Infusion of iPSC-Derived CD34 + Hematopoietic Progenitors Following Myeloablative HSC Transplantation and Assessment of Their Immunogenicity in MHC-Defined Nonhuman Primate Model

Abstract Bone marrow suppression and associated neutropenia that renders the patient more susceptible to infection is a major complication and limiting factor for current chemotherapy. Strategies aimed at neutrophil reconstitution using transfusions or accelerating neutrophil recovery with G-CSF show a limited effect in reducing the rate of infection. To overcome these limitations, administration of ex vivo expanded somatic hematopoietic progenitors (HPs) has been explored as a way to facilitate a more rapid myeloid recovery and improve overall survival following myeloablation. Recent advances in reprogramming and induced pluripotent stem cell (iPSC) technologies have created alternative platforms for off-the-shelf supply of immunologically compatible HPs, including cellular products derived from MHC homozygous superdonors which can increase the degree of MHC matching and may provide a maximum utility for stem cell banking. In these studies, we developed Mauritian cynomolgus macaque (MCM) model to evaluate the utility and safety of CD34 +CD45 + hematopoietic progenitors derived from iPSCs (iHPs) generated from MHC homozygous animals in the treatment of cytopenia following myeloablative stem cell transplantation. MCM iPSCs were generated from MHC homozygous animals and used to generate iHPs in coculture with OP9. Three groups of MCMs underwent myeloablative total body irradiation (TBI) followed by transplantation of cryopreserved autologous CD34 + HSPCs. Four days after autologous HSPC transplantation, animals received transfusion of 30x10 6/kg cryopreserved iCD34 + cells tagged with eGFP or tdTomato from homozygous MHC-matched iPSCs (group 2) or homozygous MHC mismatched iPSCs (group 3). We have demonstrated that infusion of iHPs is safe and well tolerated. No teratoma or tumor formation was observed in animals one year after infusion of iHPs. Although we detected few iPSC derived hematopoietic cells in the blood within 1-week post-infusion, we did not see any significant differences in blood counts or other peripheral blood parameters between all animal groups. Intriguingly we found footprints of iPSC-derived cells in the colon, lymph node, skin and brain specimens collected 25 days after iHP infusion from one animal. This suggests a possibility of migration and retention within these tissues of iHP and iHP-derived myeloid cells. To assess immunogenicity, MHC homozygous iHPs were infused into immunocompetent MCMs across different MHC barriers. 10x10 6/kg of cryopreserved iCD34+ from homozygous iPSCs were transfused every week for 3 weeks, into animals that were divided into 4 groups namely autologous (group I), MHC matched homozygous (group II), MHC matched heterozygous (group III) and MHC mismatch (group IV). Overall, these studies revealed low immunogenicity of MHC homozygous iHPs in MHC matched homozygous and heterozygous animals, while weak immune response was detected following iHP infusion in some MHC mismatched animals. Disclosures Slukvin: Cynata Therapeutics: Consultancy, Current equity holder in publicly-traded company.

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Structurally Specific Heparan Sulfates Support Primitive Human Hematopoiesis by Formation of a Multimolecular Stem Cell Niche

Blood ◽

10.1182/blood.v92.12.4641 ◽

1998 ◽

Vol 92 (12) ◽

pp. 4641-4651 ◽

Cited By ~ 105

Author(s):

Pankaj Gupta ◽

Theodore R. Oegema ◽

Joseph J. Brazil ◽

Arkadiusz Z. Dudek ◽

Arne Slungaard ◽

...

Keyword(s):

