Large-Scale Ex Vivo Generation of Human Red Blood Cells from Cord Blood CD34+ Cells.
ABSTRACT: The ex vivo generation of human red blood cells on a large scale from hematopoietic stem and progenitor cells has been considered as a potential method to overcome blood supply shortages. Here, we report that functional human erythrocytes can be efficiently produced from cord blood (CB) CD34+ cells using a bottle turning device culture system. Safety and efficiency studies were performed in murine and nonhuman primate (NHP) models. With the selected optimized culture conditions, one human CB CD34+ cell could be induced ex vivo to produce up to 200 million erythrocytes with a purity of 90.1%?±?6.2% and 50%?±?5.7% (mean?±?SD) for CD235a+ cells and enucleated cells, respectively. The yield of erythrocytes from one CB unit (5 million CD34+ cells) could be, in theory, equivalent to 500 blood transfusion units in clinical application. Moreover, induced human erythrocytes had normal hemoglobin content and could continue to undergo terminal maturation in the murine xenotransplantation model. In NHP model, xenotransplantation of induced human erythrocytes enhanced hematological recovery and ameliorated the hypoxia situation in the primates with hemorrhagic anemia. These findings suggested that the ex vivo-generated erythrocytes could be an alternative blood source for traditional transfusion products in the clinic. Stem Cells Translational Medicine 2017;6:1698-1709.
Project description:Currently, there is a growing need for culturing hematopoietic stem/progenitor cells (HSPCs) in vitro for various clinical applications including gene therapy. Compared with cord blood (CB) CD34+ HSPCs, it is more challenging to maintain or expand CD34+ peripheral blood mobilized stem/progenitor cells (PBSCs) ex vivo. To fill this knowledge gap, we have systematically surveyed 466 small-molecule drug compounds for their potential in cytokine-dependent expansion of human CD34+CD90+ HSPCs. We found that epigenetic modifiers, especially histone deacetylase inhibitors (HDACis), could preferentially maintain and expand these cells. In particular, treatment of CD34+ PBSCs with a single dose of HDACi trichostatin A (TSA) at a concentration of 50 nmol/L ex vivo yielded the greatest expansion (11.7-fold) of CD34+CD90+ cells when compared with the control (dimethyl sulfoxide [DMSO] plus cytokines) group. Additionally, TSA-treated PBSC CD34+ cells had a statistically significant higher engraftment rate than the control-treated group in xenotransplantation experiments. Mechanistically, TSA treatment was associated with increased expression of HSPC-related genes such as GATA2 and SALL4. Furthermore, TSA-mediated CD34+CD90+ expansion was reduced by downregulation of SALL4 but not GATA2. Overall, we have developed a robust, short-term (5-day), PBSC ex vivo maintenance/expansion culture technique and found that the HDACi-TSA/SALL4 axis is important for the biological process.
Project description:The supply of red blood cells (RBCs) is not sufficient in many developing countries or in developed countries for patients who need chronic transfusion from best-matched donors. Ex vivo expansion and maturation of human erythroid precursor cells (erythroblasts) could represent a potential solution. Proliferating erythroblasts can be expanded from human umbilical cord blood mononuclear cells (CB MNCs) ex vivo for 10^6-10^7 fold (in ~50 days) before undergoing senescence. Here, we report that ectopic expression of three to four genetic factors that have been used for iPS cell derivation enables CB-derived erythroblasts to undergo extended ex vivo expansion (≥10^51 fold in ~9 months) in a defined suspension culture condition without change of cell identity or function. These vastly expanding erythroblasts maintain homogeneously immature erythroblast phenotypes, a normal diploid karyotype and dependence on specific combination of cytokines and hormone for survival and proliferation throughout the continuous expansion period. When switched to a culture condition for terminal maturation, these immortalized erythroblasts gradually exit cell cycle, decrease cell size, accumulate hemoglobin, condense nuclei and eventually give rise to enucleated hemoglobin-containing erythrocytes. Our result may ultimately lead to the development of unlimited sources of cultured RBCs for optimally-matched or personalized transfusion medicine. We compared the global gene expression profiles of different human cell types: iE: immortalized erythroblasts generated by genetic reprogramming from pCBE; pCBE: primary cord blood-derived erythroblasts; CD34+: CD34+ purified hematopoietic stem/progenitor cells from adult blood or fetal liver; TF-1: a human erythroleukemia cell line; ESC: human embryonic stem cells; iPSCs: human induced pluripotent stem cells. We want to see the relationship among these cell types. We included multiple samples (biological replicates) for most cell types.
