Category Archives: Kansas Stem Cells

Introduction

SARS-CoV-2 is the virus responsible for the COVID-19 pandemic and its damaging effects on both health and economics worldwide. Transmission and pathology of the virus appears to be mediated through the respiratory system via interaction of the viral spike protein with the ACE-2 receptor13 differentially presented on various cells within the respiratory system.4,5 Expression of ACE-2 has been reported on lung alveolar epithelial cells, enterocytes of the small intestine, circulatory endothelial cells, arterial smooth muscle cells,6 adipose tissue, bone marrow, duodenum, endometrium, heart, kidney, testis, and thyroid7 suggesting a potential direct effect of COVID-19 on those tissues. Additionally, infection with COVID-19 has been associated with significant liver injuries and altered liver function tests.8

Alterations in liver function have been attributed to secondary effects of cytokine cascade, hypoxia, underlying liver disease,911 or infection of ACE-2 positive cholangiocytes.12 There have also been reports of coronavirus particles in hepatocytes without a defined mechanism for infection.13

In a recent report studying the receptome of spike binding, ACE-2 was confirmed as the primary receptor for the spike protein via the binding domain (RBD) on the spike 1 portion of the molecule and the N-terminal-domain as the sites critical for virushost interaction.14 Additionally, the report described binding of the spike protein with ectopically expressed ASGR1 and KREMEN1 in transfected non-liver cells. The results strongly suggested the existence of additional entry points into cells for the SARS-CoV-2 virus via the spike protein. Differences in primary infection sites and clinical manifestations of SARS-CoV and SARS-CoV-2, both utilizing ACE-2 as the primary site of cellular infection, suggested that other cellular receptors may be involved in SARS-CoV-2 host interactions.14

E12 TERT-immortalized multi-lineage progenitor cells (MLPC) derived from human umbilical cord blood have been differentiated into immortalized AT2-like cells (AT2) (manuscript submitted) and fused directly with primary human hepatocytes to create immortalized hepatocyte-like cells (HLC).15 The resultant E12 AT2-like cells expressed the characteristics of small airway epithelial cells associated with alveolar type 2 cells and not alveolar type 1 cells. The E12/PHH fusion cells (HLC) expressed the characteristics of fully mature and highly differentiated hepatocytes.15

This report studied the interactions of the SARS-CoV-2 spike protein with potential receptors on human cord blood-derived MLPC differentiated AT2, HLC and primary human hepatocytes (PHH) by confocal analysis. The characteristics of spike protein binding were examined using biotinylated spike proteins and blockade of binding by un-labeled spike proteins, spike protein-directed neutralizing antibodies and an antibody directed against the hepatocyte surface membrane asialoglycoprotein receptor 1 (ASGr1). The results suggested that binding and inhibition analyses can be used to assess the potential mechanisms of viral host cell interactions with a myriad of different target cells in the body, but also to assess therapeutics designed to inhibit that binding.

Immortalized AT2 and HLC could provide accurate and reproducible tools to study the differential virushost interactions between these targets of COVID-19 infection and aid in the development of therapeutics designed to inhibit binding and infection by the SARS-CoV-2 virus. In addition, the potential binding of spike protein to the ASGr1 on hepatocytes suggested a mechanism of viral entry via the clathrin-coated pit receptor-mediated pathway and direct injury to the liver.16,17

MLPC are multi-potent non-hematopoietic stem cells isolated from human umbilical cord blood.15 Umbilical cord blood was collected as part of an FDA submission to market PrepaCyte-CB, a product to de-bulk cord blood for cryo-banking and transplantation. IRB approval of the studies was conducted by the University of Minnesota, the Saint Louis Cord Blood Bank and by Quorum Review Protocol #800, March 3, 2005. The cord blood samples were collected by the American Red Cross Cord Blood Program (Saint Paul, Minnesota) and Ridgeview Medical Center (Waconia, MN). Donations were collected with donor consent for research use only.

Briefly, isolated leukocytes were incubated overnight in MSCGM (PT-4105, Lonza, Walkerville, MD) after which non-adherent cells were removed. Cells were cultured in MSCGM until 8090% of cells had a fibroblastic morphology. These cells were transfected with the gene for TERT, as previously described15 and were cloned by limited dilution. The E12 clone was selected for both immortality and differentiating potential. The E12 MLPC, expanded and cryopreserved for over 14 years, were used as undifferentiated control cells and as the source of cells for the development of the AT2-like cells and the fusion partner in the development of MLPC/hepatocyte hybrid cells.15 For confocal analysis, E12 cells (106/mL in MSCGM, 200 L per well) were plated in non-coated 16 well chamber slides (Nalge, Nunc International, Rochester, NY) and allowed to attach overnight before use in the analysis.

AT2-like cells were developed from the differentiation of E12 MLPC. Briefly, E12 cells (3 x 105 cells/mL) in MSCGM were added to non-coated tissue culture vessels and allowed to attach overnight. Medium was then exchanged with SAGM (SAGM, Lonza, Walkerville, MD, cat # 3118) and allowed to culture for 814 days with 3 medium changes per week. Upon achieving 70% confluence, cells were harvested by treatment with Tryp-LE (12605028, Life Technologies, Grand Island, NY) allowed to dissociate from the culture vessel and used for confocal analysis, as a positive control for binding spike proteins and ACE-2 expression. Cells (106/mL in SAGM, 200 L per well) were plated in non-coated 16 well chamber slides and allowed to adhere overnight prior to confocal analysis.

Hepatocyte-like fusion cells were created by the fusion of E12 MLPC with primary human hepatocytes, as previously described.15 Equal numbers of E12 MLPC and primary hepatocytes were fused using 50% polyethylene glycol in RMPI + 0.01% EDTA. Resultant cells were plated into collagen-coated 75 cm2 tissue culture flasks and were cultured for 7 days in RPMI + 20% FBS. After 7 days, non-fused PHH were no longer viable and did not contribute to the HLC cell lines. HLC were examined for hepatocyte-specific markers including albumin and urea production. HLC were demonstrated to express markers and production consistent with fully mature and well-differentiated hepatocytes. HLC (106/mL in hepatocyte expansion medium, 200 L per well) were plated in collagen-coated 16 well chamber slides and were allowed to adhere overnight prior to confocal analysis. Hepatocyte expansion medium consisted of Williams Medium E supplemented with 2% fatty acid-free BSA (Sigma, A7030), 1% ITS solution (Lonza, 17838Z), 5mM hydrocortisone 21-hemisuccinate (Sigma, H2270) and glutamax (35050, Gibco) supplemented with FGF basic (20 ng/mL) (233-FB), FGF-4 (20 ng/mL) (7460-F4), HFG (40 ng/mL) (294-HG), SCF (40 ng/mL) (255-SC), Oncostatin M (20 ng/mL) (295-OM), BMP-4 (20 ng/mL) (314-BP), EGF (40 ng/mL) (236-EG) and IL-1 (20 ng/mL)(201-LB) all from R&D Systems (Minneapolis, MN).

Cryo-preserved primary human hepatocytes and media were obtained from Zenotech (Kansas City, KS). Cells were thawed with OptiThaw medium and enumerated with OptiCount medium in a standard hemacytometer. Hepatocytes were diluted to a final concentration of 106 cells/mL of OptiPlate medium and were plated in collagen-coated 16 well chamber slides at 200 L per well. After 4 hours of plating, the medium was changed to OptiCulture medium to allow overnight attachment and spread of cells prior to confocal analysis.