Stem Cell ◽

Cell Line ◽

Stem Cell Niche ◽

Ex Vivo ◽

Recent Observation ◽

Hematopoietic Progenitors ◽

Platelet Factor ◽

Cell Niche

Abstract Stem cell localization, conservation, and differentiation is believed to occur in niches in the marrow stromal microenvironment. Our recent observation that long-term in vitro human hematopoiesis requires a stromal heparan sulfate proteoglycan (HSPG) led us to hypothesize that such HSPG may orchestrate the formation of the stem cell niche. We compared the structure and function of HS from M2-10B4, a hematopoiesis-supportive cell line, with HS from a nonsupportive cell line, FHS-173-We. Long-term culture-initiating cell (LTC-IC) maintenance was enhanced by PG from supportive cells but not by PG from nonsupportive cells (P < .005). The supportive HS were significantly larger and more highly sulfated than the nonsupportive HS. Specifically, supportive HS contained higher 6-O-sulfation on the glucosamine residues. In agreement with these observations, purified 6-O-sulfated heparin and highly 6-O-sulfated bovine kidney HS similarly maintained LTC-IC. In contrast, completely desulfated heparin, N-sulfated heparin, and unmodified heparin did not support LTC-IC maintenance. Moreover, the supportive HS promoted LTC-IC maintenance but not differentiation of CD34+/HLA-DR−cells into colony-forming cells (CFCs) and mature blood cells. The supportive HS but not the nonsupportive HS bound both cytokines and matrix components critical for hematopoiesis, including interleukin-3 (IL-3), macrophage inflammatory protein-1 (MIP-1), and thrombospondin (TSP). Significantly more CD34+ cells adhered directly to immobilized O-sulfated heparin than to N-sulfated or desulfated heparin. Thus, hematopoiesis-supportive stromal HSPG possessing large, highly 6-O-sulfated HS mediate the juxtaposition of hematopoietic progenitors with stromal cells, specific growth-promoting (IL-3) and growth-inhibitory (MIP-1 and platelet factor 4 [PF4]) cytokines, and extracellular matrix (ECM) proteins such as TSP. We conclude that the structural specificity of stromal HSPG that determines the selective colocalization of cytokines and ECM components leads to the formation of discrete niches, thereby orchestrating the controlled growth and differentiation of stem cells. These findings may have important implications for ex vivo expansion of and gene transfer into primitive hematopoietic progenitors.

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Exploring the Mesenchymal Stem Cell Secretome for Corneal Endothelial Proliferation

Stem Cells International ◽

10.1155/2020/5891393 ◽

2020 ◽

Vol 2020 ◽

pp. 1-10

Author(s):

Bert Van den Bogerd ◽

Nadia Zakaria ◽

Steffi Matthyssen ◽

Carina Koppen ◽

Sorcha Ní Dhubhghaill

Keyword(s):

Endothelial Cells ◽

Stem Cell ◽

Mesenchymal Stem Cell ◽

Conditioned Medium ◽

Ex Vivo ◽

Growth Stimulation ◽

Soluble Proteins ◽

Size Exclusion ◽

Limiting Factor ◽

Growth Enhancement

Ex vivo grown human corneal endothelial cells (HCEnC) are a new emerging treatment option to treat visually impaired patients aimed at alleviating the current global donor shortage. Expanding HCEnC is still challenging, and obtaining cells in sufficient quantities is a limiting factor. It is already known that conditioned medium obtained from bone marrow mesenchymal stem cells can stimulate the proliferation of endothelial cells. The aim of this study was to take this work a step further to identify some of the underlying factors responsible. We confirmed the stimulatory effect of the mesenchymal stem cell secretome seen previously and separated the exosomes from the soluble proteins using size exclusion chromatography. We demonstrated the presence of exosomes and soluble proteins in the early and late fractions, respectively, with transmission electron microscopy and protein assays. Proliferation studies demonstrated that growth stimulation could be reproduced with the later protein-rich fractions but not with the exosome-rich fraction. Antibody assays revealed the presence of the secreted proteins EGF, IGFBP2, and IGFBP6 in protein-high fractions, but the growth enhancement was not seen with purified protein formulations. In conclusion, we confirmed the stimulatory effect of stem cell-conditioned medium and have determined that the effect was attributable to the proteins rather than to the exosomes. We were not able to reproduce the growth stimulation, however, with the pure recombinant protein candidates tested. Specific identification of the underlying proteins using proteomics could render a bioactive protein that can be used for ex vivo expansion of cells or as an in vivo drug to treat early corneal endothelial damage.

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Inverse Tendency between Ex Vivo Expansion Potential of Hematopoietic Progenitors and Time to Engraftment after Hematopoietic Stem Cell Transplantation

Annals of Laboratory Medicine ◽

10.3343/kjlm.2006.26.6.385 ◽

2006 ◽

Vol 26 (6) ◽

pp. 385-392

Author(s):

Ji Myung Kim ◽

Chan Jeoung Park ◽

Hyun Sook Chi ◽

Jae Hwan Lee ◽

Gyu Hyung Lee ◽

...