Project description:There is a constant shortage of red blood cells (RBCs) from sufficiently matched donors for patients who need chronic transfusion. Ex vivo expansion and maturation of human erythroid precursors (erythroblasts) from the patients or optimally matched donors could represent a potential solution. Proliferating erythroblasts can be expanded from umbilical cord blood mononuclear cells (CB MNCs) ex vivo for 10(6)-10(7)-fold (in ~50 days) before proliferation arrest and reaching sufficient number for broad application. Here, we report that ectopic expression of three genetic factors (Sox2, c-Myc, and an shRNA against TP53 gene) associated with iPSC derivation enables CB-derived erythroblasts to undergo extended expansion (~10(68)-fold in ~12 months) in a serum-free culture condition without change of cell identity or function. These expanding erythroblasts maintain immature erythroblast phenotypes and morphology, a normal diploid karyotype and dependence on a specific combination of growth factors for proliferation throughout expansion period. When being switched to a terminal differentiation condition, these immortalized erythroblasts gradually exit cell cycle, decrease cell size, accumulate hemoglobin, condense nuclei and eventually give rise to enucleated hemoglobin-containing erythrocytes that can bind and release oxygen. Our result may ultimately lead to an alternative approach to generate unlimited numbers of RBCs for personalized transfusion medicine.
Project description:Diabetes can impair wound closure, which can give rise to major clinical problems. Most treatments for wound repair in diabetes remain ineffective. This study aimed to investigate the influence on wound closure of treatments using expanded human cord blood CD34(+) cells (CB-CD34(+) cells), freshly isolated CB-CD34(+) cells and a cytokine cocktail. The test subjects were mice with streptozotocin-induced diabetes. Wounds treated with fresh CB-CD34(+) cells showed more rapid repair than mice given the PBS control. Injection of expanded CB-CD34(+) cells improved wound closure significantly, whereas the injection of the cytokine cocktail alone did not improve wound repair. The results also demonstrated a significant decrease in epithelial gaps and advanced re-epithelialization over the wound bed area after treatment with either expanded CB-CD34(+) cells or freshly isolated cells compared with the control. In addition, treatments with both CB-CD34(+) cells and the cytokine cocktail were shown to promote recruitment of CD31(+)-endothelial cells in the wounds. Both the CB-CD34(+) cell population and the cytokine treatments also enhanced the recruitment of CD68-positive cells in the early stages (day 3) of treatment compared with PBS control, although the degree of this enhancement was found to decline in the later stages (day 9). These results demonstrated that expanded CB-CD34(+) cells or freshly isolated CB-CD34(+) cells could accelerate wound repair by increasing the recruitment of macrophages and capillaries and the reepithelialization over the wound bed area. Our data suggest an effective role in wound closure for both ex vivo expanded CB-CD34(+) cells and freshly isolated cells, and these may serve as therapeutic options for wound treatment for diabetic patients. Wound closure acceleration by expanded CB-CD34(+) cells also breaks the insufficient quantity obstacle of stem cells per unit of cord blood and other stem cell sources, which indicates a broader potential for autologous transplantation.