Cells were prepared for staining with antibodies and binding of spike proteins by fixing the cells in 1% formaldehyde for 1 hour. Cells were then washed x 2 with PermaCyte permeabilization medium (WBP-1000, CMDG, St. Paul, MN). All staining took place in the presence of PermaCyte. Cells were incubated with an unlabeled primary antibody (100 ng) for 30 minutes at room temperature. ACE-2 (labeled with alexa 594, FAB9332T), albumin (MAB1456) and asialoglycoprotein receptor 1 (MAB4394) antibodies were obtained from R&D Systems (Minneapolis, MN). Unbound antibody was removed by washing with PermaCyte and the cells were counterstained with a secondary antibody specific for mouse (A-11005) antibody labelled with Alexa 594 dye (Life Technologies, (Eugene, OR)). Marker expression was confirmed by positive staining when compared to cells stained with antibody isotype controls (QTC1000, CMDG, St. Paul, MN). The nuclei of the cells were visualized by staining with DAPI.

The binding of SARS-CoV-2 spike and spike 1 proteins was analyzed by confocal microscopy using biotinylated spike proteins. Cells were prepared as described above. Cells were labelled with 250 ng of either biotinylated spike (RBD) (SPD-C8E9, ACROBiosystems, Newark, DE) or biotinylated spike 1 protein (SIN-C82E8, ACROBiosystems) for 30 minutes. Unbound spike proteins were removed by washing cells twice with PermaCyte medium. Bound spike proteins were visualized by secondary staining with streptavidin-alexa 594 (S11227 Life Technologies). Cells were counterstained with DAPI to visualize the nuclei.

Specificity of biotinylated spike proteins binding to the cells was confirmed by blockade of binding by a 5 molar excess of unlabeled spike protein. Cells were prepared as per the confocal analysis of antibody binding. Cells were incubated with 1.25 g of unlabeled spike protein (ACROBiosystems, SPD-S52H6) or spike 1 protein (ACROBiosystems, S1N-C52H3) for 1 hour. Without washing the unbound unlabeled spike protein, biotinylated spike and spike 1 proteins were added to the cells and incubated for 30 minutes. Cells were washed twice with PermaCyte medium to remove any unbound proteins. Bound biotinylated spike proteins were observed by secondary labeling with streptavidin-alexa 594. Cells were counterstained with DAPI to visualize the nucleus.

The effects of antibodies on the binding of the spike proteins to the cells were examined using two commercially available neutralizing antibodies obtained from ACROBiosystems (SAD-S35) and Novatein Biosystems (PR-nCOV-mABS1, Boston, MA) and the ASGr1-specific antibody (R&D Systems). One g of either neutralizing antibody was preincubated with the spike protein for one hour prior to the addition of the mixture to the cells prepared as described for binding of the spike proteins. The ASGr1 antibody (300 ng) was preincubated with cells prior to the addition of the spike protein. Visualization of the binding of biotinylated spike protein was accomplished by secondary staining with streptavidin-alexa 594. The nuclei of the cells were visualized with DAPI.

Cells were analyzed on the Olympus Fluoview 1000 confocal microscope. The confocal images in Figures 14 are representative of at least 3 studies done on different days.

Figure 1 Biotinylated spike and spike 1 protein binding to E12 differentiated AT2-like cells and inhibition by unlabeled spike protein and neutralizing antibodies. Bound biotinylated spike proteins were visualized by sequential labeling with streptavidin-alexa 594. Cells positive for binding are shown by red fluorescence. Blue nuclei were visualized by counterstaining with DAPI. (A) Binding of biotinylated spike protein (containing RBD). (B) Inhibition of biotinylated spike protein binding by co-incubation with a 5 molar excess of unlabeled spike protein (RBD). (C) Binding of spike 1 protein. (D) Inhibition of biotinylated spike protein binding by preincubation with a neutralizing antibody from ACROBiosystems. (E) Lack of binding inhibition by neutralizing antibody from Novatein Bio. (F) Inhibition of biotinylated spike 1 binding by unlabeled spike (RBD) protein.

Abbreviation: RBD, receptor-binding domain.

Figure 2 Expression of ACE-2, ASGr1 and serum albumin. Undifferentiated E12 MLPC data are shown in (AD). E12 HLC fusion cell results are presented in (EH). Primary human hepatocytes (PHH) are shown in (IL). Cells were incubated with unlabeled primary antibody and sequentially stained with secondary antibody labeled with alexa-594. Positive binding is shown by red fluorescence. Blue nuclei were visualized by DAPI counterstaining. Figures (A, E and I) were stained with isotype control antibodies. Figures (B, F and J) were stained with antibody specific for ACE-2. Figures (C, G and K) were stained with antibody specific for the asialoglycoprotein receptor 1 (ASGr1). Figures (D, H and L) were stained with antibody specific for serum albumin.

Figure 3 Undifferentiated E12 MLPC confocal microscopy is shown in (AD). E12 HLC fusion cell data are presented in (E-H). Primary human hepatocytes (PHH) are shown in (IL). Positive binding is indicated by red fluorescence. Blue nuclei were visualized with DAPI counterstaining. Figures (A, E and I) were labeled with Sav-594. Figures (B, F and J) were labeled with biotinylated spike protein followed by sequential staining with streptavidin-alexa 594. Figures (C, G and K) were labeled with biotinylated spike 1 protein followed by sequential staining with streptavidin-alexa 594. Figures (D, H and L) biotinylated spike protein binding was blocked by a 5 molar excess of unlabeled spike protein (RBD) followed by sequential staining with streptavidin-alexa 594.

Figure 4 Inhibition of biotinylated spike binding by neutralizing antibodies to spike 1, spike and ASGr1. E12 HLC fusion cell data are shown in (AD). Primary human hepatocytes (PHH) are shown in (EH). Positive binding of biotinylated spike proteins is shown by red fluorescence. Blue nuclei are visualized by counterstaining with DAPI. Figures (A and E) confocals show the inability of a 5 molar excess of unlabeled spike 1 protein to block the binding of biotinylated spike protein. Figures (B and F) show inhibition of binding of biotinylated spike protein by neutralizing antibody from ACROBiosystems. Figures (C and G) show binding inhibition of biotinylated spike protein by neutralizing antibody from Novatein Bio. Figures (D and H) demonstrate inhibition of any detectable binding of biotinylated spike protein by antibody specific for the hepatocyte membrane ASGr1.

In a parallel study that surveyed the differentiation of E12 MLPC to AT2-like cells, it was demonstrated that AT2-like cells were positive for markers associated with AT2 cells (surfactant protein C, ACE2, TM4SF1, HT2-280), negative for markers associated with AT1 cells (AGER, caveolin 1 and aquaporin) and positive for markers not unique to AT2 cells but known to be expressed on AT2 cells (CK19, CD26 and EpCAM). These results were identical to primary small airway epithelial cells. Both cell types were also shown to bind spike and spike 1 proteins. Biotinylated spike proteins could be blocked by unlabeled spike protein (RBD). Pre-incubation with neutralizing antibodies prevented the binding of biotinylated spike protein by the ACROBiosystems neutralizing antibody, but not the Novatein antibody. Expressions of ACE-2, spike protein binding and inhibition were repeated for this study to confirm the involvement of the ACE-2 receptor (Figure 1).

The expressions of ACE-2, ASGr1 and albumin in control E12 MLPC, HLC and PHH were studied by antibody staining. E12 MLPC were shown to be negative for ACE-2, ASGr1 and albumin expression. In contrast, ASGr1 and albumin were shown to be strongly expressed by both HLC and PHH. ACE-2 was not detectible in either cell type (Figure 2).

The ability of E12 MLPC, HLC and PHH to bind spike and spike 1 proteins was studied using biotinylated spike proteins. E12 MLPC were unable to bind either spike or spike 1 proteins. HLC and PHH were able to bind spike protein but not spike 1 protein. The binding of biotinylated spike protein could be blocked by pre-incubation with unlabeled spike protein (Figure 3). The binding of biotinylated spike protein could not be blocked by spike 1, but could be blocked by ACROBiosystems and Novatein neutralizing antibodies and also an antibody directed against the ASGr1 (Figure 4).

