mice were inoculated intraperitoneally with 2 105 Luc-expressing Raji cells, and tumor engraftment was assessed by IVIS imaging 3 days later for a baseline pretreatment reading. On day 4 after transplant, mice were left untreated or were treated with 1 107 PB-NK cells or hnCD16-iNK cells, alone or in combination with 300 g of rituximab weekly for 4 weeks. NK cells were supported by injection of IL-15 for the first week and by injection of IL-2 for 3 weeks. IVIS imaging was done weekly to monitor tumor progression. (B) Tumor burden was determined by BLI over the first 28 days. (C) Time course of IVIS imaging. Data are mean SEM for the mice in panel B. Anti-CD20 vs PB-NK+anti-CD20 not significant for all data points, 2-tailed Student t test. (D) Kaplan-Meier curve representing the percent survival of the experimental groups. The median survival for the untreated group and the groups treated with Anti-CD20, PB-NK+anti-CD20, and hnCD16-iNK+anti-CD20 are 25, 47, 61, and 76 days, respectively. Anti-CD20 vs PB-NK+anti-CD20, P = .0185; anti-CD20 vs hnCD16-iNK+anti-CD20, P = .0065; PB-NK+anti-CD20 vs hnCD16-iNK+anti-CD20, P = .0485; 2-tailed log-rank test. *P < .05, **P < .01, anti-CD20 vs hnCD16-iNK+anti-CD20, 2-tailed Student t test.

Interestingly, of the 3 mice in the PB-NK cells + anti-CD20 mAb group that did not exhibit detectable tumor at day 14, all exhibited tumor relapse by day 28 (). However, only 1 of the 4 mice that had undetectable tumor at day 14 in the hnCD16-iNK cells + anti-CD20 mAb group experienced tumor relapse at day 28 (), and 2 mice maintained complete remission (>200 days), demonstrating a more durable antitumor response (). Furthermore, the survival rate of mice receiving multiple dosing of hnCD16-iNK cells + anti-CD20 mAb treatment was significantly better than for PB-NK cells + anti-CD20 mAb treatment (P = .0485; ). These results support the strategy of multidosing of hnCD16-iNK cells to maximize ADCC in vivo to enable long-term survival and possible complete tumor elimination.

To evaluate the in vivo ADCC activity of hnCD16-iNK cells in a more clinically relevant model, we used an in vivo systemic tumor model in which Raji-Luc tumor cells and NK cells were dosed IV (). In this model, tumor distribution is disseminated, and disease progression is more aggressive than when tumor cells are delivered intraperitoneally. The median survival of mice in untreated groups was 17 days (). Consistent with previous studies, treatment with PB-NK cells, unmodified iNK cells, or hnCD16-iNK cells alone did not inhibit tumor growth (). A single dose of anti-CD20 mAb alone decreased tumor burden () and improved the median survival from 17 to 35 days (; P = .021). Combination treatment using PB-NK cells or unmodified iNK cells + anti-CD20 mAb did not improve tumor control in comparison with anti-CD20 mAb treatment alone (). As with the intraperitoneal injection model, combination treatment with hnCD16-iNK cells + anti-CD20 mAb mediated improved antitumor activity with significantly better survival than anti-CD20 mAb alone (P = .0269), PB-NK cells + anti-CD20 mAb (P = .0342), or unmodified iNK cells + anti-CD20 mAb (P = .0350; ). Notably, 3 mice in the hnCD16-iNK cells + anti-CD20 mAb group maintained complete remission at 100 days posttumor transplant, whereas no mice in other groups survived beyond 60 days (). These results further confirmed that hnCD16-iNK cells can effectively mediate ADCC and provide a more durable antitumor response against human lymphoma.

hnCD16-iNK cells effectively mediate ADCC in a human lymphoma systemic tumor model. (A) Flow scheme of IV NK cell infusion in vivo study. NSG mice were inoculated IV with 2 105 Luc-expressing Raji cells. On day 1 after transplant, mice were left untreated or were treated with 1 107 PB-NK cells, unmodified iNK cells, or hnCD16-iNK cells, alone or in combination with 300 g of anti-CD20 antibody. NK cells were supported by injection of IL-15 for the first week and by injection of IL-2 for 3 weeks; IVIS imaging was performed weekly to track tumor progression. (B) Tumor burden was determined by BLI over the first 35 days. (C) IVIS imaging time course. Data are mean SEM for the mice in panel B. (D) Kaplan-Meier curve representing the percent survival of the experimental groups. The median survival was not reached in the hnCD16-iNK + anti-CD20 group. Anti-CD20 vs untreated, P = .0021; anti-CD20 vs hnCD16-iNK+anti-CD20, P = .0269; hnCD16-iNK+anti-CD20 vs PB-NK+anti-CD20, P = .0342; hnCD16-iNK+anti-CD20 vs umnodified-iNK+anti-CD20, P = .0350; 2-tailed log-rank test. *P < .05, **P < .01, ***P < .001, hnCD16-iNK+anti-CD20 vs umnodified-iNK+anti-CD20, 2-tailed Student t test.

We then investigated the in vivo persistence and homing of NK cells in a separate group of tumor-bearing mice by examining blood, bone marrow, spleen, kidney, liver, and heart for the presence of NK cells over 21 days postinjection. On day 7, human NK cells were detected in all of the organs examined (supplemental Figure 6A-B), although some of the cells seen in organs may be from blood perfusing those organs, because this cannot be distinguished when organs are processed for analysis. The infused NK cells reached a peak at day 7 and persisted for up to 21 days (supplemental Figure 6C). PD-1 expression was not detected on iNK cells before or after adoptive transfer (supplemental Figure 6D). Unmodified iNK cells, hnCD16-iNK cells, and PB-NK cells show similar persistence, and homing was confirmed by immunohistochemistry staining in organs (supplemental Figure 6E), indicating that the improved antitumor effect mediated by hnCD16-iNK cells was due to increased ADCC rather than differences in persistence or homing.

To test whether hnCD16-iNK cells can also elicit antitumor effects against solid tumors in vivo, we used a mouse xenograft model and SKOV-3 ovarian carcinoma cells (). Combination treatment with hnCD16-iNK cells + anti-HER2 mAb led to significantly lower tumor burden at all time points between day 18 and day 60 (). Moreover, hnCD16-iNK cells + anti-HER2 mAb significantly improved survival (P = .0040; ). These results demonstrate that hnCD16-iNK cells can also mediate an antitumor response in an in vivo ovarian cancer model when combined with anti-HER2.

hnCD16-iNK cells mediate improved ADCC in vivo against ovarian cancer. NSG mice were inoculated intraperitoneally with 1 105 Luc-expressing SKOV-3 cells, and tumor engraftment was assessed by IVIS imaging 4 days later. On day 5 after tumor transplant, mice were left untreated or were treated with 100 g of anti-HER2 alone or in combination with 5 106 hnCD16 iNK cells. NK cells were supported by twice weekly injections of IL-2, and IVIS imaging was done weekly to track tumor load. (A) IVIS imaging. (B) Quantification of geometric mean standard deviation for the mice in panel A. (C) Kaplan-Meier curve representing the percent survival of the experimental groups. Untreated vs anti-HER2, P = .0140; anti-HER2 vs anti-HER2 + hnCD16 iNK+, P = .004; 2-tailed log-rank test. (D) Mice injected with Luc-expressing SKOV-3 cells were treated with 1 107 (1e7) or 2 107 (2e7) cryopreserved hnCD16-iNK cells or with 2 107 fresh hnCD16-iNK cells + anti-HER2 antibody. (D) IVIS imaging. (E) Quantification of the geometric mean standard deviation for the mice in panel D.

A key challenge in developing off-the-shelf adoptive cell therapies is getting cells from the manufacturing site to the patient without compromising safety or efficacy.49 Cryopreservation provides the best opportunity to deliver multiple doses, which can augment the antitumor effect while maintaining the high levels of killing seen in previous studies,50 provided that the cell viability and function are not negatively impacted upon thawing. To test the anticancer activity of hnCD16-iNK cells after cryopreservation, we compared cryopreserved cells and fresh cells in the SKOV-3 cell ovarian tumor xenograft model (). Notably, one-time dosing of cryopreserved hnCD16-iNK cells demonstrated similar antitumor activity as fresh hnCD16-iNK cells ().