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell Transplantation ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Hematopoietic Stem Cell ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem

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Protein kinase B (PKB/c-akt) regulates homing of hematopoietic progenitors through modulation of their adhesive and migratory properties

Blood ◽

10.1182/blood-2009-10-250258 ◽

2010 ◽

Vol 116 (13) ◽

pp. 2373-2384 ◽

Cited By ~ 17

Author(s):

Miranda Buitenhuis ◽

Edwin van der Linden ◽

Laurien H. Ulfman ◽

Frans M. Hofhuis ◽

Marc B. Bierings ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Protein Kinase ◽

Ex Vivo ◽

Protein Kinase B ◽

Cell Manipulation ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem ◽

Immunodeficient Mice

Abstract Limited number of hematopoietic stem cells in umbilical cord blood (UCB) presents a problem when using UCB for stem cell transplantation. Improving their homing capacity could reduce the need for high initial cell numbers during transplantation procedures. Although it is evident that protein kinase B (PKB/c-Akt) plays an important role in regulation of migration of various cell types, a role for PKB in regulation of migration and homing of human hematopoietic stem and progenitor cells remains to be determined. PKB activity was found to be required for induction of adhesion to bone marrow–derived stromal cells and detrimental for migration of UCB-derived CD34+ hematopoietic progenitors. In addition, PKB activity was found to positively regulate integrin expression. CD34+ hematopoietic progenitors, and their capacity to form colonies in vitro, were not affected by transient inhibition of PKB. Finally, transplantation of β2-microglobulin−/− nonobese diabetic/severe combined immunodeficient mice with CD34+ cells ectopically expressing constitutively active PKB resulted in reduced migration to the bone marrow, whereas inhibition of PKB activity resulted in an induction in bone marrow homing and engraftment. These results indicate that transient inhibition of PKB activity may provide a means for ex vivo stem cell manipulation to improve bone marrow transplantation regimes.

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Structurally Specific Heparan Sulfates Support Primitive Human Hematopoiesis by Formation of a Multimolecular Stem Cell Niche

Blood ◽

10.1182/blood.v92.12.4641.424k10_4641_4651 ◽

1998 ◽

Vol 92 (12) ◽

pp. 4641-4651 ◽

Cited By ~ 3

Author(s):

Pankaj Gupta ◽

Theodore R. Oegema ◽

Joseph J. Brazil ◽

Arkadiusz Z. Dudek ◽

Arne Slungaard ◽

...

Keyword(s):

Stem Cell ◽

Cell Line ◽

Stem Cell Niche ◽

Ex Vivo ◽

Recent Observation ◽

Hematopoietic Progenitors ◽

Platelet Factor ◽

Cell Niche

Stem cell localization, conservation, and differentiation is believed to occur in niches in the marrow stromal microenvironment. Our recent observation that long-term in vitro human hematopoiesis requires a stromal heparan sulfate proteoglycan (HSPG) led us to hypothesize that such HSPG may orchestrate the formation of the stem cell niche. We compared the structure and function of HS from M2-10B4, a hematopoiesis-supportive cell line, with HS from a nonsupportive cell line, FHS-173-We. Long-term culture-initiating cell (LTC-IC) maintenance was enhanced by PG from supportive cells but not by PG from nonsupportive cells (P < .005). The supportive HS were significantly larger and more highly sulfated than the nonsupportive HS. Specifically, supportive HS contained higher 6-O-sulfation on the glucosamine residues. In agreement with these observations, purified 6-O-sulfated heparin and highly 6-O-sulfated bovine kidney HS similarly maintained LTC-IC. In contrast, completely desulfated heparin, N-sulfated heparin, and unmodified heparin did not support LTC-IC maintenance. Moreover, the supportive HS promoted LTC-IC maintenance but not differentiation of CD34+/HLA-DR−cells into colony-forming cells (CFCs) and mature blood cells. The supportive HS but not the nonsupportive HS bound both cytokines and matrix components critical for hematopoiesis, including interleukin-3 (IL-3), macrophage inflammatory protein-1 (MIP-1), and thrombospondin (TSP). Significantly more CD34+ cells adhered directly to immobilized O-sulfated heparin than to N-sulfated or desulfated heparin. Thus, hematopoiesis-supportive stromal HSPG possessing large, highly 6-O-sulfated HS mediate the juxtaposition of hematopoietic progenitors with stromal cells, specific growth-promoting (IL-3) and growth-inhibitory (MIP-1 and platelet factor 4 [PF4]) cytokines, and extracellular matrix (ECM) proteins such as TSP. We conclude that the structural specificity of stromal HSPG that determines the selective colocalization of cytokines and ECM components leads to the formation of discrete niches, thereby orchestrating the controlled growth and differentiation of stem cells. These findings may have important implications for ex vivo expansion of and gene transfer into primitive hematopoietic progenitors.

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