It is critical to elucidate the mechanisms of virus/host interactions of the SARS-CoV-2 for the development of therapeutics designed to inhibit the binding and internalization of the virus to a myriad of cell types. The overarching strategy for the development of vaccines or therapeutics has involved the interaction between the S1 portion of the viral spike protein and the ACE-2 cellular receptor found in the respiratory tract and in various other tissues.47 Differences in transmission, pathology and organ involvement between SARS-CoV and SARS-CoV-2 (both dependent upon ACE-2 binding) suggested that additional receptors may contribute to the attachment and internalization of the SARS-CoV-2 virus14 in both the respiratory lungs and other organ systems.

The potential infection of tissues that are ACE-2 negative has spurred the search for additional receptor interactions of the spike protein. Some of these potential spike protein receptor targets include neuropilin-1,18 ASGR1 and KREMEN1.14 The observation of SARS-CoV-2 particles in hepatocytes8 and the robust expression of ASGr1 receptors and neuropilin-1 on hepatocytes suggested that altered liver function associated with COVID-19 infection may be directly caused by infection with the virus and mediated by binding to one or both receptors.

We investigated the potential virus: receptor interactions via the spike protein using fluorescent confocal microscopy and biotinylated spike (RBD) and spike 1 proteins. In a parallel study, E12 MLPC were differentiated to AT2-like cells (manuscript submitted). These cells expressed markers associated with AT2 cells (surfactant protein C, ACE2, TM4SF1, HT2-280), negative for markers associated with AT1 cells (AGER, caveolin 1 and aquaporin) and positive for markers not unique to AT2 cells but known to be expressed on AT2 cells (CK19, CD26 and EpCAM). These results were identical to primary small airway epithelial cells. The binding of biotinylated spike proteins and specific blocking by unlabeled protein and neutralizing antibodies confirmed that the primary interaction of spike protein with AT2-like cells and primary small airway epithelial cells was via the S1 portion of the protein with the ACE-2 receptor. We repeated those studies in support of our findings with the HLC and PHH. The differential inhibition of spike protein binding with two different antibodies suggested that viral neutralization could result from mechanisms other than direct inhibition of S1 (RBD) binding to ACE-2.

The characteristics of SARS-CoV-2 interactions with hepatocytes were studied by observing the binding of biotinylated spike (RBD) and spike 1 proteins to HLC and PHH using the undifferentiated E12 as a known negative control. It was observed that HLC and PHH were both negative for ACE-2, precluding that as a potential site of viral binding. This was confirmed by the inability of the cells to bind S1 protein. The binding of biotinylated spike protein and blockade by unlabeled spike protein on HLC and PHH suggested that the binding was specific, and via a mechanism distinct from ACE-2. The complete inhibition of spike binding by an antibody directed against the ASGr1 is strongly suggestive that ASGr1 is a binding site for the spike protein on hepatocytes. Interestingly, blockade of spike binding by both neutralizing antibodies on HLC and PHH was distinct from AT2 cells where inhibition occurred solely with the antibody that was directed against the RBD. This is suggestive of neutralizing activity that can occur outside the RBD.

Utilization of multiple cell types to study the interactions of spike protein binding will help identify additional receptor pathways for infection with COVID-19. They could also provide a powerful tool to aid in the development of therapeutics against multiple sites on the spike protein or receptors of the host cells. With the existent emergence of new variants and mutations of the SARS-CoV-2 exhibiting enhanced transmissibility, it is especially important to expand our repertoire of cellular models to investigate the effects of the mutations on the binding characteristics of the virus to host cells. The availability of immortalized cells with the stable characteristics of human alveolar type 2 cells and mature well-differentiated hepatocytes could provide an accurate and reproducible tool to effectively study the various virushost interactions via spike proteins by providing potential viral receptors that are segregated according to cell type. We believe that AT2 and HLC provide such a tool.

Dr Daniel P Collins reports personal fees from BioE, LLC, during the conduct of the study. In addition, Dr Daniel P Collins has a patent Composition for an in vitro culture medium to maintain and expand stem cell-derived hepatocyte-like cells pending, as well as,a patent Methods to develop immortalized hybrid hepatocyte-like cells, also pending. The authors report no other conflicts of interest in this work.

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2. Lan J, Ge J, Yu J, et al. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature. 2020;581(7807):215220. doi:10.1038/s41586-020-2180-5

3. Premkumar L, Segovia-Chumbez B, Jadi R, et al. The receptor-binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in SARS-CoV-2 patients. Sci Immunol. 2020;5(48):eabc8413. doi:10.1126/sciimmunol.abc8413

4. Hikmet F, Mar L, Edvinsson , et al. The protein expression profile of ACE2 in human tissues. Mol Sys Biol. 2020;16(7):e9610. doi:10.15252/msb.20209610

5. Sungnak W, Huang N, Bcavin C, et al. SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes. Nat Med. 2020;26(5):681687. doi:10.1038/s41591-020-0868-6

6. Hamming I, Timens W, Bulthuis MLC, Lely AT, Navis GJ, van Goor H. Tissue distribution of ACE2 protein, the functional receptor for SARS corona virus. A first step in understanding SARS pathogenesis. J Pathol. 2004;203(2):631637. doi:10.1002/path.1570

7. Wang D, Eraslan B, Weiland T, et al. A deep proteome and transcriptome abundance atlas of 29 healthy human tissues. Mol Syst Biol. 2019;15(2):e8503. doi:10.15252/msb.20188503

8. Wang Y, Liu S, Liu H, et al. SARS-CoV-2 infection of the liver directly contributes to hepatic impairment in patients with COVID-19. J Hepatol. 2020;73(4):807816. doi:10.1016/jhep.2020.05.002

9. Desai N, Neyaz A, Szabolcs A, et al. Temporal and spatial heterogeneity of host response to SARS-CoV pulmonary infection. Nat Comm. 2020;11(1):6319. doi:10.1038/s41467-020-20139-7

10. Mehta P, McAuley DF, Brown M, et al. COVID-19: consider cytokine storm syndromes and immunosuppression. Lancet. 2020;395(10229):10331034. doi:10.1016/S0140-6736(20)30628-0

11. Chu H, Chan JF, Wang Y, et al. Comparative replication and immune activation profiles of SARS-CoV-2 and SARS-CoV in human lungs: an ex vivo study with implications for the pathogenesis of COVID-19. Clin Infect Dis. 2020;71(6):14001409. doi:10.1093/cid/ciaa410

12. Chai X, Hu L, Zhang Y, et al. Specific ACE2 expression in cholangiocytes may cause liver damage after 2019-nCoV infection. bioRxiv. 2020. doi:10.1107/2020.02.03.931/766

13. Lozano-Sepulveda SA, Galan-Huerta K, Martnez-Acua N, Arellanos-Soto D, Rivas-Estilla AM. SARS-CoV-2 another kind of liver aggressor, how does it do that? Ann Hepatol. 2020;19(6):592596. doi:10.1016/j.aohep.2020.08.062

14. Gu Y, Cao J, Zhang X, et al. Interaction network of SARS-CoV-2 with host receptome through spike protein. bioRxiv. 2020. doi:10.1101/2020.09.09.28/508

15. Collins DP, Hapke JH, Aravalli RN, Steer CJ. Development of immortalized human hepatocyte-like hybrid cells by fusion of multi-lineage progenitor cells with primary hepatocytes. PLoS One. 2020;15(6):e234002. doi:10.1371/journal.pone.0234002

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[Full text] Binding of the SARS-CoV-2 Spike Protein | HMER - Dove Medical Press

NEW YORK, Dec. 03, 2020 (GLOBE NEWSWIRE) -- IN8bio, Inc., a clinical-stage biotechnology company focused on developing innovative allogeneic, autologous and genetically modified gamma-delta T cell therapies for the treatment of cancers (IN8bio or the Company), today announced an upcoming presentation that provides an update of the ongoing Phase I clinical trial of their product candidate INB-100 at the 62nd American Society of Hematology Annual Meeting & Exposition (ASH), which will take place virtually from December 5 to 8, 2020. INB-100 is designed for the treatment of patients with leukemia undergoing hematopoietic stem cell transplantation with haploidentical donors.