The ability to improve targeting and specificity are key requirements to better enable NK cellmediated killing of solid tumors and lymphoid malignancies that are typically more resistant to this therapeutic modality. Here, we demonstrate the ability to use human pluripotent stem cells as a platform to produce engineered NK cells that can be effectively combined with therapeutic mAbs to successfully target and kill typically NK cellresistant tumors. Specifically, creation and use of a novel CD16 molecule that contains the 158V high-affinity variant, combined with an S197P mutation that confers resistance to ADAM17-mediated cleavage, allows us to produce NK cells with improved ADCC activity in vitro and in vivo. In this model, CD16 engagement and signaling provide an important strategy to make NK cells antigen specific. These engineered iPSC-derived NK cells can now be produced at a clinical scale,20,24,51 as well as cryopreserved (), to enable upcoming clinical trials of hnCD16-iNK cells.

NK cellbased adoptive immunotherapy provides a promising therapeutic option for allogeneic cancer therapy, with many clinical trials underway for a variety of hematological malignancies and solid tumors.3-5 Most of these clinical trials use allogeneic PB-NK cells, and significant remissions have been observed when they are used to treat acute myeloid leukemia.2,19 However, as noted, the efficacy of PB-NK cells in the treatment of solid tumors, such as ovarian carcinoma and lung cancer, has been limited,10,15 likely as a result of the poor infiltration, inefficient homing, lack of specificity, and decreased persistence of NK cells in these patients.52 The solid tumor microenvironment can also act to decrease immune cell functions, including the elicitation of CD16 shedding.53 Human pluripotent stem cell-derived NK cells provide a novel option for adoptive immune cell therapy that may avoid some of the limitations of PB-NK cells and UCB-NK cells.20,24 Specifically, iPSCs can be routinely genetically modified on a clonal level to produce a homogeneous population of uniform engineered NK cells, rather than the heterogeneous NK cells that are typically obtained from peripheral blood or umbilical cord blood.21 Additionally, iNK cells can be expanded into a clinically scalable cell population that is suitable to treat hundreds or thousands of patients simultaneously. Indeed, our studies demonstrate that repeat dosing, combined with a targeting mAb, leads to long-term elimination of otherwise refractory tumor cells in these xenograft models ( and ).

Unlike autologous CAR-T cells that persist and remain functional for years posttransplantation,54 allogeneic NK cells normally survive in the host for only a few weeks in the adoptive transfer setting.2,7,10,19 However, considering the toxicities seen with CAR T-cell therapies,55 this property of allogeneic NK cells may be advantageous to enable more precise dosing strategies without significant concern for limiting toxicities. The use of PB-NK cells and UCB-NK cells does not typically allow for repeat dosing, because all of the cells collected or produced are used for the initial treatment, often after a dose of lymphodepleting chemotherapy. Indeed, clinical trials using genetically unmodified iPSC-derived NK cells have been initiated with repeat cell dosing on a weekly basis, for a total of 3 doses (clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT03841110","term_id":"NCT03841110"}}NCT03841110). Other treatment schedules, such as monthly dosing, possibly combined with chemotherapy to treat solid tumors, can also be envisioned. Because previous trials of allogeneic NK cellbased therapies utilizing PB-NK cells, UCB-NK cells, or NK92 cells did not show complications, such as cytokine release syndrome, neurotoxicity, or graft-versus-host disease, that are seen with CAR T cells, these trials will be essential to demonstrate the safety and suitability of this multidosing strategy.

A recent study demonstrated that ADAM17-mediated CD16a shedding plays a role in the disassembly of the NK cell immune synapse during ADCC and regulates NK cell motility and detachment from target cells, potentially leading to improved NK cellmediated serial killing of tumor targets.56 However, we did not observe the inhibition of detachment from targets by hnCD16 in hnCD16-iNK cells (supplemental Figure 5). Furthermore, a direct comparison in long-term assays showed superior killing with hnCD16 compared with wild-type CD16. The discrepancy might be caused by different NK cells used in these studies. Srpan et al56 used NK-92 cells, which do not express endogenous CD16 and cannot mediate ADCC. Moreover, those studies did not test the effects of blocking CD16a shedding on NK cell effector functions in vivo and, in particular, in the tumor microenvironment. Notably, NK cells from patients with solid tumors have been shown to have lower CD16 expression and function compared with healthy controls.57 For these studies, we used multiple in vivo tumor models that all demonstrate the benefits of stable high-level noncleavable CD16a expression by NK cells.

In conclusion, this platform therapy provides high impact for immediate translation because it can be combined with essentially any readily available anti-tumor antibody (eg, rituximab, trastuzumab, or cetuximab). This offers several advantages over CAR T-cell therapy and avoids the complexities of developing individualized products with single specificity. Our data demonstrate the advantages of multidosing strategies and the ability to use cryopreserved iNK cell products to provide a new off-the-shelf therapeutic strategy for improved cancer control when used in combination with anti-cancer mAbs that mediate ADCC.

Contribution: H.Z. and R.B. designed and implemented studies, acquired and analyzed data, and wrote the manuscript; R.H.B. acquired and analyzed data and wrote the manuscript; S.G., P.R., G.B.B., and P.-F.T. acquired data; T.T.L. and R.A. designed and implemented studies and acquired and analyzed data; J.W. designed studies, generated the noncleavable human CD16 construct, and reviewed and revised the manuscript; J.S.M. and B.W. designed studies and reviewed and revised the manuscript; and B.V. and D.S.K. designed studies, analyzed data, and reviewed and revised the manuscript.

Conflict-of-interest disclosure: R.B., S.G., P.R., T.T.L., R.A., G.B.B., P.-F.T., and B.V. are employees of Fate Therapeutics with stock holdings and options. J.S.M. consults for and hold stock options in Fate Therapeutics, a company which may commercially benefit from the results of this research project. These interests have been reviewed and managed by the University of Minnesota in accordance with its conflict of interest policy. B.W. collaborates with Fate Therapuetics with a sponsored research agreement. D.S.K. is a consultant for Fate Therapeutics, has equity and receives income. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies.The remaining authors declare no competing financial interests.

Correspondence: Dan S. Kaufman, University of California, San Diego, 9500 Gilman Dr, MC 0695, La Jolla, CA 92093; e-mail: ude.dscu@namfuaksd.

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Pluripotent stem cellderived NK cells with high-affinity noncleavable ...

Auburn University student Rachel Ruhlin never expected the lessons she learned in her audiology class might potentially save her fathers life, but thats exactly what happened earlier this year.

Ruhlinan incoming senior from Eden Prairie, Minnesota, studying Speech, Language and Hearing Sciences in the College of Liberal Artswill eventually apply what shes learned in the program to her career. But Ruhlin is different in that shes already taken what she learned from the classroom all the way to the Mayo Clinic after she set a chain of events in motion that led to the discovery of her fathers brain tumor.

In class, Ruhlin learned about parts of the ear, the importance of hearing aids and tumors like acoustic neuromas that can cause hearing loss. Meanwhile, her father, Joe, had struggled with worsening hearing loss for yearsonly talking on the phone on one side, not hearing anything said near his left earso Ruhlin urged him to set up an appointment with an audiologist.

Once I started taking these classes, it put it more into perspective, Rachel Ruhlin said. My professor would talk about how many people have hearing loss, and if you dont get hearing aids, your hearing will just get worse and worse. Finally, I texted my dad and I said, We have to go. I didnt really give him an option.

Originally, the appointment was simply to test if Joe Ruhlin would be a candidate for hearing aids. He thought his worsening hearing was just a result of aging, but after Rachel explained the many social, emotional and psychological implications of hearing loss, he was persuaded.

Plus, Joe Ruhlin said, it would be interesting for his daughter to see a real hearing test, so he agreed to go.

She strongly encouraged me to set up an appointment while she was home, Joe Ruhlin said. And I think she knew I would go with her, partly because it was going to be interesting for her to see up close what an audiology test looks like, what a hearing test would look like and participate in it and ask questions.

At the first audiology appointment, it was no surprise to Ruhlin that her fathers hearing test indicated serious hearing loss on one side. The next step in the process was for Rachels father to visit an ear, nose and throat doctor, who would conduct a more comprehensive test, including ordering an MRI.

