scholarly journals Selection for CD26- and CD49A+ cells from pluripotent stem cells-derived islet-like clusters improves therapeutic activity in diabetic mice

2021 ◽  
Author(s):  
Kfir Molakandov ◽  
Denise A. Berti ◽  
Avital Beck ◽  
Ofer Elhanani ◽  
Michael D. Walker ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Therapy ◽  
Pluripotent Stem Cells ◽  
Islet Cells ◽  
Diabetic Mice ◽  
Cell Capture ◽  
Physiological Control ◽  
C Peptide ◽  
Selection For

Abstract Background. Cell therapy of diabetes aims at restoring the physiological control of blood glucose by transplantation of functional pancreatic islet cells. A potentially unlimited source of cells for such transplantations would be islet cells derived from an in vitro differentiation of human pluripotent stem cells (hESC/hiPSC). The islet-like clusters (ILC) produced by the known differentiation protocols contain various cell populations. Among these, the β-cells that express both insulin and the transcription factor Nkx6.1 seem to be the most efficient to restore normoglycemia in diabetes animal models. Our aim was to find markers allowing selection of these efficient cells. Methods. Functional Cell-Capture Screening (FCCS) was used to identify markers that preferentially capture the cells expressing both insulin and Nkx6.1, from hESC-derived ILC cells. In order to test whether selection for such markers could improve cell therapy in diabetic mouse models, we used ILC produced from a clinical-grade line of hESC by a refined differentiation protocol adapted to up-scalable bioreactors. Re-aggregated MACS sorted cells were encapsulated in microspheres made of alginate modified to reduce foreign body reaction. Implantation was done intraperitoneally in STZ-treated C57BL/6 immuno-competent mice. Results. CD49A (integrin alpha1) was identified by FCCS as a marker for cells that express insulin (or C-peptide) as well as Nkx6.1 in ILC derived by hESC differentiation. The ILC fraction enriched in CD49A+ cells rapidly reduced glycemia when implanted in diabetic mice, whereas mice receiving the CD49A depleted population remained highly diabetic. CD49A-enriched ILC cells also produced higher levels of human C-peptide in the blood of transplanted mice. However, the difference between CD49A-enriched and total ILC cells remained small. Another marker, CD26 (DPP4), was identified by FCCS as binding insulin-expressing cells which are Nkx6.1 negative. Depletion of CD26+ cells followed by enrichment for CD49A+ cells increased insulin+/Nkx6.1+ cells fraction to ~70%. The CD26-/CD49A+ enriched ILC exhibited improved function over non-sorted ILC or CD49A+ cells in diabetic mice and maintain prolonged blood C-peptide levels.Conclusions. Refining the composition of ILC differentiated from hPSC by negative selection to remove cells expressing CD26 and positive selection for CD49A expressing cells could enable more effective cell therapy of diabetes.

scholarly journals Selection for CD49A+ and CD26- cells in pancreatic islet-like clusters differentiated from human pluripotent stem cells improves their therapeutic activity in diabetic mice

2020 ◽  
Author(s):  
Kfir Molakandov ◽  
Denise A. Berti ◽  
Avital Beck ◽  
Ofer Elhanani ◽  
Michael D. Walker ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Therapy ◽  
Pancreatic Islet ◽  
Pluripotent Stem Cells ◽  
Islet Cells ◽  
Single Cells ◽  
Human Pluripotent Stem Cells ◽  
Diabetic Mice ◽  
C Peptide ◽  
Selection For