The poster and accompanying narrated slide presentation is titled, First-in-Human Phase I Trial of Adoptive Immunotherapy with Ex Vivo Expanded and Activated gamma delta T-Cells Following Haploidentical Bone Marrow Transplantation and Post-BMT Cyclophosphamide and reviews the study design and provides a brief update on enrollment and patient status.

The company reported that, as of abstract submission, three female subjects with acute leukemia had been enrolled in the INB-100 Phase 1 trial, of whom two had been dosed, and that no treatment-related adverse events had been recorded. The trial is continuing to enroll and treat patients. The abstract for the presentation can be found at https://ash.confex.com/ash/2020/webprogram/Paper142876.html.

The poster and slide presentation are jointly authored by the scientific and physician investigators from IN8bio and The University of Kansas Cancer Center (KU Cancer Center), and will be presented by the studys Principal Investigator, Dr. Joseph McGuirk, Schutte-Speas Professor of Hematology-Oncology, Division Director of Hematological Malignancies and Cellular Therapeutics and Medical Director, Blood and Marrow Transplant at KU Cancer Center.

This preliminary data report from KU Cancer Center with our allogeneic product candidate, INB-100, demonstrates the absence of significant GvHD in these initial patients, said William Ho, Chief Executive Officer of IN8bio. This suggests that gamma delta T-cells delivered as an off-the-shelf allogeneic cell therapy may be well tolerated and have significant potential to treat patients with serious and life-threatening cancers.

Dr. McGuirk, commented, Potentially curative stem cell transplants using partially matched donors -- called haploidentical transplants have greatly expanded access to stem cell transplantation. The infusion of donor-derived gamma delta T-cells from the stem cell donor, offers the hope of diminishing this risk of relapse and curing more patients.

About IN8bioIN8bio is a clinical-stage biotechnology company focused on developing novel therapies for the treatment of cancers, including solid tumors, by employing allogeneic, autologous and genetically modified gamma-delta T cells. IN8bios technology incorporates drug-resistant immunotherapy (DRI), which has been shown in preclinical studies to function in combination with therapeutic levels of chemotherapy. IN8bio is currently conducting two investigator-initiated Phase 1 clinical trials for its lead gamma-delta T cell product candidates: INB-200 for the treatment of newly diagnosed glioblastoma, which is a difficult to treat brain tumor that progresses rapidly, and INB-100 for the treatment of patients with acute leukemia undergoing hematopoietic stem cell transplantation. For more information about the Company and its programs, visit http://www.IN8bio.com.

Forward Looking StatementsCertain statements herein concerning the Companys future expectations, plans and prospects, including without limitation, the Companys current expectations regarding the curative potential of its product candidates, constitute forward-looking statements. The use of words such as may, might, will, should, expect, plan, anticipate, believe, estimate, project, intend, future, potential, or continue, the negative of these and other similar expressions are intended to identify such forward looking statements. Such statements, based as they are on the current expectations of management, inherently involve numerous risks and uncertainties, known and unknown, many of which are beyond the Companys control. Consequently, actual future results may differ materially from the anticipated results expressed in such statements. Specific risks which could cause actual results to differ materially from the Companys current expectations include: scientific, regulatory and technical developments; failure to demonstrate safety, tolerability and efficacy; final and quality controlled verification of data and the related analyses; expense and uncertainty of obtaining regulatory approval, including from the U.S. Food and Drug Administration; and the Companys reliance on third parties, including licensors and clinical research organizations. Do not place undue reliance on any forward-looking statements included herein, which speak only as of the date hereof and which the Company is under no obligation to update or revise as a result of any event, circumstances or otherwise, unless required by applicable law.

Contact:IN8bio, Inc.Kate Rochlin, Ph.D.+1 646.933.5605info@IN8bio.com

Investor Contact:Julia Balanova+ 1 646.378.2936jbalanova@soleburytrout.com

Media Contact:Ryo Imai / Robert Flamm, Ph.D.Burns McClellan, Inc.212-213-0006 ext. 315 / 364Rimai@burnsmc.com/rflamm@burnsmc.com

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IN8bio announces first-in-human Phase 1 trial Update from The University of Kansas Cancer Center using INB-100, IN8bio's Gamma Delta T-cell product...

NEW YORK, Aug. 24, 2020 (GLOBE NEWSWIRE) -- Incysus Therapeutics, Inc., a clinical-stage biopharmaceutical company focused on delivering innovative gamma-delta () T cell immunotherapies for the treatment of cancer, today announced that it has changed its name to IN8bio, Inc. (IN8bio or the Company). The Companys new name reflects its novel approach to cell therapy, focused on the development of gamma-delta T cells for anti-cancer therapies. These powerful immune cells possess properties of both innate and adaptive immune cells and can serve as a functional bridge between the two systems to impact tumor killing.

IN8bio was founded to develop novel immunotherapies to treat cancer. Our new name, IN8bio, reflects that focus, commented William Ho, President, Chief Executive Officer and co-founder of IN8bio. As we continue to treat patients in our ongoing clinical programs, we are focused on delivering the next generation of innovative cancer therapies.

IN8bio is using autologous, allogeneic and genetically modified gamma-delta T cells to address the high unmet need in both solid and liquid tumors. IN8bio entered the clinic in 2020 with two Phase 1 clinical trials which are currently enrolling patients. In February 2020, IN8bio initiated enrollment in a Phase 1 clinical trial of gamma-delta T cell immunotherapy in leukemia patients undergoing allogeneic stem cell transplantation. That trial, the first clinical trial of an expanded and activated allogeneic gamma-delta T cell immunotherapy, is being conducted with its partners at the University of Kansas Cancer Center. Additionally, in February 2020, IN8bio initiated enrollment in a Phase 1 clinical trial of patients with newly diagnosed glioblastoma, which is a difficult to treat brain tumor that progresses rapidly. This trial is being conducted at the ONeal Comprehensive Cancer Center at the University of Alabama at Birmingham. IN8bios proprietary Drug Resistant Immunotherapy (DRI), which is being used in the glioblastoma trial, is the first genetically engineered gamma-delta T cell therapy to be administered to patients.

About IN8bioIN8bio is focused on delivering novel immunotherapies for the treatment of cancer. By using allogeneic and genetically modified gamma-delta () T cells, IN8bios technology addresses certain challenges that immunotherapies face targeting cold, low mutation cancers. IN8bios immuno-oncology programs include activated and gene-modified adoptive cellular therapies that are designed to protect cells from chemotherapy and may allow novel combinations of drugs to disrupt the tumor microenvironment and increase immunogenicity. IN8bios first clinical program is targeted to address leukemia in patients who are undergoing hematopoietic stem cell transplant (HSCT) and its second program is targeted to the treatment of newly diagnosed glioblastoma in combination with chemotherapy. For more information about the Company and its programs, visit http://www.IN8bio.com.

Forward Looking StatementsCertain statements herein concerning the Companys future expectations, plans and prospects, including without limitation, the Companys current expectations regarding its business strategy, product candidates, and clinical development process and timing, constitute forward-looking statements. The use of words such as may, might, will, should, expect, plan, anticipate, believe, estimate, project, intend, future, potential, or continue, the negative of these and other similar expressions are intended to identify such forward looking statements. Such statements, based as they are on the current expectations of management, inherently involve numerous risks and uncertainties, known and unknown, many of which are beyond the Companys control. Consequently, actual future results may differ materially from the anticipated results expressed in such statements. In the case of forward-looking statements regarding investigational product candidates and continuing further development efforts, specific risks which could cause actual results to differ materially from the Companys current expectations include: scientific, regulatory and technical developments; failure to demonstrate safety, tolerability and efficacy; final and quality controlled verification of data and the related analyses; expense and uncertainty of obtaining regulatory approval, including from the U.S. Food and Drug Administration; and the Companys reliance on third parties, including licensors and clinical research organizations. Do not place undue reliance on any forward-looking statements included herein, which speak only as of the date hereof and which the Company is under no obligation to update or revise as a result of any event, circumstances or otherwise, unless required by applicable law.