When the MRI results came back showing a large tumor, Rachel knew exactly what it was: an acoustic neuroma, an extremely rare, but serious brain tumor. It was one of the worst-case scenarios she was familiar with through her classes at Auburn.

In Auburns Speech, Language and Hearing Sciences, or SLHS, program, all students must take Professor Sridhar Krishnamurtis audiology class, where they learn foundational knowledge about hearing loss.

This class teaches them what the field is about, its our trademark class in audiology for undergraduates, Krishnamurti said. Very few people, like Rachel, take it to that next level where they translate it to help their family. This is the first time in 25 years that Ive experienced a student affecting their familys future.

Because of that class, Ruhlin said she had a firm grasp on everything that was happening.

When we went to the audiologist the first time, I knew exactly what was going on with his audiogram and how bad it was because I had seen audiograms in class all the time, Ruhlin said. Then we went to the surgeons, and they were talking about parts of the ear, and Ive learned all those. Before surgery, I knew everything that was going on because we learned about this specific type of tumor in class. So, if I hadnt had these classes, I definitely wouldve been a lot more confused and in the dark.

An acoustic neuroma, also known as a vestibular schwannoma, is a benign tumor that grows in the cells surrounding the hearing and balance nerves. Common symptoms include hearing loss on one side, ringing in one ear, dizziness and facial numbness when the tumor gets large.

Acoustic neuromas are rare, affecting only about three people in 100,000, but are risky because of their proximity to the brain stem. Left untreated, they can grow large enough to compress the brain stem and become life-threatening.

Dr. Michael Link is the neurosurgeon who took care of Joe Ruhlin at the Mayo Clinic, one of the best facilities in the world for vestibular schwannoma treatment. Link has seen thousands of patients with this type of tumor and said the longer it goes untreated, the more dangerous it can become.

Even though its benign, having something growing inside your head is somewhat of a risk. And where this tumor arises, theres a lot of important things, especially the brain stem, right next to it. So, as these tumors slowly enlarge, they can start to push on some critical structures, Link said. The other issue is that right with the hearing and balance nerve runs the facial nerves, which innervates all the muscles of facial expression. As the tumor gets bigger, the risk that the facial nerve will be injured or wont work well after surgery goes up.

The treatment options for a vestibular schwannoma include surgery, radiation and observation. Because of the size of Joe Ruhlins tumor, Link conducted a successful surgical removal on Feb. 16 at the Mayo Clinic in Rochester, Minnesota.

Because the tumor is so embedded in the hearing nerves, total hearing loss on the affected side is expected when a large tumor is removed. Link said patients also experience temporary balance issues and facial weakness following the surgery.

Link said while acoustic neuromas are rare, any hearing issues should be taken seriously.

The great, great majority of the time, when people have unilateral hearing loss or unilateral ringing in the ear, it is not a tumor. But once again, its always worth getting it checked out, Link said. Its been fascinating to me that so many people have hearing loss and refuse to get it checked out. It is a big issue for quality of life if youre missing a lot of whats going on around you. So, I think for all of our family members, we have to be vigilant and say if youre not hearing well, you need to get it checked out.

For the next few months, Joe Ruhlin will recover from the surgery and slowly regain his balance, content with the knowledge that the tumor is completely removed and he is no longer at risk of further damage.

Ruhlin said hes grateful his daughter had the knowledge to help guide him through the process.

It means a lot that she helped me get through this, Ruhlin said. Im very grateful that she pushed me to go see the doctor. It did take some encouragement, and shes very good at encouraging me to do things. Daughters can be that way.

So, Im very grateful that she was in that audiology class at the time. She was talking to her audiology professors, and they were backing up what we were hearing, so it was very comforting to have her support.

Throughout the process, Rachel Ruhlin consulted her SLHS professors, who also worked with her schedule to ensure that she could be at home with her father around the time of the surgery.

The SLHS program was so supportive prior to the surgery and during the surgery, she said. I could just tell them my dad had an acoustic neuroma, and they knew exactly what it was. I didnt have to explain anything, and they still ask me how hes doing. The support from the SLHS faculty, especially being thousands of miles away, I couldnt be happier to be a part of this program.

Krishnamurti said what really sets Rachel apart from her peers is that she had the conviction to immediately apply what she learned in class, from the first hearing test through treatment.

All credit goes to Rachel because she was conscientious, she listened in class, she wrote it down and went home and took her father to the doctor, Krishnamurti said. She made sure that he went to one of the best places in the world and got the best treatment, and Im sure hes a lot happier man today. Its heartening for us as faculty members that we were able to give her the information she needed to do the right things.

Despite some of her professors encouragement to pursue audiology, Ruhlin still looks forward to becoming a speech-language pathologist who works with Spanish-speaking families. She said this experience has given her a new appreciation for audiology.

A lot of times, you learn something in the classroom and you just kind of leave it in the classroom, but it was so impactful that I was able to apply it to something so meaningful in my life, Ruhlin said. This will have an impact for years and years, because now the tumors gone and hell be fine. What I learned potentially saved his life.

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Life-saving lecture: Auburn student uses lessons from class to help discover father's brain tumor - Office of Communications and Marketing

After years of research and billions of dollars spent, Bluebird bio is on the cusp of a milestone only a few drugmakers have ever reached.

Two gene therapies its developed are under review by the Food and Drug Administration and, if approved, would become only the third and fourth cleared by the agency for use in treating inherited diseases.

But Bluebird, a pioneer in the field, simultaneously faces financial peril. The Massachusetts-based biotech is quickly running out of cash and earlier this year warned investors that it may struggle to stay solvent. Its already cutting costs.

Approvals, should they come, could mean Bluebirds survival. They would also help restore confidence in a field thats been recently shaken by safety scares and regulatory surprises.

Most importantly, FDA clearances would bring new treatment options for patients with few. One of Bluebirds therapies treats the rare blood condition beta thalassemia, which in its severe form requires blood transfusions every few weeks for life. The other is meant to halt the progression of a devastating neurological disorder called CALD, or cerebral adrenoleukodystrophy, that affects young boys.

On Thursday and again on Friday, the FDA will convene a panel of outside experts to review Bluebirds data and advise it on the treatments respective benefits and risks. Documents published Tuesday indicate agency scientists are cautiously supportive of the beta thalassemia treatment, called beti-cel, but are more skeptical of the CALD therapy, eli-cel.

The FDA is due to make its decisions by Aug. 19 and Sept. 16, respectively. Its verdicts could determine Bluebirds fate, shape how other gene therapies are developed and, potentially, change how two genetic diseases are treated.

I think there really is a spotlight on how this is handled by the FDA, said Christine Duncan, a physician at Boston Childrens Hospital and medical director of the gene therapy program there.

Four years ago, Bluebird was riding high. The biotechs progress developing gene therapies for rare genetic diseases early last decade had helped propel the field forward, and Bluebird, as a result, had become one of the industrys most valuable companies.

That January, Nick Leschly, Bluebirds longtime CEO, took the stage at the pharmaceutical industrys most closely watched conference and told the gathered crowd his company would soon ask regulators for approval of three experimental drugs.

It was a noteworthy declaration, meant to signal Bluebirds coming transition into a commercial-stage drugmaker after nearly three decades of laboratory and clinical research. Leschlys forum, the grand ballroom of the Westin St. Francis at that years J.P. Morgan Healthcare Conference, seemed to match the moment, and reflect Bluebirds ascension.

We believe we're in a leadership position in an important field that's emerging, Leschly said, according to a transcript of his 2018 speech by market data firm Koyfin.

But Bluebirds transition has been rockier than anticipated. While the company did win milestone approvals of beti-cel and eli-cel in Europe, manufacturing hurdles and difficulties securing reimbursement forced Bluebird to later pull both from market and shut down its operations there. Only a handful of patients received either treatment.

In the U.S., disagreements with the FDA over production tests twice delayed Bluebirds plans to seek approval of beti-cel, and slowed progress with another gene therapy for the blood condition sickle cell. The FDA also initially refusedBluebird and partner Bristol Myers Squibbs application for the third drug referred to by Leschly, a cell therapy for multiple myeloma, before eventually approving it in early 2021.

Its the challenge of being [on] a frontier. You hit all the waves first, Leschly said in a February interview, a few months after leaving to head 2seventybio, a new biotech that Bluebird created by spinning out its oncology business. (Andrew Obenshain, formerly Bluebirds genetic disease head, succeeded Leschly as Bluebirds CEO.)