Project description:Cord blood (CB) cells that express CD34 have extensive hematopoietic capacity and rapidly divide ex vivo in the presence of cytokine combinations; however, many of these CB CD34+ cells lose their marrow-repopulating potential. To overcome this decline in function, we treated dividing CB CD34+ cells ex vivo with several histone deacetylase inhibitors (HDACIs). Treatment of CB CD34+ cells with the most active HDACI, valproic acid (VPA), following an initial 16-hour cytokine priming, increased the number of multipotent cells (CD34+CD90+) generated; however, the degree of expansion was substantially greater in the presence of both VPA and cytokines for a full 7 days. Treated CD34+ cells were characterized based on the upregulation of pluripotency genes, increased aldehyde dehydrogenase activity, and enhanced expression of CD90, c-Kit (CD117), integrin ?6 (CD49f), and CXCR4 (CD184). Furthermore, siRNA-mediated inhibition of pluripotency gene expression reduced the generation of CD34+CD90+ cells by 89%. Compared with CB CD34+ cells, VPA-treated CD34+ cells produced a greater number of SCID-repopulating cells and established multilineage hematopoiesis in primary and secondary immune-deficient recipient mice. These data indicate that dividing CB CD34+ cells can be epigenetically reprogrammed by treatment with VPA so as to generate greater numbers of functional CB stem cells for use as transplantation grafts.
Project description:Cord blood (CB) is an attractive source of hematopoietic stem cells (HSCs) for hematopoietic cell transplantation. However, its application remains limited due to the low number of HSCs/progenitors in a single CB unit and its notoriously difficulty in expanding ex vivo. Here, we demonstrated that the human fetal liver sinusoidal endothelial cells engineered to constitutively express the adenoviral E4orf1 gene (hFLSECs-E4orf1) is capable of efficient expansion ex vivo for human CB hematopoietic stem and progenitor cells (HSPCs). Coculture of CD34+ hCB cells with hFLSECs-E4orf1 resulted in generation of substantially more total nucleated cells, CD34+CD38- and CD34+ CD38-CD90+ HSPCs in comparison with that of cytokines alone after 14 days. The multilineage differentiation potential of the expanded hematopoietic cells in coculture condition, as assessed by in vitro colony formation, was also significantly heightened. The CD34+ hCB cells amplified on hFLSECs-E4orf1 were capable of engraftment in vivo. Furthermore, hFLSECs-E4orf1 highly expressed hematopoiesis related growth factor and Notch receptors. Accordingly, the CD34+ hCB cells amplified on hFLSECs-E4orf1 exhibited Notch signaling activation. Taken together, our findings indicated that FLSECs may potentially be the crucial component of the microenvironment to support recapitulation of embryonic HSC amplification in vitro and allow identification of new growth factors responsible for collective regulation of hematopoiesis.
Project description:Advantages associated with the use of cord blood (CB) transplantation include the availability of cryopreserved units, ethnic diversity and lower incidence of graft-versus-host disease compared with bone marrow or mobilized peripheral blood. However, poor engraftment remains a major obstacle. We and others have found that ex vivo fucosylation can enhance engraftment in murine models, and now ex vivo treatment of CB with fucosyltransferase (FT) VI before transplantation is under clinical evaluation (NCT01471067). However, FTVII appears to be more relevant to hematopoietic cells and may alter acceptor substrate diversity. The present study compared the ability of FTVI and FTVII to improve the rapidity, magnitude, multi-lineage and multi-tissue engraftment of human CB hematopoietic stem and progenitor cells (HSPCs) in vivo.CD34-selected CB HSPCs were treated with recombinant FTVI, FTVII or mock control and then injected into immunodeficient mice and monitored for multi-lineage and multi-tissue engraftment.Both FTVI and FTVII fucosylated CB CD34? cells in vitro, and both led to enhanced rates and magnitudes of engraftment compared with untreated CB CD34? cells in vivo. Engraftment after treatment with either FT was robust at multiple time points and in multiple tissues with similar multi-lineage potential. In contrast, only FTVII was able to fucosylate T and B lymphocytes.Although FTVI and FTVII were found to be similarly able to fucosylate and enhance the engraftment of CB CD34? cells, differences in their ability to fucosylate lymphocytes may modulate graft-versus-tumor or graft-versus-host effects and may allow further optimization of CB transplantation.