Contact:IN8bio, Inc.William Ho, President & CEOwho@IN8bio.com+1 646.600.6GDTinfo@IN8bio.comwww.IN8bio.com

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Incysus Therapeutics Announces Name Change to IN8bio, Inc. - GlobeNewswire

On August 23, 2019, the FamilieSCN2A Foundation held their biennial SCN2A Professional and Family meeting, in Seattle, Washington. The gathering brought together 37 families of individuals with mutations in the SCN2A gene, 60 investigators, eight clinicians and five industry groups that conduct research and/or clinical work on conditions related to this genetic change. A number of SFARI scientists and staff also attended the event.

The SCN2A family meeting was one of many events that family organizations of rare, neurodevelopmental disorders organized last summer. These meetings help families connect with others similarly affected as well as professionals working to better understand these conditions and develop new therapeutics. SFARI often attends and facilitates research opportunities carried on at these events.

SCN2A is a high-confidence autism risk gene, which encodes a subunit of a sodium channel in the brain called Nav1.2. When the channel malfunctions, conditions like epilepsy and autism follow. As part of its mission to understand the genetics and neurobiological underpinnings of autism, SFARI has awarded about $3 million for research on SCN2A, and some of this research was presented at the meeting. SFARI also supports a genetics first initiative called Simons Searchlight (formerly known as Simons VIP), which enrolls people with a genetic diagnosis showing rare genetic changes associated with autism and related neurodevelopmental conditions, such as SCN2A.

Many stories that may reflect the different ways SCN2A can be disabled were told at the meeting. One child had his first seizure when he was days old, and now spends many of his days irritable and immobilized by dystonia. Another developed normally until his first seizure as a toddler, which seemed to wipe out all of his skills; his milestones are now hard won in the face of continuing seizures and an autism diagnosis. Another had a sudden regression at 1 year of age, and after a misdiagnosis and seizure medication, she goes to a school for children with autism. Still another suffered from relentless seizures, which robbed her of speech; she died last year at the age of 12.

So far, about 300 different variants of the SCN2A gene have beendocumented, and the functional consequences of many are unclear. Some researchers have developed high-throughput experiments to systematically test each of thesevariants, and to screen compounds that could normalize their function2. Another approach may use genetherapy to boostexpression of the remaining good copy of SCN2A. Either way, finding appropriate in vitro testing grounds for these SCN2A variants is essential and may help personalize treatment approaches or identify more homogeneous patient groups for drug trials.

The meeting also underscored the power of family gatherings to push the science ahead. Investigators could see multiple examples of a rare genetic condition and engage new participants in research studies such as The Investigation of Genetic Exome Research (TIGER), a project of the University of Washington that compares phenotypes of single-gene conditions. In turn, families had the opportunity to express their concerns to scientists and infuse the research proceedings with urgency.

My biggest takeaway from this years conference was the mutual inspiration between the scientists and the families, says Leah Schust, meeting organizer and executive director of the FamilieSCN2A Foundation. Her son has a mutation in SCN2A.

Meeting the researchers working on a cure for our kids motivates us to fight on, Schust says. Then the scientists all say that meeting the families inspires them to go back to their labs and work even harder.

Family focus. The family meeting helped researchers reconsider what would be meaningful clinical endpoints for potential treatments. Schust says that most researchers and industry groups had thought seizure control was the most important issue. After listening to us, they realized that quality of life, movement disorders and autonomic dysfunction are higher on our list of where we would like to see improvement, she says.

When SCN2A mutations were first linked to autism, the gene stood out because it encodes a relatively well-understood protein, unlike many of the other identified genes. Nav1.2 is a voltage-gated channel found exclusively on excitatory neurons in the brain, where it controls the flow of sodium ions into the neuron, and thus its propensity for firing action potential. Experiments have revealed detailed pictures of Nav1.2s structure3, and known drugs alter its function4.

SCN2A also stands out because of its high recurrence rate in autism: unlike other autism genes, SCN2A is mutated with somewhat regular frequency5 (Figure 1).

Just as understanding why a car wont start is critical to fixing it, researchers need to understand how these SCN2A mutations alter the Nav1.2 channel. A current model1 posits that some mutations are gain-of-function, rendering the channel too active and the brain hyperexcitable, leading to infantile epilepsy; conversely, loss-of-function mutations reduce excitability and seem associated with autism and/or intellectual disability, as well as childhood-onset (as opposed to neonatal) seizures.

Yet the functional consequences of most SCN2A mutations remain unknown, and some may not fall neatly into a loss-of-function or gain-of-function category. A way of making sense of these mutations may come from looking at the working parts of Nav1.2, said Arthur Campbell of the Broad Institute of MIT and Harvard. For example, missense SCN2A variants linked to epilepsy seem to hit the channel randomly. But when marking their location on a crystal structure model of the channel, the missense variants cluster in several places: on the voltage sensor, on the linker helix responsible for conveying voltage sensor movement to the channel pore, on an area thought to interact with the beta-subunits involved in chaperoning the channel to the right place, and on the inactivation gate, which closes the pore off from sodium ion flow. He suggested that this knowledge, combined with the structural similarities between all sodium channels, may help drug development for SCN2A-related conditions.

High-throughput systems that can assay hundreds of cells at a time are helping researchers systematically explore SCN2A mutation, explained SFARI Investigator Al George of Northwestern University. While conventional electrophysiology would require weeks of work to characterize a single SCN2A variant, Georges group uses an automated patch-clamp system that can characterize multiple variants transfected into non-neuronal cell lines in a day. Using this system, two variants associated with neonatal seizures both exhibited an exceptional willingness to activate and a slowness to inactivate, which are properties consistent with a gain-of-function interpretation.

The high-throughput set up also promises to expedite the hunt for drugs to normalize SCN2A function: George described a 384-well plate design that allows measurement of the effects of two different drugs, at four different concentrations, on the SCN2A variant and control channels simultaneously. A known drug (carbamazepine) and an experimental drug (PRX-330) shifted channel inactivation to more hyperpolarized voltages, which could help quiet channels with gain-of-function mutations.

To narrow in on potentially therapeutic compounds, Jeff Cottrell and colleagues at the Broad Institute of MIT and Harvard have come up with a two-stage screen to find small molecule activators or inhibitors of Nav1.2 channels. First, compounds are initially tested on non-neural cells transfected with Nav1.2 sodium channels and potassium channels, which enables them to spike. The cells in 384-well plates are stimulated in parallel, and voltage-sensitive dyes give a readout of spiking activity; remarkably, Cottrells system allows data collection from up to 96 wells simultaneously. Any compounds that modulate spiking would then be subjected to the second stage, in a high-throughput electrophysiology assay similar to that described by George. Compounds with helpful mechanisms would then be tested for selectivity for Nav1.2 versus other sodium channels. A selective compound would then be tested in neurons, first in vitro then in vivo. This step-wise process has identified an activating compound that makes Nav1.2 more likely to open at rest and has potent effects on action potentials in brain slices and on electroencephalogram (EEG) traces from mice engineered to carry a disabled copy of SCN2A; however, Cottrell said this particular compound is not a therapeutic candidate in part because it broadens the action potential in a way that could promote seizures. A full screen is underway, and so far has identified 378 modulators from a library of 77,000 compounds.