Other gene therapy developers have struggled to cross the FDAs finish line as well, or have run into unexpected roadblocks with their products

Whats happening to them is not unique to Bluebird, said Nicole Paulk, an assistant professor of AAV gene therapy at the University of California, San Francisco. Any gene therapy company you can name off the top of your head theyre all facing the same problems around manufacturing and [production controls].

For Bluebird, though, the delays and setbacks have been especially costly. Its market value, near $10 billion in early 2018, has dwindled to just above $250 million currently. The company moved its headquarters from Cambridge to Somerville, Massachusetts to save money and in April said it will lay off about a third of its employees.

The cost-cutting might not be enough, either. Bluebird, which regularly records quarterly losses totaling hundreds of millions of dollars, now has just $267 million in available funds. In March and again in May, the biotech warned there was substantial doubt it will be able to stay afloat for the next year.

Bluebirds survival may come down to whether the FDA clears beti-cel and eli-cel. Approval of either would give Bluebird the chance to make money not only from commercial sales, but also from special regulatory vouchers the FDA awards that typically sell for about $100 million each.

Yet Bluebirds treatments arent a sure bet to win the FDAs blessing. Looming over both are safety concerns that have cropped up over the past year.

Last February, Bluebird suspended testing of its sickle cell gene therapy after a study participant developed a form of leukemia. While no cases were reported for beti-cel, which uses similar technology, the FDA subsequently placed study holds on both therapies.

An investigation by the company found no clear link between treatment and the leukemia case, and in June the FDA lifted its mandated trial pauses.

But then in August Bluebird reported a case of myelodysplastic syndrome, a cancer-like condition of the bone marrow that can evolve into leukemia, in a CALD patient treated with eli-cel.

Two more eli-cel patients were subsequently diagnosed with myelodysplastic syndrome, or MDS. Two of the cases were directly linked to treatment, while the third was judged highly likely to be related.

In documents published Tuesday, FDA scientists made clear their concerns, noting that, as all three cases occurred long after treatment, they expect more to emerge from recently treated study participants. Laboratory testing also revealed troubling genetic signs in many of the other patients that could portend but not necessarily cause future cancer.

Both eli-cel and beti-cel, as well as Bluebirds sickle cell treatment, are based around a similar therapeutic concept. Stem cells are taken from each patient and, in a laboratory, genetically modified using a type of virus known as a lentivirus. Once reinfused, the engineered stem cells mature and express proteins that replace ones mutated or missing in beta-thalassemia, CALD and sickle cell.

The question is whether the insertion of the virus into the genome of the cells has resulted in the clonal expansion of that population of cells and, potentially, evolution towards leukemia, said Paul Orchard, a pediatric transplant specialist at the University of Minnesota who helped run a study of eli-cel.

The FDA and its advisers will have to weigh the therapies safety risks against their benefits. In the case of eli-cel, the treatments effectiveness was primarily measured as the percent of treated patients who survived two years without major functional disabilities like tube-feeding or wheelchair dependence.

Twenty-nine of the 32 study participants assessed reached this goal, a rate thats near what Bluebird found for similar CALD patients treated with donor-derived stem cell transplants in two natural history studies.

Donor, or allogeneic, transplants can be an effective treatment for CALD, but work best when the stem cells come from a sibling. Unfortunately, only about 30% of boys with CALD have matched sibling donors, according to the FDA.

Aside from donor factors, it is unclear if there is a CALD population for whom the benefit of treatment with eli-cel outweighs the significant and unknown long-term risk of MDS, FDA staff wrote in the documents.

Duncan, of Boston Childrens, agrees the tradeoffs are difficult to weigh but argues that, for some at least, eli-cel would be a needed additional option.

Its not black and white, said Duncan, who is an investigator in an eli-cel study and will present to the FDAs advisers Thursday on behalf of Bluebird. This is an area of gray in that for some patients, if they dont have an allogeneic stem cell transplant match, they dont have a choice. Their disease progresses, theyre neurologically devastated and they die.

The FDA appears more supportive of beti-cel, which in testing showed a powerful ability to free beta thalassemia patients from needing regular blood transfusions. Agency staff agreed the data show it to provide a meaningful benefit for those patients and noted treatment could reduce serious health risks associated with chronic transfusions.

Even so, the FDA is asking its advisers to weigh the cancer risk observed in testing of eli-cel and Bluebirds sickle cell therapy, and whether that should impact its decision on beti-cel.

Neither eli-cel or beti-cel, if approved, are expected to become top-selling drugs, despite expectations that Bluebird could price them similarly to other gene therapies available in the U.S. and Europe that cost more than $1 million.

Bluebird estimates there are between 1,000 and 1,300 beta thalassemia patients in the U.S. who require regular blood transfusions. The number of boys with CALD is far less, perhaps as few as 50 in the U.S, according to analysts from RBC Capital Markets.

But the impact of each treatment could still be significant, both for patients that might receive them and for the broader gene therapy field.

Only two gene therapies for inherited diseases are approved in the U.S. Luxturna for a form of childhood blindness and Zolgensma for the neurodegenerative disease spinal muscular atrophy. (Six CAR-T cell therapies, which are sometimes classified as gene therapy, are also approved for a range of blood cancers.)

Previously FDA officials had predicted that, by 2025, theyd be reviewing between 10 and 20 gene and cell therapies each year a figure that now looks likely to be an overestimate. The agency also appears to be moving cautiously as more safety concerns beyond Bluebird emerge, freezing a number of gene therapy studies over the past year.

And while investment in gene therapy has boomed, a large number of companies are now restructuring their research, cutting costs or laying off employees amid a broader biotech market downturn.

We need a catalyst moment to bring us out of this sentiment slump, said Paulk.

FDA approval of beti-cel and eli-cel could provide one. If the agency decides to hold off, expectations for more gene therapy clearances in the near future might be further tempered.

For Bluebird, the consequences are clearer. The FDAs decisions could determine whether the company remains around long enough to provide either therapy to patients.

It would be extremely unfortunate to get to the point where [weve] developed efficacious therapies and then the companies dont have the finances to move forward, said Orchard, of the University of Minnesota.

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Bluebird's future in balance as FDA weighs gene therapy approvals - BioPharma Dive

In Communities that KARE, a 9-year-old who needs a blood stem cell transplant is traveling the country to find donors, not just for him but for others too.

BLOOMINGTON, Minnesota Nine-year-old Alfredo Torres has a big appetite for food, Chicago Bulls basketball, and music. He loves to wrestle and play soccer with his cousins and run.

But what sets this 4th grader from Chicago apart from other kids his age is that he lives with a blood disorder and needs a blood stem cell transplant.

A blood stem cell transplant replaces a patient's unhealthy blood-forming cells with healthy ones from their donor. The cells used in transplants come from peripheral blood stem cells (PBSC), marrow, and umbilical cord blood. A successful transplant can cure or treat more than 75 diseases, including leukemia and lymphoma, aplastic anemia, sickle cell, and immune-deficiency disorders.

Alfredo and his mom Natalia Torres traveled to Minnesota to raise awareness for Be the Match, a Twin Cities-based nonprofit supporting patients battling blood cancers and disorders.

"He's full of life. He wants to live," said Torres. "He gets infections; he gets sick real quick because his immune system is suppressed."

Be the Match runs a national registry of potential donors for bone marrow and blood stem transplants. They also search international registries with access to more than 39 million people.

Still, some patients have a hard time finding a match. "We're searching for [a] possible donor from a Hispanic donor because we're Hispanics," said Torres.

The likelihood of finding a matching donor ranges from 29% to 70%, depending on ethnic background. "When we look across the spectrum, our lowest chance of finding a match is for Black and African American patients at 29%," explained Alex Mensing, director of benefactor engagement for Be the Match. "Really, the answer to that is more diverse registry members joining."

The Mall of America is joining the cause by partnering with Be the Match for a unique campaign called 30,000 lifesavers.

Shoppers can join the registry at on-site events throughout the year or make a financial donation. "We fully fund patient assistance for our patients and their families. As everybody knows, cancer treatment, [and] blood disease treatment is not cheap," said Mensing.