Abstract BackgroundCell therapy of diabetes aims at restoring the physiological control of blood glucose by transplantation of functional pancreatic islet cells. Human islets from post-mortem donations have shown efficiency but the demand for islets vastly exceeds the availability of donations. A potentially unlimited source of cells for such transplantations would be islet cells derived from in vitro differentiation of human pluripotent stem cells (hPSC), such as embryonic stem cells (hESC). The islet-like clusters (ILC) produced by the known differentiation protocols contain various cell populations. Among these, the beta cells that express both insulin and the transcription factor Nkx6.1 seem to be the most efficient to restore normoglycemia in diabetes animal models. Our aim was to find markers allowing selection of these efficient cells.MethodsFunctional Cell-Capture Screening (FCCS), using an array of antibodies to cell surface proteins, was used to identify markers that preferentially capture the cells expressing insulin, or expressing both insulin and Nkx6.1, from hESC-derived ILC cells. In order to test whether selection for such markers could improve cell therapy in diabetic mouse models, we used ILC produced from a clinical-grade line of hESC by a refined differentiation protocol adapted to up-scalable bioreactors. The ILC, dissociated to single cells, were fractionated by Magnetic Activated Cell Sorting (MACS) for presence of the marker. The sorted cells, re-aggregated into clusters, were encapsulated in microspheres made of alginate modified to reduce foreign body reaction. Implantation was done intraperitoneally in C57BL/6 immuno-competent mice that were made diabetic by prior injections of Streptozotocin (STZ).ResultsCD49A (integrin alpha1) was identified by FCCS as a marker for cells double positive (DP) for insulin (and C-peptide) as well as Nkx6.1 in ILC derived by hESC differentiation. After sorting by MACS with CD49A antibodies, the ILC fraction enriched in CD49A+ cells rapidly reduced glycemia when implanted in the diabetic mice, whereas mice receiving the CD49A depleted population remained highly diabetic. CD49A-enriched ILC cells also produced significantly higher levels of human C-peptide in mouse blood. Another marker, CD26 (DPP4, dipeptidyl peptidase-4), was identified by FCCS as binding insulin-expressing cells which are Nkx6.1-negative. Depletion of CD26+ cells followed by enrichment for CD49A+ cells increased DP cells to over 70%. After this double selection, the CD26 depleted/CD49A enriched ILC were more active than non-sorted ILC to reduce glycemia in the diabetic mice.ConclusionsRefining the composition of ILC differentiated from hPSC by negative selection to remove cells expressing CD26 and positive selection for CD49A expressing cells can enable more effective cell therapy of diabetes.

scholarly journals Generation of hypoimmunogenic human pluripotent stem cells

2019 ◽  
Vol 116 (21) ◽  
pp. 10441-10446 ◽  
Author(s):  
Xiao Han ◽  
Mengning Wang ◽  
Songwei Duan ◽  
Paul J. Franco ◽  
Jennifer Hyoje-Ryu Kenty ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Therapy ◽  
Immune Responses ◽  
Pluripotent Stem Cells ◽  
Nk Cell ◽  
Immune Surveillance ◽  
Human Pluripotent Stem Cells ◽  
Innate Immune

Polymorphic HLAs form the primary immune barrier to cell therapy. In addition, innate immune surveillance impacts cell engraftment, yet a strategy to control both, adaptive and innate immunity, is lacking. Here we employed multiplex genome editing to specifically ablate the expression of the highly polymorphic HLA-A/-B/-C and HLA class II in human pluripotent stem cells. Furthermore, to prevent innate immune rejection and further suppress adaptive immune responses, we expressed the immunomodulatory factors PD-L1, HLA-G, and the macrophage “don’t-eat me” signal CD47 from the AAVS1 safe harbor locus. Utilizing in vitro and in vivo immunoassays, we found that T cell responses were blunted. Moreover, NK cell killing and macrophage engulfment of our engineered cells were minimal. Our results describe an approach that effectively targets adaptive as well as innate immune responses and may therefore enable cell therapy on a broader scale.

scholarly journals Differentiation of Multipotent Stem Cells to Insulin-Producing Cells for Treatment of Diabetes Mellitus: Bone Marrow- and Adipose Tissue-Derived Cells Comparison

2021 ◽  
Author(s):  
Zahra Eydian ◽  
Alaleh Mohammad Ghasemi ◽  
Samira Ansari ◽  
Ali Naghi Kamali ◽  
Maryam Khosravi ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Stem Cells ◽  
Bone Marrow ◽  
Adipose Tissue ◽  
Islet Cells ◽  
Human Adipose Tissue ◽  
Multipotent Stem Cells ◽  
C Peptide ◽  
Treatment Of Diabetes