Beyond academia, J.P. Johnson Jr. of Xenon in Burnaby, British Columbia, discussed the companys work to create sodium channel inhibitors for treating epilepsy. To obtain selective compounds, the group targets the voltage-sensing domain because its structure is the most diverse region of sodium channels. Xenon uses a trial-and-error method to optimize sodium channel inhibitor potency and selectivity. The methodical process has yielded some interesting compounds, including both selective Nav1.6 inhibitors and dual Nav1.6 and Nav1.2 inhibitors. Both quashed spiking in mouse excitatory pyramidal neurons, which contain only Nav1.2 and Nav1.6, but they did not alter spiking in Nav1.1-containing inhibitory neurons. A Nav1.6 selective inhibitor, XEN901, is currently undergoing safety trials in humans.

Kathrin Meyer of Nationwide Childrens Hospital in Columbus, Ohio, addressed the possibility of using gene therapy to normalize malfunctioning Nav1.2 channels. Meyer has been involved in several gene-therapy trials for neuromuscular disorders, including a successful one for infant-onset spinal muscular atrophy type6. Gene therapy for brain diseases was spurred by the discovery of adeno-associated virus 9 (AAV9), which can cross the bloodbrain barrier to deliver genetic material to the central nervous system. AAV9 is small, cannot replicate, does not integrate into host DNA and seems not to cause disease in humans. In considering gene therapy for SCN2A-related conditions, Meyer emphasized an approach that adds back a working copy of the gene, thus sidestepping the need for gene editing to make mutation-specific corrections. Such a treatment would only apply to those with loss-of-function mutations.

The large size of the SCN2A gene precludes its delivery by AAV9, however. As a workaround, Meyer suggested that SCN2As mRNA transcript could be targeted in an attempt to replace only the affected area of the mRNA. So far, such strategies have not been very efficient, but there are new ideas that might address some of the difficulties. Because access to tissue samples of patients with neurological disorders is limited, the development and testing of new therapies is complicated. Meyer suggested developing gene therapies in vitro using neurons reprogrammed from skin cells of patients. This might help identify which patients would react best to a certain treatment. There is likely not a one-fit-for-all situation, she said.

SFARI deputy scientific director John Spiro underscored the need for in vitro systems, citing the organizations initiative to bank blood cells to systematically generate induced pluripotent stem cells from individuals with autism. Simons Searchlight is also a resource of many different biospecimens for researchers. So far, 186 families with SCN2A-related changes have registered, and 83 of these have completed consent, lab reports and medical histories with a large number of blood samples as well. (On the sidelines of the meeting, 18 parents, 11 of their children with SCN2A mutations, and three unaffected siblings donated blood toward this initiative.) Spiro also stressed a need to come up with more quantitative methods of phenotyping, such as wearable electronics that can monitor sleep and circadian rhythms. Data that can be collected longitudinally and at home might provide sensitive outcome measures for clinical trials.

A new role for Nav1.2 has been revealed in recent work described by SFARI Investigator Kevin Bender of the University of California, San Francisco: the channels mediate back-propagating action potentials, which travel into the dendritic trees of neurons. Mice engineered to lack one copy of SCN2A a situation that mimics people with truncating SCN2A mutations that render the resulting Nav1.2 channels useless had cortical neurons with slower action potentials, reduced dendritic excitability and immature synapses based on their shape and function7. This role for Nav1.2 was particularly important later in development: when conditional knockout mice lost an SCN2A copy later in life, their cortical neurons exhibited immature synapses, though their density remained normal. Preliminary experiments suggest that adding back a working copy of SCN2A later in life through transgenic methods or by upregulating transcription of the remaining good copy of SCN2A via CRISPR techniques can restore action potential velocity and synaptic maturity.

Bender stressed how interacting with the SCN2A family group helped focus his research on important aspects of their childrens conditions. For example, parents have noted sensory hypersensitivity in their children, leading Bender to collaborate with colleague Evan Feinberg to use an eye-tracking assay in mice to measure their visual responses. He noted that SCN2A haploinsufficient mice were more sensitive to certain visual stimuli than control mice; if the assay is robust, it could help bridge the gap between SCN2A-related phenotypes in humans and behaviors measured in mice.

As meeting attendees sorted through the new findings, therapeutic questions lingered. An important issue for any therapy, whether drug or gene, will be how early in development one will have to intervene to help someone with an SCN2A mutation. Bender noted that synaptic properties could be rescued in his mice when they were 30 days old equivalent to a 10-year-old human but these and other experiments will have to probe the time periods during which therapies will be maximally effective. To find good measures of efficacy also means understanding the full complement of conditions that beset people with SCN2A mutations. For example, though seizures afflict many, Keith Coffman of Childrens Mercy Hospital in Kansas City, Missouri, suggested that, in some cases, these represent a movement disorder rather than epilepsy. Basic descriptive knowledge like this is imperative for guiding future treatment approaches.

Another smaller SCN2A meeting is planned for this year from July 30 to August 2, in Columbus, Ohio. This will be more family focused, says Schust, and there will be opportunities to participate in research.

There is clearly a lot more work to do before all the terrific basic research that was discussed at this meeting produces meaningful results for families, but it is extremely gratifying to see how much progress has been made on so many fronts and how many new good ideas are emerging, Spiro says. And its terrific to witness firsthand the positive cycle of how families drive researchers and vice versa.

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Seeing through a forest of SCN2A gene variation - SFARI News

Brynn Niblock, FHSU junior in pre-med from Hoxie, swabs her cheek as part of the sign up for the Be the Match bone marrow registry Feb. 6 at Gross Memorial Coliseum.

By CRISTINA JANNEYHays Post

Usually Tiger basketball games are a time to have fun cheer the home team, eat some popcorn but students and community members at Feb. 6 game took a few minutes to stop and potentially save a life.

FHSU student health sponsored a Be the Match bone marrow registry drive.

Potential donors ages 18 to 44 answered a list of qualifying health questions on their smartphones and then swabbed their cheeks to be matched with a potential cancer sufferer in need of bone marrow transplants.

Kathy Pyke of Hays knows too the well the importance of the registry. Pyke was at Gross Memorial Coliseum the night of the drive as a volunteer handing out information to potential donors.

Her husband, Tom, was diagnosed with leukemia on March 1, 2014. Family members were tested, and they were not matches. Doctors were also unable to find a bone marrow match on the national registry. There were 6.2 million people in the registry at the time.

In lieu of a bone marrow transplant, Pyke was given donated umbilical cord blood.

Initially the treatment improved Pyke's condition. However, he ultimately died as a result of the disease on Feb. 12, 2015 at the age of 62.

Kathy said the family was rocked by Tom's illness. He was playing golf and went fishing the week before he was diagnosed with cancer.

Kathy said she wishes she could be on the registry to help another family, but her age prevents her from doing so.

"Not only for my husband," she said of the importance of the registry. "I did pray there had been a match. We stayed at the Hope Lodge that was run by the American Cancer Society in Kansas City. There were 45 apartments there and everyone there has someone who has cancer plus a caregiver in it. You just see so many lives being touched. ...

"If this is something that can help somebody, it is just an easy thing to do."

Kathy said she had a good friend who had a family member sign up for the registry, and he was able to donate to someone who had cancer in England.

Pyke said she would also like to see more hospitals participate in the cord blood bank, which is what helped her husband. At the time of Tom's illness, HaysMed was not participating in the umbilical cord blood bank.

Michelle Toogood, BSN, RN, supervisor of Hays Meds Women's andInfant Care Center/NICU, said parents wishing to participate in cord blood donation should initiate the process prior to delivery. HaysMed staff will then aid in the collection of the specimen.

"I just can't express how much people need to do this," Pyke said of signing up for the registry. "It is just so easy to swab test and they could potentially save more than one person's life. It is so easy to do and so important."