Every dollar Be the Match raises helps more patients afford transplants, adds potential blood stem cell donors to the registry, and funds research. In 2021, Be The Match provided $6.1 million in patient assistance to 2,600 families.

The burden is heavy for families, and instead of just waiting for a call, the Torres family calls for action. "We just need to raise awareness for these patients in need."

Adults can donate one of two ways. First, about 85% of the time, a patient's doctor requests a PBSC donation, a non-surgical, outpatient procedure similar to donating platelets or plasma.

About 15% of the time, a patient's doctor requests marrow, a surgical, outpatient procedure at a hospital. General or regional anesthesia is always used.

Adults between the ages of 18-40 who meet health guidelines can join the Be The Match Registry for no cost by visiting BeTheMatch.org. Registration involves completing a health history form and giving a swab of cheek cells. People between 18 and 35 are most urgently needed since they are requested by transplant doctors most often, and research shows that these donors provide the best chance for transplant success.

Another way Be the Match helps patients is by organizing couriers who hand-deliver the donated cells, transported as quickly and safely as possible to a patient in need across the country and the world. That life-saving transport happens around 15 times a day.

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Be the Match is looking for more ethnically diverse donors to help patients with blood cancers and diseases - KARE11.com

Every year, more than 18,000 patients search the Be The Match registry hoping to connect with someone who can provide a life-saving donation. The organization locates blood stem-cell or bone marrow transplants for patients with blood cancer or disease.

Matches are found across the country and the world.

I didnt think anything was going to come of it, said donor Haven Davis of Minneapolis.

The teacher added her name to the registry in 2017 after hearing a podcast about the life-saving program. Once someone signs up to be a donor, the organization sends a kit in the mail and the donor provides a cheek swab. The DNA sample is then analyzed.

I think it was less than two months later that I got a phone call I had matched, said Davis. It was just kind of hard to believe.

Be The Match provided limited details about the person who would receive of her donation, including the age and diagnosis.

When I found out that she was only 14, that was when I was really convinced, she said. How can I not help this teenager who needs this?

The 14-year-old patient was Gwen Cinquemani, who lived more than a thousand miles away in New Rochelle, New York. Her life had recently been upended by an MDS diagnoses. Its a rare disorder that affects the bodys red blood cell supply.

I was very active, I had just made the JV cheer team, she told us. The diagnosis kind of halted everything so fast. I went from what felt like everything to nothing.

Prior to her diagnosis, Tiffany Cinquemani noticed her daughter seemed more tired and bruised more easily than normal. Blood work at an annual appointment confirmed she wasnt well.

Your whole world stops, said Tiffany Cinquemani. You get a call from the doctor and youre basically told get your daughter to the hospital immediately so you know its not good. We took her there, and you know, after a series of tests they diagnosed her with MDA and it was terrible. Theres nothing worse.

She added, It was very scary. I mean obviously I knew something was wrong but had no idea how serious it really was.

Doctors determined Gwen needed a bone marrow transplant. Despite being a triplet, her brothers werent a match. In Minneapolis, Davis answered the call.

It really didnt feel like a difficult process or a difficult decision, Davis said.

In November 2018, Davis had a surgical procedure to extract bone marrow from her hip. She went home from the hospital on the same day.

In New York, Gwens recovery from the transplant took longer.

It took two days to get the cells, the teenager explained. Its given to you like a blood transfusion so you just kind of wait for it all to go in. Its done really precisely where the nurses and doctors are kind of timing how fast it goes into you.

Gwen stayed in the hospital until her body started making its own blood cells. After 56 days, she went home. The procedure was successful.

About a year and a half later, the teenager was able to connect with Davis through e-mail.

Before I even opened the email, I could see it said Thank you for saving our daughters life, said Davis. I think that was the moment I realized how impactful this whole situation was.

They stayed in touch during the pandemic, sharing life updates. Last Thursday, they finally met in-person at the Be The Match Gala in New York City.

It was surreal, said Tiffany Cinquemani. It was just amazing to meet the person who saved her life and shes just a wonderful person.

The family embraced Davis on stage.

I think my biggest takeaway from the entire experience has just been a feeling of gratitude, said Davis. I feel really grateful to have such a beautiful connection with such a great family.

The moment also highlighted Gwens progress.

I can go to school, cheer practice hang out with my friends, said Gwen, who is now 16-years-old. Id say Im fully healthy now.

Doctors will monitor her closely for five years post-transplant. She explained she still sees a hematologist every couple of months to track her progress.

Be The Match estimates about only 25 to 30 percent of patients have a family member who can provide a donation. Last year, the organization facilitated about 6,600 transplants.

We need people to join the registry, said Joy King, the chief advancement officer. We need young people to join the registry because the younger you are, the healthier your cells are that create blood that the patient needs. We also need individuals that are ethnically diverse to join the registry as well so we can make sure that all of our patients have equal outcomes.

King explained its critical that donors follow through once they start the process as well.

For many patients, their journey to receive a blood stem cell or bone marrow transplant is their only chance at cure, she said. Its really important people follow through and once you say yes, and start your process to donate to a patient, that you dont back out. Thats because patients start regiment a treatment regimen that absolutely gets rid of their immune system and we need that to fight off disease. Its can be life-threatening if that donation process doesnt match up with the timing that the patient needs to receive those lifesaving cells.

If you are interested in joining the Be The Match registry, click here.

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Minnesota woman reunites with teenager she saved through Be The Match transplant - KSTP

Curr Cardiol Rev. 2013 Feb; 9(1): 6372.

1Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota, USA

2Stem Cell Institute, University of Minnesota Medical School, Minneapolis, Minnesota, USA

1Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota, USA

1Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota, USA

2Stem Cell Institute, University of Minnesota Medical School, Minneapolis, Minnesota, USA

3Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota, USA

1Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota, USA

2Stem Cell Institute, University of Minnesota Medical School, Minneapolis, Minnesota, USA

3Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota, USA

Received 2012 Jun 11; Revised 2012 Jul 31; Accepted 2012 Aug 27.

Induced pluripotent stem (iPS) cells, are a type of pluripotent stem cell derived from adult somatic cells. They have been reprogrammed through inducing genes and factors to be pluripotent. iPS cells are similar to embryonic stem (ES) cells in many aspects. This review summarizes the recent progresses in iPS cell reprogramming and iPS cell based therapy, and describe patient specific iPS cells as a disease model at length in the light of the literature. This review also analyzes and discusses the problems and considerations of iPS cell therapy in the clinical perspective for the treatment of disease.

Keywords: Cellular therapy, disease model, embryonic stem cells, induced pluripotent stem cells, reprogramm.

Induced pluripotent stem (iPS) cells, are a type of pluripotent stem cell derived from adult somatic cells that have been genetically reprogrammed to an embryonic stem (ES) cell-like state through the forced expression of genes and factors important for maintaining the defining properties of ES cells.

Mouse iPS cells from mouse fibroblasts were first reported in 2006 by the Yamanaka lab at Kyoto University [1]. Human iPS cells were first independently produced by Yamanakas and Thomsons groups from human fibroblasts in late 2007 [2, 3]. iPS cells are similar to ES cells in many aspects, including the expression of ES cell markers, chromatin methylation patterns, embryoid body formation, teratoma formation, viable chimera formation, pluripotency and the ability to contribute to many different tissues in vitro.

The breakthrough discovery of iPS cells allow researchers to obtain pluripotent stem cells without the controversial use of embryos, providing a novel and powerful method to "de-differentiate" cells whose developmental fates had been traditionally assumed to be determined. Furthermore, tissues derived from iPS cells will be a nearly identical match to the cell donor, which is an important factor in research of disease modeling and drug screening. It is expected that iPS cells will help researchers learn how to reprogram cells to repair damaged tissues in the human body.

The purpose of this paper is to summarize the recent progresses in iPS cell development and iPS cell-based therapy, and describe patient specific iPS cells as a disease model, analyze the problems and considerations of iPS therapy in the clinical treatment of disease.

The methods of reprogramming somatic cells into iPS cells are summarized in Table . It was first demonstrated that genomic integration and high expression of four factors, Oct4/Sox2/Klf4/c-Myc or Oct4/Sox2/Nanog/LIN28 by virus, can reprogram fibroblast cells into iPS cells [1-3]. Later, it was shown that iPS cells can be generated from fibroblasts by viral integration of Oct4/Sox2/Klf4 without c-Myc [4]. Although these iPS cells showed reduced tumorigenicity in chimeras and progeny mice, the reprogramming process is much slower, and efficiency is substantially reduced. These studies suggest that the ectopic expression of these three transcription factors (Oct4/Klf4/Sox2) is required for reprogramming of somatic cells in iPS cells.