Abstract Background: Mesenchymal stem cells (MSCs) from human adipose tissue and bone marrow have a great potential for use in cell therapy due to their ease of isolation, expansion, and differentiation. Our intention was to isolate and promote in vitro expansion and differentiation of MSCs from human adipose and bone marrow tissue into cells with a pancreatic endocrine phenotype and to compare the potency of these cells together.Methods and Results: MSCs were pre-induced with nicotinamide, mercaptoethanol, B-27 and b-FGF in L-DMEM for 2 days and re-induced again in supplemented H-DMEM for another 3 days. Expression of five genes in differentiated beta cells was evaluated by Real-time PCR and western blotting and the potency of insulin release in response to glucose stimulation was evaluated by insulin and C-peptide ELISA kit.Quantitative RT-PCR results showed up-regulation of four genes in differentiated beta-islet cells (Insulin, Ngn-3, Pax-4 and Pdx-1) compared with the control. Western blot analysis showed that MSCs cells mainly produced proinsulin and insulin after differentiation but nestin was more expressed in pre-differentiated stem cells. Glucose and insulin secretion assay showed that insulin levels and C-peptide secretion were significantly increased in response to 10 mM glucose.Conclusions: Our study showed that both adipose and bone marrow stem cells could differentiate into functional beta-islet cells but it seems that adipose stem cells could be a better choice for treatment of diabetes mellitus according to their more safety and potency.

4. Therapy using pluripotent stem cells

2021 ◽  
pp. 52-74
Author(s):  
Jonathan Slack
Keyword(s):  
Stem Cells ◽  
Cell Therapy ◽  
Pluripotent Stem Cells ◽  
Source Material ◽  
Age Related Macular Degeneration ◽  
Cell Therapies ◽  
Differentiated Cells ◽  
Age Related ◽  
Existing Form

‘Therapy using pluripotent stem cells’ examines some of the diseases that have been the first to be treated by cell therapy using pluripotent stem cells as the source material. Proposed cell therapies involve making the required differentiated cells in vitro and then implanting them into the appropriate site in the patient. The biggest success story so far is the treatment of the retina for a condition called age-related macular degeneration (ARMD). Diabetes is one of the top targets for cell therapy based on pluripotent stem cells, building on an existing form of cell therapy called islet transplantation.

P2571Differentiation of iPS-derived cardiac mesoderm cells into cardiomyocytes using inducible shRNA technology

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
A Garcia Duran ◽  
T Nazari-Shafti ◽  
A Kurtz ◽  
V Falk ◽  
M Gossen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Cell Therapy ◽  
Pluripotent Stem Cells ◽  
Wnt Signalling ◽  
Severe Side Effect ◽  
Cardiomyocyte Differentiation ◽  
Cardiac Mesoderm