If you are identified as a match to someone suffering from cancer, you would be contacted through the registry and asked if you are willing to donate,Amanda McCord, RN at the FHSU student health center.

"Finding the perfect match is essential for people who are fighting this type of cancer," McCord said. "The closer the match the better their chances of remission and beating whatever cancer they are fighting."

There are over 70 diseases that can be treated by bone marrow transplants, according to Be the Match.

Physicians will usually look for matches among relatives first, but only 70 percent of the time are matches made from family members, McCord said.

Statistics also indicate minority patients are less likely to find matches than Caucasian patients. Be the Match is trying to boost minority participation as there are fewer minority participants in the registry at this time, McCord said.

Donating bone marrow is a little bit different for every donor, McCord said.

Most give through a Peripheral Blood Stem Cell (PBSC) donation. A machine draws blood from one arm, extracts the cells it needs, and returns the remaining blood through your other arm, according to the Be the Match website.

Others give through a marrow donation. Liquid marrow is withdrawn from the back of your pelvic bone with a needle. In this case, youll receive anesthesia and feel no pain during the procedure, the Be the Match website said.

According to Be the Match,PBSC donors may experience headaches or body aches several days before collection, but these disappear shortly after donation. Most donors feel completely recovered within a few weeks.

If you missed the Be the Match event at FHSU last week, you can contact Be the Match though its website, and the organization will send you the cheek swab kit to sign up for the registry.

The Be the Match website also has information on the donation process and a link to make monetary donations to the Be the Match program.

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FHSU partners with Be the Match for bone marrow registry event - hays Post

WICHITA, Kan. (KAKE) -

There's a major medical break-through in the world of cancer treatment. No more chemotherapy or radiation; doctors in Wichita are using patients' own blood cells to fight off cancer, and it's working.

62-year-old David Butler's appointments at the Cancer Center of Kansas are coming to an end. It's been two long years for him - in 2017, stomach pain led doctors to discover 13 tumors inside his abdomen. Butler went through months of chemotherapy, then stem cell therapy. But the cancer kept coming back.

The news could have been grim. But not for Butler. The Cancer Center of Kansas is one of just nine facilities in the nation chosen to participate in a study using the patient's own cells to fight off the disease.

It's called Car T-cell Therapy. A patient's own immune cells are harvested, then genetically modified and inserted back into the body. Those t-cells then search out and kill the cancer. And the Car T-cell Therapy can be given as outpatient treatment, with no hospital stay required.

David Butler is now cancer-free, and his doctors are hopeful the t-cells will continue to stave off the disease.

The Cancer Center of Kansas has treated two patients, and so far so good. Once the study is finished, doctors and the drug company will go to the FDA for full approval to use the therapy to treat cancer across the country.

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Trial cancer treatment in Wichita - KAKE

SAN FRANCISCO (ChurchMilitant.com) - Bombshell testimony from the trial of Center for Medical Progress undercover journalists David Daleiden and Sandra Merritt has revealed that infanticide is commonplace insideU.S. abortion mills.

Earlier this month, attorneys for Daleiden submitted a closing argument brief detailing that live births are occurring inside the facilitiesand that these newborns are routinely killed their organs harvested while still alive.

Describing Daleiden's research into the practice, the brief recounts that hediscovered "a mainstream media expos produced and aired in 2000 by Chris Wallace for the program '20/20.'"

From Wallace's report, Daleiden learned of Dean Alberty, who worked as a fetal tissue procurement technician inside a Planned Parenthood facilityin suburban Kansas City.

According to the brief:

From the "20/20" video he learned that Alberty had been handed whole fetuses from Planned Parenthood doctors and had harvested beating hearts. Alberty had also testified before Congress and described a live birth of twins who were actually cuddling each other. He would not harvest from them and the abortion doctor drowned them in a pan of water.

The practice was not isolated to Kansas City, Daleiden discovered.

In the course of his research, he came across the bookBeyond Abortion: A Chronicle of Fetal Experimentation, which documents experiments performed on unborn babies, as well as the removal of organs from infant abortion survivors.

In Beyond Abortion, Daleiden found an article titled "Artificial Placenta," which described "obtaining live fetuses as old as 24 weeks from unnamed abortionists and keeping them alive in a machine for study but letting them drown in the machine after obtaining data."

In the summer of 2011, he learned of a company named StemExpress, which specializes in providing "biospecimens" to researchers across the country; adeeper look at the firm revealed the scaleof tissue harvestingoccurring inside American abortion mills.

Daleiden discovered that StemExpress required "tissue procurers to service 48 universities and 8 private entities with fetal organs and tissues."

He later uncovered"a StemExpress order form with a list of organs and tissues for sale, including whole hearts, hearts with veins and arteries attached, as well as brains, livers and other organs."

He then found"aStanford study using whole human fetal hearts obtained from StemExpress which they put on a Langendorff perfusion machine."

According to the brief:

Mr. Daleiden learned through his own research and by consulting experts, including Dr. Theresa Deisher, that in order to use the Langendorff machine the heart had to either still be beating when it was placed on the machine or a beating heart had to be arrested in a relaxed state in a potassium solution and then quickly transported to the machine. ... Dr. Deisher testified that she told Mr. Daleiden that the "most horrifying aspect of the use of the remains of aborted fetuses was that some of the babies had to be alive, have beating hearts when they were harvested."

Deisher, a stem cell research scientist,testified that based on her experience with stem cell research on hearts, this was a frequent occurrence. Thebabies' hearts haveto be harvested while still beating, she explained, as otherwise the organ would have no research value because once in "contracture," the heart's cells would no longer be capable of regenerative growth.

The brief detailedadditional evidence of infanticide including testimony by a former StemExpress employee:

Mr. Daleiden continued to gather evidence for his investigation. He met Holly O'Donnell who had worked for StemExpress and told him she left after seeing a late gestated fetus. She was directed to dissect its brain. She did so. She also told him that her superior Jessica tapped the fetuses'heart and it started beating. She also told him of seeing a message stating that an intact fetus was being sent to the StemExpress facility.

Daleiden later learned that "StemExpresstechnicians had to work very closely with the abortion doctors at [Planned Parenthood] MarMonte who increased dilation on thepatients in order to obtain intact fetuses with beating hearts."

California Attorney General Xavier Becerra a self-identified Catholic is prosecuting Daleiden and Merritt for their undercover work. In his preliminary hearing closing argument, he made no effort to rebut testimony about the harvesting of infant abortion survivors' organs.

Instead, Becerra suggested that harvesting organs from newly born infants is protected by the state's abortion statutes.

The "defendants willfully misrepresent the law on homicide in California," he argued. "California law is clear that therapeutic abortion is not homicide."

Pro-life advocates counter that there is nothing "therapeutic" about harvesting beating hearts from live infants.

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Infanticide: Live Organ Harvesting Commonplace in US Abortion Mills - Church Militant

1How much is the seminar?

This is a complimentary seminar. No cost and no obligation. The doctor will be reviewing the latest research on how stem cells are able to help revive and rejuvenate worn, damaged joints and tissues (such as knees, shoulders and hips). Please contact us if you have any questions!

2How much downtime should I expect after regenerative cell therapy?

Typically, there is no little to no downtime from regenerative cellular therapy.

3Is more than one treatment needed?

Not every person is the same. Obviously that possibility exists and thats why we take our patients through our advanced assessment so we can carefully determine if you are one of those special cases. It is important to understand that once a joint regenerates, there would be no need for further treatment unless a new injury occurred or over-time,that joint degenerated again.

4How much does it cost?

Every person is unique and would require a one on one consultation with a provider to see if you are a candidate. The good news is, we've made it a goal to never let price get in the way of your health. If together we find regenerative therapy is right for you, and will improve your quality of life, there are several flexible payment options available.

5What determines the outcome of my regenerative cell therapy?