Various growth factors and chemical compounds have recently been found to improve the induction efficiency of iPS cells. Shi et al., [5] demonstrated that small molecules, able to compensate for Sox2, could successfully reprogram mouse embryonic fibroblasts (MEF) into iPS cells. They combined Oct4/Klf4 transduction with BIX-01294 and BayK8644s and derived MEF into iPS cells. Huangfu et al., [6, 7] reported that 5-azacytidine, DNA methyltransferase inhibitor, and valproic acid, a histone deacetylase inhibitor, improved reprogramming of MEF by more than 100 folds. Valproic acid enables efficient reprogramming of primary human fibroblasts with only Oct4 and Sox2.

Kim et al. showed that mouse neural stem cells, expressing high endogenous levels of Sox2, can be reprogrammed into iPS cells by transduction Oct4 together with either Klf4 or c-Myc [19]. This suggests that endogenous expression of transcription factors, that maintaining stemness, have a role in the reprogramming process of pluripotency. More recently, Tsai et al., [20] demonstrated that mouse iPS cells could be generated from the skin hair follicle papilla (DP) cell with Oct4 alone since the skin hair follicle papilla cells expressed endogenously three of the four reprogramming factors: Sox2, c-Myc, and Klf4. They showed that reprogramming could be achieved after 3 weeks with efficiency similar to other cell types reprogrammed with four factors, comparable to ES cells.

Retroviruses are being extensively used to reprogram somatic cells into iPS cells. They are effective for integrating exogenous genes into the genome of somatic cells to produce both mouse and human iPS cells. However, retroviral vectors may have significant risks that could limit their use in patients. Permanent genetic alterations, due to multiple retroviral insertions, may cause retrovirus-mediated gene therapy as seen in treatment of severe combined immunodeficiency [25]. Second, although retroviral vectors are silenced during reprogramming [26], this silencing may not be permanent, and reactivation of transgenes may occur upon the differentiation of iPS cells. Third, expression of exogenous reprogramming factors could occur. This may trigger the expression of oncogenes that stimulate cancer growth and alter the properties of the cells. Fourth, the c-Myc over-expression may cause tumor development after transplantation of iPS derived cells. Okita et al. [10] reported that the chimeras and progeny derived from iPS cells frequently showed tumor formation. They found that the retroviral expression of c-Myc was reactivated in these tumors. Therefore, it would be desirable to produce iPS cells with minimal, or free of, genomic integration. Several new strategies have been recently developed to address this issue (Table ).

Stadtfeld et al. [16] used an adenoviral vector to transduce mouse fibroblasts and hepatocytes, and generated mouse iPS cells at an efficiency of about 0.0005%. Fusaki et al. [22] used Sendai virus to efficiently generate iPS cells from human skin fibroblasts without genome integration. Okita et al. [27] repeatedly transfected MEF with two plasmids, one carrying the complementary DNAs (cDNAs) of Oct3/4, Sox2, and Klf4 and the other carrying the c-Myc cDNA. This generated iPS cells without evidence of plasmid integration. Using a polycistronic plasmid co-expressing Oct4, Sox2, Klf4, and c-Myc, Gonzalez et al., [28] reprogrammed MEF into iPS cells without genomic integration. Yu et al. [29] demonstrated that oriP/EBNA1 (EpsteinBarr nuclear antigen-1)-based episomal vectors could be used to generate human iPS cells free of exogenous gene integration. The reprogramming efficiency was about 36 colonies/1 million somatic cells. Narsinh et al., [21] derived human iPS cells via transfection of human adipocyte stromal cells with a nonviral minicircle DNA by repeated transfection. This produced hiPS cells colonies from an adipose tissue sample in about 4 weeks.

When iPS cells generated from either plasmid transfection or episomes were carefully analyzed to identify random vector integration, it was possible to have vector fragments integrated somewhere. Thus, reprogramming strategies entirely free of DNA-based vectors are being sought. In April 2009, it was shown that iPS cells could be generated using recombinant cell-penetrating reprogramming proteins [30]. Zhou et al. [30] purified Oct4, Sox2, Klf4 and c-Myc proteins, and incorporated poly-arginine peptide tags. It allows the penetration of the recombinant reprogramming proteins through the plasma membrane of MEF. Three iPS cell clones were successfully generated from 5x 104 MEFs after four rounds of protein supplementation and subsequent culture of 2328 days in the presence of valproic acid.

A similar approach has also been demonstrated to be able to generate human iPS cells from neonatal fibroblasts [31]. Kim et al. over-expressed reprogramming factor proteins in HEK293 cells. Whole cell proteins of the transduced HEK293 were extracted and used to culture fibroblast six times within the first week. After eight weeks, five cell lines had been established at a yield of 0.001%, which is one-tenth of viral reprogramming efficiency. Strikingly, Warren et al., [24] demonstrated that human iPS cells can be derived using synthetic mRNA expressing Oct3/4, Klf4, Sox2 and c-Myc. This method efficiently reprogrammed fibroblast into iPS cells without genome integration.

Strenuous efforts are being made to improve the reprogramming efficiency and to establish iPS cells with either substantially fewer or no genetic alterations. Besides reprogramming vectors and factors, the reprogramming efficiency is also affected by the origin of iPS cells.

A number of somatic cells have been successfully reprogrammed into iPS cells (Table ). Besides mouse and human somatic cells, iPS cells from other species have been successfully generated (Table ).

The origin of iPS cells has an impact on choice of reprogramming factors, reprogramming and differentiation efficiencies. The endogenous expression of transcription factors may facilitate the reprogramming procedure [19]. Mouse neural stem cells express higher endogenous levels of Sox2 and c-Myc than ES cells. Thus, two transcription factors, exogenous Oct4 together with either Klf4 or c-Myc, are sufficient to generate iPS cells from neural stem cells [19]. Ahmed et al. [14] demonstrated that mouse skeletal myoblasts endogenously expressed Sox2, Klf4, and c-Myc and can be easily reprogrammed to iPS cells.

It is possible that iPS cells may demonstrate memory of parental source and therefore have low differentiation efficiency into other tissue cells. Kim et al. [32] showed that iPS cells reprogrammed from peripheral blood cells could efficiently differentiate into the hematopoietic lineage cells. It was found, however, that these cells showed very low differentiation efficiency into neural cells. Similarly, Bar-Nur et al. found that human cell-derived iPS cells have the epigenetic memory and may differentiate more readily into insulin producing cells [33]. iPS cells from different origins show similar gene expression patterns in the undifferentiated state. Therefore, the memory could be epigenetic and are not directly related to the pluripotent status.

The cell source of iPS cells can also affect the safety of the established iPS cells. Miura et al. [54] compared the safety of neural differentiation of mouse iPS cells derived from various tissues including MEFs, tail-tip fibroblasts, hepatocyte and stomach. Tumorigenicity was examined. iPS cells that reprogrammed from tail-tip fibroblasts showed many undifferentiated pluripotent cells after three weeks of in vitro differentiation into the neural sphere. These cells developed teratoma after transplantation into an immune-deficient mouse brain. The possible mechanism of this phenomenon may be attributable to epigenetic memory and/or genomic stability. Pre-evaluated, non-tumorigenic and safe mouse iPS cells have been reported by Tsuji et al. [55]. Safe iPS cells were transplanted into non-obese diabetic/severe combined immunodeficiency mouse brain, and found to produce electrophysiologically functional neurons, astrocytes, and oligodendrocytes in vitro.

The cell source of iPS cells is important for patients as well. It is important to carefully evaluate clinically available sources. Human iPS cells have been successfully generated from adipocyte derived stem cells [35], amniocytes [36], peripheral blood [38], cord blood [39], dental pulp cells [40], oral mucosa [41], and skin fibroblasts (Table ). The properties and safety of these iPS cells should be carefully examined before they can be used for treatment.