Abstract Introduction The development of robust and scalable cardiomyocyte differentiation protocols from human pluripotent stem cells (hPSCs) offers the opportunity to derive large number of autologous cardiomyocytes for cell therapy. However, human induced pluripotent stem cells-derived cardiomyocytes (hiPSCs-CM) do not proliferate, are susceptible to ischemia and poorly retained in the recipient myocardium. In addition, arrhythmias are a severe side effect of cell therapy with hiPSC-CM, probably due to their immature phenotype. Based on previous observations in our lab, we speculate that the heart environment might be an important factor to enable full maturation and in situ coupling of the implanted cells in vivo. We therefore speculate that transplantation of robust, proliferative, partially committed, non-tumorigenic cardiac mesoderm cells and subsequent differentiation in situ may overcome these limitations. Purpose We seek to engineer hiPSC-derived cardiac mesoderm cells for implantation in the failing myocardium that, once they have settled, consolidated, and proliferated in situ, can be selectively induced to mature into cardiomyocytes. Methods Wnt signalling induces mesoderm lineage differentiation of hiPSCs and inhibits further maturation towards CM. To induce Wnt signalling inhibition, we designed a customized biomimetic shRNA targeting the Wnt agonist β-catenin fused to a fluorescent reported protein controlled by a Tet-ON promoter. This transgene was transferred to the safe harbour locus AAVS1 of the OPTI-OX hiPS cell line constitutively expressing the reverse tetracycline transactivator protein (rtTA) in the ROSA26 locus. In this configuration, gene expression can be activated in trans upon doxycycline administration. Results We studied the kinetics of gene expression activation on the engineered undifferentiated hiPSCs and observed that a maximal reduction of 80–90% β-catenin correlating with reporter expression was achieved upon a 4-day treatment with doxycycline. Importantly, this activation could be fully reverted 6 days upon withdrawal. Furthermore, in vitro differentiation of cardiac mesoderm cells into cardiomyocytes was enhanced upon doxycycline administration as compared to the untreated cells as assessed by the upregulation of cardiomyocyte specific genes and the increased percentage of cTNT+ cells. Conclusion An inducible system for targeted β-catenin downregulation by shRNA allows for significant, sustained, and reversible Wnt signalling modulation, which is sufficient to induce cardiomyocyte differentiation of cardiac mesoderm cells in vitro. This technology may be implemented to differentiate the transplanted cells in the failing myocardium, opening new prospects to overcome the current limitations of hiPSC-based cardiac cell therapy. Acknowledgement/Funding BMBF funding (FKZ 13GW0098, FKZ 13GW0099) DFG funding through the BSRT GSC 203

Cellular Aggregates Induced from Monolayer Cultures of Human Pancreatic Cells Correct Hyperglycemia in Diabetic Mice and Demonstrate Physiological Production of Human C-Peptide.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4184-4184
Author(s):  
Fred D. Wu ◽  
Michael R. Jagir ◽  
Thomas T. Luu ◽  
Jerry S. Powell
Keyword(s):  
Pancreatic Islet ◽  
Islet Cells ◽  
Severe Combined Immunodeficiency ◽  
Cell Phenotype ◽  
Pancreatic Islet Cells ◽  
Diabetic Mice ◽  
C Peptide ◽  
Extracellular Matrix Components ◽  
Cellular Aggregates

Abstract Diabetes is due to loss of appropriate insulin production by pancreatic islet cells, resulting in hyperglycemia and significant morbidity. We have developed cell cultures of pancreas-derived precursor cells that can be maintained for more than 20 population doublings in culture, growing as monolayers of undifferentiated cells. These monolayer cells can then be induced to differentiate into cellular aggregates that resemble islets when grown on different extracellular matrix components, including matrigel, or poly-d-lysine. The induced cellular aggregates, but not monolayer cells, correct hyperglycemia in diabetic (streptozotocin) SCID (severe combined immunodeficiency) mice after implantation under the kidney capsule. This animal model can be used to follow the process of differentiation from undifferentiated precursor cell to fully functional insulin producing beta cells, and to identify key proteins and genes involved in differentiation of pancreatic islet cells. Here we report that in the mice, after implantation of cellular aggregates, human c-peptide concentrations are controlled physiologically appropriately in response to glucose tolerance testing (challenged with 2 grams of glucose injected intraperitoneally) in the treated diabetic SCID mice (n = 6) as well as in normal mice that received implanted cellular aggregates (n = 6). Human c peptide is not detected in any of the control animals, nor in animals implanted with monolayer cells. Comparative RNA microarray data analyses, using U133 arrays (Affymetrix), were compared between the monolayer cells in culture and the cellular aggregates 48 hours after induction with poly-d-lysine. We consider of particular interest the up-regulation of BMP, CXCR4, and HGF in the cell aggregates, and the down-regulation of VCAM-1. We believe that these genes are necessary for either aggregation or for physiologically functional insulin secretion. When examined for greater than two-fold gene expression differences between monolayer and aggregated cells, 424 gene sequences were upregulated and 690 genes down regulated. In addition, RAGE display analysis for tyrosine kinases also showed that the kinases Erb-B2 and PDGFR-B were upregulated at 48 hours in the aggregating process. In contrast, DDR2, a collagen I receptor tyrosine kinase, was expressed equally in both monolayer and aggregated cells. In summary, the results suggest: 1) that a population of pancreatic stem cells can be isolated and cultured in vitro, 2) that these cells can be induced to form functional islet cells that correct hyperglycemia in diabetic mice, 3) that human c-peptide is physiologically regulated in aggregate implanted mice in response to glucose challenge, and 4) HGF, along with other genes of potential interest, is upregulated in the process of differentiation from monolayer to aggregated cell phenotype.