Various factors will determine the outcome of your Regenerative Cell Therapy treatment, such as the extent of damage, disease and the location being treated. Most people respond well to this therapy option and experience relief from pain in just a short period of time. For instance, this therapy has been known provide full relief to patients after only one treatment.

6How are regenerative cells collected?

Our Regenerative Cell Treatment is a revolutionary breakthrough treatment option for people suffering from inflammation, reduced mobility, sports injuries, tissue and ligament damage, or chronic pain. Regenerative Cell Therapy is an injectable regenerative tissue matrix solution, that oftentimes leaves the patient feeling relief after only ONE treatment. This cutting edge treatment takes the best components from all the current non-invasive treatment options and puts them into one. This Regenerative Cell Treatment is collected from mothers who have donated their placental tissue after delivering a child by c-section birth.

7Is there anything else I can do to increase the effectiveness of my therapy?

Yes! Our treatment plans are comprehensive. Not only will we provide you with the most cutting-edge treatment options, but we will also assist you through rehabilitation. Following the custom program created for your specific needs will thoroughly increase the effectiveness.

8How long does the repair process take?

Generally, the repair process begins immediately and the good news is that it can continue to repair for up to eight additional months from the date of the initial procedure.

9Can the procedure fail?

Like any other procedure, there is no 100% guarantee. In certain cases, it is possible that you may need additional treatments or your stem cells do not have enough repair potential relative to your personal injury.

10If the regenerative cell therapy does not work can I still have surgery?

Yes. There is nothing about these procedures that would preclude you from having traditional surgery. We evaluate each case carefully, however, so if its possible to tell that the best course of action is truly conventional surgery we will advise you on that.

11Will insurance cover it

Unfortunately insurance and Medicare do not cover stem cell therapy (yet). We have made this treatment option extremely affordable because of this.

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Kansas Regenerative Stem Cell Seminar - Stem Cell Centers ...

key benefits ofstem cell treatment

NOBEL PRIZE IN PHYSIOLOGY & MEDICINE

The Nobel Prize in physiology and medicine was awarded to Drs. Gurdon and Yamanaka in 2012 for achieving the regress mature cells of living human beings into these pluripotent cells. These cells are known as induced pluripotent stem cells (iPSC) and are in their infancy as part of the landscape of orthopedic biologics.

Combining Treatment Elements

In addition to the mesenchymal and hematopoietic stem cells, the concentrate includes similar growth factors found in PRP as well as substances known as cytokines, which recruit more healing cells to the site. The concentrate is injected into the affected site under ultrasound-guidance to ensure accurate placement.

Common Uses

Bone marrow and fat aspirate are commonly used in degenerative conditions such as osteoarthritis and cartilage defects. A retrospective study performed in 2014 by Centeno et al found significant improvements in pain and function at all follow-up intervals after treatment of 840 knees with osteoarthritis using bone marrow aspirate with fat grafting.

Same Day In-Office Procedure

Eliminating the risks or costs associated with anesthesia. Procedures are conveniently performed in a comfortable office setting. PRP procedures take less time in this setting.

Less Cost & Risk

Total knee replacement surgery costs between $56,000 and $58,000. Not including pre-operative costs for office visits,xrays, laboratory work, or the post-surgical costs for physical therapy which can average $2,500 to $4,500. This treatment is conservative, minimally invasive, able to be performed in patients with comorbid conditions.

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Stem Cell Procedures | Motus Biologics of Kansas City

Stem Cell therapy is now offered at Rejuvenate Mind-Body Wellness Center. We have an MD and a Nurse Practitioner that will be doing the treatments usingumbilical cord blood stem cells, from healthy live births.

Click here to register for our next Stem Cell Presentation

Stem Cell and Regenerative Medicine is an exciting advancement in medicine! Mesenchymal Stem Cells (MSCs) occur naturally in every person and are cells the body uses naturally to heal and repair. Injured tissues in the body give off signals, and MSCs hone in on those areas, and begin the process of repairing and regenerating the damaged and injured tissues. As we age, we have less and less MSCs in our body. This is why when we are younger, we can do certain activities and recover. However, when we are older, it takes much longer to recover (if at all!).

There lots of MSCs in our younger years. MSCs repair muscle, bone, cartilage, and tendons. Unfortunately, MSCs rapidly decline with age. This results in longer repair and recovery times, and individuals are more prone to aging and disease. That is whyour clinic uses umbilical cord stem cells in our treatment. We not only get a higher number of stem cells with each injection, but we also know the exact dosage of stem cells within injection (compared with bone marrow stem cells, adipose [fat] stem cells, and amniotic stem cells).

We use stem cells that are taken from umbilical cords. Some families choose to bank their childs umbilical blood in case they need it in the future. Other families choose to donate their childs umbilical blood our stem cells come from the donated umbilical cords of live, healthy, and full-term births. Donations are screened for communicable diseases just like a blood donation.

The stem cells are undifferentiated and immunonaive. This means the cells will not be seen as foreign by your body. Stem cells from one cord can go into several patients without the need for typing. These stem cells will not induce a reaction from your body, and not cause side effects. This also means the cells are free to become the specific type of cell that your body most needs to repair itself.

You might be wondering why you should choose umbilical cord stem cell therapy over treatment with your own bone marrow-derived stem cells. Harvesting stem cells from your bone marrow is both an uncomfortable and expensive procedure, and bone marrow stem cells mainly become blood cells. Umbilical cord (Mesenchymal) stem cells are predisposed to turn into bone cells (osteophytes), cartilage cells (chondrocytes) and muscle cells (myocytes).

Another common stem cell therapy is derived from your own adipose (fat) cells by having liposuction performed. Similar to bone marrow-derived stem cells, the cost, invasiveness, and discomfort of the procedure are all higher with adipose-derived stem cells. Umbilical cord stem cells dont have the invasiveness, as high of a cost, or the amount of post-procedure discomfort compared to adipose-derived stem cells.

Stem cells are smart cells they know where to go! Stem cells are attracted to the inflammatory signals your body is exerting. When injected, the stem cells will seek out the source of the inflammatory signal and adhere to the site. For this reason, you will be asked to avoid taking anti-inflammatory medications (Ibuprofen, Advil, Aleve, Naproxen, etc) for one week prior to your injection and to avoid taking these medications for 2-3 weeks after your injection. We dont want to quiet down the signal the cells are looking for! Acetaminophen based medications like Tylenol or Hydrocodone are fine to take.

Stem cells are live cells! And they are most plentiful in umbilical cords. On average 1 cc of stem cells from an umbilical cord will yield 10,000,000 (10 million) active stem cells, compared to ~250,000 active stem cells taken from a 30-year-old patient. Not only that, but the cells will begin multiplying once theyve been injected into your body.

Umbilical stem cells replicate every 28 hours. If you receive 1 cc, you will be injected with 10,000,000 (10 million) stem cells that will double in number every 28 hours. They will do this for up to 8 weeks. While the stem cells are replicating they are recruiting your own bodys stem cells to regenerate and heal as well.

It is not uncommon for patients to see noticeable improvement at their first follow up, 4-6 weeks after the injection. However, the entire regeneration process can take up to 6-8 months. It is at this point that patients are able to fully appreciate their improvement.

There are no drug interactions or side effects with stem cells. Other than avoiding anti-inflammatory medications before and after your injection you may continue your normal routine. The only exception to that is with exercise. You will be asked to avoid strenuous exercise and repetitive motions for up to a month following your injection (depending on site). At 4 weeks you may resume all normal activity.

The large majority of cases who are good candidates for stem cell therapy, only need one (1) injection for their condition. Only a small minority of patients need a 2nd injection 6-8 months later. Each patients condition is unique, and you will discuss any future possible injections with your provider at your follow up appointments.

Click here to register for our next Kansas City Stem Cell Presentation.

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Stem Cell Therapy in Kansas City- Rejuvenate KC | Stem ...