Project description:Although hematopoietic stem cells (HSC) are the best characterized and the most clinically used adult stem cells, efforts are still needed to understand how to best ex vivo expand these cells. Here we present our unexpected finding that OCT4 is involved in the enhancement of cytokine-induced expansion capabilities of human cord blood (CB) HSC. Activation of OCT4 by Oct4-activating compound 1 (OAC1) in CB CD34(+) cells enhanced ex vivo expansion of HSC, as determined by a rigorously defined set of markers for human HSC, and in vivo short-term and long-term repopulating ability in NSG mice. Limiting dilution analysis revealed that OAC1 treatment resulted in 3.5-fold increase in the number of SCID repopulating cells (SRCs) compared with that in day 0 uncultured CD34(+) cells and 6.3-fold increase compared with that in cells treated with control vehicle. Hematopoietic progenitor cells, as assessed by in vitro colony formation, were also enhanced. Furthermore, we showed that OAC1 treatment led to OCT4-mediated upregulation of HOXB4. Consistently, siRNA-mediated knockdown of HOXB4 expression suppressed effects of OAC1 on ex vivo expansion of HSC. Our study has identified the OCT4-HOXB4 axis in ex vivo expansion of human CB HSC.
Project description:The generation of cultured red blood cells from stem cell sources may fill an unmet clinical need for transfusion-dependent patients, particularly in countries that lack a sufficient and safe blood supply. Cultured red blood cells were generated from human CD34+ cells from adult peripheral blood or cord blood by ex vivo expansion, and a comprehensive in vivo survival comparison with standard red cell concentrates was undertaken. Significant amplification (>105-fold) was achieved using CD34+ cells from both cord blood and peripheral blood, generating high yields of enucleated cultured red blood cells. Following transfusion, higher levels of cultured red cells could be detected in the murine circulation compared to standard adult red cells. The proportions of cultured blood cells from cord or peripheral blood sources remained high 24 hours post-transfusion (82±5% and 78±9%, respectively), while standard adult blood cells declined rapidly to only 49±9% by this time. In addition, the survival time of cultured blood cells in mice was longer than that of standard adult red cells. A paired comparison of cultured blood cells and standard adult red blood cells from the same donor confirmed the enhanced in vivo survival capacity of the cultured cells. The study herein represents the first demonstration that ex vivo generated cultured red blood cells survive longer than donor red cells using an in vivo model that more closely mimics clinical transfusion. Cultured red blood cells may offer advantages for transfusion-dependent patients by reducing the number of transfusions required.
Project description:Pigs are used as potential donor animals for xenotransplantation. However, porcine endogenous retrovirus (PERV), shown to infect both human and non-human primate (NHP) cells in vitro, presents a risk of transmission to humans in xenotransplantation. In this study, we analyzed PERV transmission in various organs after pig-to-NHP xenotransplantation. We utilized pig-to-NHP xenotransplant tissue samples obtained using two types of transgenic pigs from the National Institute of Animal Science (NIAS, Republic of Korea), and examined them for the existence of PERV genes in different organs via PCR and RT-PCR with specific primers. To determine PERV insertion into chromosomes, inverse PCR using PERV long terminal repeat (LTR) region-specific primers was conducted. The PERV gene was not detected in NHP organs in cardiac xenotransplantation but detected in NHP bladders in renal xenotransplantation. The insertion experiment confirmed that PERVs originate from porcine donor cells rather than integrated provirus in the NHP chromosome. We also demonstrate the presence of pig cells in the NHP bladder after renal xenotransplantation using specific-porcine mitochondrial DNA gene PCR. The PERV sequence was detected in the bladder of NHPs after renal xenotransplantation by porcine cell-microchimerism but did not integrate into the NHP chromosome.