scholarly journals Preconditioning the Initial State of Feeder-free Human Pluripotent Stem Cells Promotes Self-formation of Three-dimensional Retinal Tissue

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Atsushi Kuwahara ◽  
Suguru Yamasaki ◽  
Michiko Mandai ◽  
Kenji Watari ◽  
Keizo Matsushita ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Therapy ◽  
Pluripotent Stem Cells ◽  
Retinal Pigment ◽  
Three Dimensional ◽  
Human Pluripotent Stem Cells ◽  
Retinal Tissue ◽  
Self Formation ◽  
Self Organizing

AbstractA three-dimensional retinal tissue (3D-retina) is a promising graft source for retinal transplantation therapy. We previously demonstrated that embryonic stem cells (ESCs) can generate 3D-retina in vitro using a self-organizing stem cell culture technique known as SFEBq. Here we show an optimized culture method for 3D-retina generation from feeder-free human pluripotent stem cells (hPSCs). Although feeder-free hPSC-maintenance culture was suitable for cell therapy, feeder-free hPSC-derived aggregates tended to collapse during 3D-differentiation culture. We found that the initial hPSC state was a key factor and that preconditioning of the hPSC state by modulating TGF-beta and Shh signaling improved self-formation of 3D-neuroepithelium. Using the preconditioning method, several feeder-free hPSC lines robustly differentiated into 3D-retina. In addition, changing preconditioning stimuli in undifferentiated hPSCs altered the proportions of neural retina and retinal pigment epithelium, important quality factors for 3D-retina. We demonstrated that the feeder-free hiPSC-derived 3D-retina differentiated into rod and cone photoreceptors in vitro and in vivo. Thus, preconditioning is a useful culture methodology for cell therapy to direct the initial hPSC state toward self-organizing 3D-neuroepithelium.

Autologous transplantation of mesenchymal stem cells into the portal vein of the miniature pig; a preliminary experiment for NOTES approach

2019 ◽  
Vol 98 (9) ◽  
pp. 350-355
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Cell Therapy ◽  
Portal Vein ◽  
Liver Parenchyma ◽  
Miniature Pig ◽  
Hepatic Diseases ◽  
Study Results

Introduction: There is evidence that mesenchymal stem cells (MSCs) could trans-differentiate into the liver cells in vitro and in vivo and thus may be used as an unfailing source for stem cell therapy of liver disease. Combination of MSCs (with or without their differentiation in vitro) and minimally invasive procedures as laparoscopy or Natural Orifice Transluminal Endoscopic Surgery (NOTES) represents a chance for many patients waiting for liver transplantation in vain. Methods: Over 30 millions of autologous MSCs at passage 3 were transplanted via the portal vein in an eight months old miniature pig. The deposition of transplanted cells in liver parenchyma was evaluated histologically and the trans-differential potential of CM-DiI labeled cells was assessed by expression of pig albumin using immunofluorescence. Results: Three weeks after transplantation we detected the labeled cells (solitary, small clusters) in all 10 samples (2 samples from each lobe) but no diffuse distribution in the samples. The localization of CM-DiI+ cells was predominantly observed around the portal triads. We also detected the localization of albumin signal in CM-DiI labeled cells. Conclusion: The study results showed that the autologous MSCs (without additional hepatic differentiation in vitro) transplantation through the portal vein led to successful infiltration of intact miniature pig liver parenchyma with detectable in vivo trans-differentiation. NOTES as well as other newly developed surgical approaches in combination with cell therapy seem to be very promising for the treatment of hepatic diseases in near future.

Sign in / Sign up

Export Citation Format

Share Document