Shimada et al. [17] demonstrated that combination of chemical inhibitors including A83-01, CHIR99021, PD0325901, sodium butyrate, and Y-27632 under conditions of physiological hypoxia human iPS cells can be rapidly generated from adipocyte stem cells via retroviral transduction of Oct4, Sox2, Klf4, and L-Myc. Miyoshi et al., [42] generated human iPS cells from cells isolated from oral mucosa via the retroviral gene transfer of Oct4, Sox2, c-Myc, and Klf4. Reprogrammed cells showed ES-like morphology and expressed undifferentiated markers. Yan et al., [40] demonstrated that dental tissue-derived mesenchymal-like stem cells can easily be reprogrammed into iPS cells at relatively higher rates as compared to human fibroblasts. Human peripheral blood cells have also been successfully reprogrammed into iPS cells [38]. Anchan et al. [36] described a system that can efficiently derive iPS cells from human amniocytes, while maintaining the pluripotency of these iPS cells on mitotically inactivated feeder layers prepared from the same amniocytes. Both cellular components of this system are autologous to a single donor. Takenaka et al. [39] derived human iPS cells from cord blood. They demonstrated that repression of p53 expression increased the reprogramming efficiency by 100-fold.

All of the human iPS cells described here are indistinguishable from human ES cells with respect to morphology, expression of cell surface antigens and pluripotency-associated transcription factors, DNA methylation status at pluripotent cell-specific genes and the capacity to differentiate in vitro and in teratomas. The ability to reprogram cells from human somatic cells or blood will allow investigating the mechanisms of the specific human diseases.

The iPS cell technology provides an opportunity to generate cells with characteristics of ES cells, including pluripotency and potentially unlimited self-renewal. Studies have reported a directed differentiation of iPS cells into a variety of functional cell types in vitro, and cell therapy effects of implanted iPS cells have been demonstrated in several animal models of disease.

A few studies have demonstrated the regenerative potential of iPS cells for three cardiac cells: cardiomyocytes, endothelial cells, and smooth muscle cells in vitro and in vivo. Mauritz [56] and Zhang [57] independently demonstrated the ability of mouse and human iPS cells to differentiate into functional cardiomyocytes in vitro through embryonic body formation. Rufaihah [58], et al. derived endothelial cells from human iPS cells, and showed that transplantation of these endothelial cells resulted in increased capillary density in a mouse model of peripheral arterial disease. Nelson et al. [59] demonstrated for the first time the efficacy of iPS cells to treat acute myocardial infarction. They showed that iPS cells derived from MEF could restore post-ischemic contractile performance, ventricular wall thickness, and electrical stability while achieving in situ regeneration of cardiac, smooth muscle, and endothelial tissue. Ahmed et al. [14] demonstrated that beating cardiomyocyte-like cells can be differentiated from iPS cells in vitro. The beating cells expressed early and late cardiac-specific markers. In vivo studies showed extensive survival of iPS and iPS-derived cardiomyocytes in mouse hearts after transplantation in a mouse experimental model of acute myocardial infarction. The iPs derived cardiomyocyte transplantation attenuated infarct size and improved cardiac function without tumorgenesis, while tumors were observed in the direct iPS cell transplantation animals.

Strategies to enhance the purity of iPS derived cardiomyocytes and to exclude the presence of undifferentiated iPS are required. Implantation of pre-differentiation or guided differentiation of iPS would be a safer and more effective approach for transplantation. Selection of cardiomyocytes from iPS cells, based on signal-regulatory protein alpha (SIRPA) or combined with vascular cell adhesion protein-1 (VCAM-1), has been reported. Dubois et al. [60] first demonstrated that SIRPA was a marker specifically expressed on cardiomyocytes derived from human ES cells and human iPS cells. Cell sorting with an antibody against SIRPA could enrich cardiac precursors and cardiomyocytes up to 98% troponin T+ cells from human ESC or iPS cell differentiation cultures. Elliott et al. [61] adopted a cardiac-specific reporter gene system (NKX2-5eGFP/w) and identified that VCAM-1 and SIRPA were cell-surface markers of cardiac lineage during differentiation of human ES cells.

Regeneration of functional cells from human stem cells represents the most promising approach for treatment of type 1 diabetes mellitus (T1DM). This may also benefit the patients with type 2 diabetes mellitus (T2DM) who need exogenous insulin. At present, technology for reprogramming human somatic cell into iPS cells brings a remarkable breakthrough in the generation of insulin-producing cells.

Human ES cells can be directed to become fully developed cells and it is expected that iPS cells could also be similarly differentiated. Stem cell based approaches could also be used for modulation of the immune system in T1DM, or to address the problems of obesity and insulin resistance in T2DM.

Tateishi et al., [62] demonstrated that insulin-producing islet-like clusters (ILCs) can be generated from the human iPS cells under feeder-free conditions. The iPS cell derived ILCs not only contain C-peptide positive and glucagon-positive cells but also release C-peptide upon glucose stimulation. Similarly, Zhang et al., [63] reported a highly efficient approach to induce human ES and iPS cells to differentiate into mature insulin-producing cells in a chemical-defined culture system. These cells produce insulin/C-peptide in response to glucose stimuli in a manner comparable to that of adult human islets. Most of these cells co-expressed mature cell-specific markers such as NKX6-1 and PDX1, indicating a similar gene expression pattern to adult islet beta cells in vivo.

Alipo et al. [64] used mouse skin derived iPS cells for differentiation into -like cells that were similar to the endogenous insulin-secreting cells in mice. These -like cells were able to secrete insulin in response to glucose and to correct a hyperglycemic phenotype in mouse models of both T1DM and T2DM after iPS cell transplant. A long-term correction of hyperglycemia could be achieved as determined by hemoglobin A1c levels. These results are encouraging and suggest that induced pluripotency is a viable alternative to directing iPS cell differentiation into insulin secreting cells, which has great potential clinical applications in the treatment of T1DM and T2 DM.

Although significant progress has been made in differentiating pluripotent stem cells to -cells, several hurdles remain to be overcome. It is noted in several studies that the general efficiency of in vitro iPS cell differentiation into functional insulin-producing -like cells is low. Thus, it is highly essential to develop a safe, efficient, and easily scalable differentiation protocol before its clinical application. In addition, it is also important that insulin-producing b-like cells generated from the differentiation of iPS cells have an identical phenotype resembling that of adult human pancreatic cells in vivo.

Currently, the methodology of neural differentiation has been well established in human ES cells and shown that these methods can also be applied to iPS cells. Chambers et al. [65] demonstrated that the synergistic action of Noggin and SB431542 is sufficient to induce rapid and complete neural conversion of human ES and iPS cells under adherent culture conditions. Swistowsk et al. [66] used a completely defined (xenofree) system, that has efficiently differentiated human ES cells into dopaminergic neurons, to differentiate iPS cells. They showed that the process of differentiation into committed neural stem cells (NSCs) and subsequently into dopaminergic neurons was similar to human ES cells. Importantly, iPS cell derived dopaminergic neurons were functional as they survived and improved behavioral deficits in 6-hydroxydopamine-leasioned rats after transplantation. Lee et al. [67] provided detailed protocols for the step-wise differentiation of human iPS and human ES into neuroectodermal and neural crest cells using either the MS5 co-culture system or a defined culture system (Noggin with a small-molecule SB431542), NSB system. The average time required for generating purified human NSC precursors will be 25 weeks. The success of deriving neurons from human iPS cells provides a study model of normal development and impact of genetic disease during neural crest development.

Wernig et al., [68] showed that iPS cells can give rise to neuronal and glial cell types in culture. Upon transplantation into the fetal mouse brain, the cells differentiate into glia and neurons, including glutamatergic, GABAergic, and catecholaminergic subtypes. Furthermore, iPS cells were induced to differentiate into dopamine neurons of midbrain character and were able to improve behavior in a rat model of Parkinson's disease (PD) upon transplantation into the adult brain. This study highlights the therapeutic potential of directly reprogrammed fibroblasts for neural cell replacement in the animal model of Parkinsons disease.

Tsuji et al., [55] used pre-evaluated iPS cells derived for treatment of spinal cord injury. These cells differentiated into all three neural lineages, participated in remyelination and induced the axonal regrowth of host 5HT+ serotonergic fibers, promoting locomotor function recovery without forming teratomas or other tumors. This study suggests that iPS derived neural stem/progenitor cells may be a promising cell source for treatment of spinal cord injury.

Hargus et al., [69] demonstrated proof of principle of survival and functional effects of neurons derived from iPS cells reprogrammed from patients with PD. iPS cells from patients with Parkinsons disease were differentiated into dopaminergic neurons that could be transplanted without signs of neuro-degeneration into the adult rodent striatum. These cells survived and showed arborization, and mediated functional effects in an animal model of Parkinsons disease. This study suggests that disease specific iPS cells can be generated from patients with PD, which be used to study the PD development and in vitro drug screen for treatment of PD.

Reprogramming technology is being applied to derive patient specific iPS cell lines, which carry the identical genetic information as their patient donor cells. This is particularly interesting to understand the underlying disease mechanism and provide a cellular and molecular platform for developing novel treatment strategy.

Human iPS cells derived from somatic cells, containing the genotype responsible for the human disease, hold promise to develop novel patient-specific cell therapies and research models for inherited and acquired diseases. The differentiated cells from reprogrammed patient specific human iPS cells retain disease-related phenotypes to be an in vitro model of pathogenesis (Table ). This provides an innovative way to explore the molecular mechanisms of diseases.

Disease Modeling Using Human iPS Cells

Recent studies have reported the derivation and differentiation of disease-specific human iPS cells, including autosomal recessive disease (spinal muscular atrophy) [70], cardiac disease [71-75], blood disorders [13, 76], diabetes [77], neurodegenerative diseases (amyotrophic lateral sclerosis [78], Huntingtons disease [79]), and autonomic nervous system disorder (Familial Dysautonomia) [80]. Patient-specific cells make patient-specific disease modeling possible wherein the initiation and progression of this poorly understood disease can be studied.

Human iPS cells have been reprogrammed from spinal muscular atrophy, an autosomal recessive disease. Ebert et al., [70] generated iPS cells from skin fibroblast taken from a patient with spinal muscular atrophy. These cells expanded robustly in culture, maintained the disease genotype and generated motor neurons that showed selective deficits compared to those derived from the patients' unaffected relative. This is the first study to show that human iPS cells can be used to model the specific pathology seen in a genetically inherited disease. Thus, it represents a promising resource to study disease mechanisms, screen new drug compounds and develop new therapies.

Similarly, three other groups reported their findings on the use of iPS cells derived cardiomyocytes (iPSCMs) as disease models for LQTS type-2 (LQTS2). Itzhaki et al., [72] obtained dermal fibroblasts from a patient with LQTS2 harboring the KCNH2 gene mutation and showed that action potential duration was prolonged and repolarization velocity reduced in LQTS2 iPS-CMs compared with normal cardiomyocytes. They showed that Ikr was significantly reduced in iPS-CMs derived from LQTS2. They also tested the potential therapeutic effects of nifedipine and the KATP channel opener pinacidil (which augments the outward potassium current) and demonstrated that they shortened the action potential duration and abolished early after depolarization. Similarly, Lahti et al., [73] demonstrated a more pronounced inverse correlation between the beating rate and repolarization time of LQTS2 disease derived iPS-CMs compared with normal control cells. Prolonged action potential is present in LQT2-specific cardiomyocytes derived from a mutation. Matsa et al., [74] also successfully generated iPS-CMs from a patient with LQTS2 with a known KCNH2 mutation. iPS-CMs with LQTS2 displayed prolonged action potential durations on patch clamp analysis and prolonged corrected field potential durations on microelectrode array mapping. Furthermore, they demonstrated that the KATP channel opener nicorandil and PD-118057, a type 2 IKr channel enhancer attenuate channel closing.

LQTS3 has been recapitulated in mouse iPS cells [75]. Malan et al. [75] generated disease-specific iPS cells from a mouse model of a human LQTS3. Patch-clamp measurements of LQTS 3-specific cardiomyocytes showed the biophysical effects of the mutation on the Na+ current, withfaster recovery from inactivation and larger late currents than observed in normal control cells. Moreover, LQTS3-specific cardiomyocytes had prolonged action potential durations and early after depolarizations at low pacing rates, both of which are classic features of the LQTS3 mutation.

Human iPS cells have been used to recapitulate diseases of blood disorder. Ye et al. [13] demonstrated that human iPS cells derived from periphery blood CD34+ cells of patients with myeloproliferative disorders, have the JAK2-V617F mutation in blood cells. Though the derived iPS cells contained the mutation, they appeared normal in phenotypes, karyotype, and pluripotency. After hematopoietic differentiation, the iPS cell-derived hematopoietic progenitor (CD34+/CD45+) cells showed the increased erythropoiesis and expression of specific genes, recapitulating features of the primary CD34+ cells of the corresponding patient from whom the iPS cells were derived. This study highlights that iPS cells reprogrammed from somatic cells from patients with blood disease provide a prospective hematopoiesis model for investigating myeloproliferative disorders.

Raya et al., [76] reported that somatic cells from Fanconi anaemia patients can be reprogrammed to pluripotency after correction of the genetic defect. They demonstrated that corrected Fanconi-anaemia specific iPS cells can give rise to haematopoietic progenitors of the myeloid and erythroid lineages that are phenotypically normal. This study offers proof-of-concept that iPS cell technology can be used for the generation of disease-corrected, patient-specific cells with potential value for cell therapy applications.

Maehr et al., [77] demonstrated that human iPS cells can be generated from patients with T1DM by reprogramming their adult fibroblasts. These cells are pluripotent and differentiate into three lineage cells, including insulin-producing cells. These cells provide a platform to assess the interaction between cells and immunocytes in vitro, which mimic the pathological phenotype of T1DM. This will lead to better understanding of the mechanism of T1DM and developing effective cell replacement therapeutic strategy.

Lee et al., [80] reported the derivation of human iPS cells from patient with Familial Dysautonomia, an inherited disorder that affects the development and function of nerves throughout the body. They demonstrated that these iPS cells can differentiate into all three germ layers cells. However gene expression analysis demonstrated tissue-specific mis-splicing of IKBKAP in vitro, while neural crest precursors showed low levels of normal IKBKAP transcript. Transcriptome analysis and cell-based assays revealed marked defects in neurogenic differentiation and migration behavior. All these recaptured familial Dysautonomia pathogenesis, suggesting disease specificity of the with familial Dysautonomia human iPS cells. Furthermore, they validated candidate drugs in reversing and ameliorating neuronal differentiation and migration. This study illustrates the promise of disease specific iPS cells for gaining new insights into human disease pathogenesis and treatment.

Human iPS cells derived reprogrammed from patients with inherited neurodegenerative diseases, amyotrophic lateral sclerosis [78] and Huntingtons disease 79, have also been reported. Dimos et al., [78] showed that they generated iPS cells from a patient with a familial form of amyotrophic lateral sclerosis. These patient-specific iPS cells possess the properties of ES cells and were reprogrammed successfully to differentiate into motor neurons. Zhang et al., [79] derived iPS cells from fibroblasts of patient with Huntingtons disease. They demonstrated that striatal neurons and neuronal precursors derived from these iPS cells contained the same CAG repeat expansion as the mutation in the patient from whom the iPS cell line was established. This suggests that neuronal progenitor cells derived from Huntingtons disease cell model have endogenous CAG repeat expansion that is suitable for mechanistic studies and drug screenings.

Disease specific somatic cells derived from patient-specific human iPS cells will generate a wealth of information and data that can be used for genetically analyzing the disease. The genetic information from disease specific-iPS cells will allow early and more accurate prediction and diagnosis of disease and disease progression. Further, disease specific iPS cells can be used for drug screening, which in turn correct the genetic defects of disease specific iPS cells.

iPS cells appear to have the greatest promise without ethical and immunologic concerns incurred by the use of human ES cells. They are pluripotent and have high replicative capability. Furthermore, human iPS cells have the potential to generate all tissues of the human body and provide researchers with patient and disease specific cells, which can recapitulate the disease in vitro. However, much remains to be done to use these cells for clinical therapy. A better understanding of epigenetic alterations and transcriptional activity associated with the induction of pluripotency and following differentiation is required for efficient generation of therapeutic cells. Long-term safety data must be obtained to use human iPS cell based cell therapy for treatment of disease.

These works were supported by NIH grants HL95077, HL67828, and UO1-100407.

The authors confirm that this article content has no conflicts of interest.

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