Studies On Producing Mature Red Blood Cells From Human Embryonic Stem Cells

Blood cells transfusion is a widely used method for cell therapy. However, its availability for use is limited by quantity and the risk of infectious disease synchronously with blood transfusion. Fo this reason, people try to obtain more safe, sufficient and economic resource of blood. Human embryonic stem cells (hES cells) are pluripotent cells derived from the inner cell mass of blastocyst-stage human embryos, which can be maintained and expanded indefinitely as pure populations of undifferentiated cells for extended periods of time, and possessing the capacity to generate every cell type in the body. To date, many of the studies have highlighted the successes in differentiating cell populations from hES cells and in demonstrating the potential of using the model to investigate early development and generating cells for replacement therapy. hES cells now have been regarded as an alternative source for blood cells to resolve the problems exists in the transfusion medicine.However, several additional problems must be further addressed to facilitate the derivation of red blood cells from hES cells, such as the risk of mouse-related disease, high cost of hES cells expansion, low differentiation efficiency from hES cells to erythroid cells, limitedexpansion of the erythroid cells, low efficiency of erythroid progenitors enucleation to fully mature red blood cells, and the immunological rejection of the host immune system, etc.In the present study, we tried to solve two key issues that limited the large scale derivation of red blood cells from hES cells. The one was the risk of mouse-related diseaseand high cost of hES cells expansion. The other was the limited expansion of erythroid progenitors and low efficiency enucleation of erythroid progenitors. By resolving these two issues, it might be possible to produce large quantities of mature red blood cells from human ES cells much more safely and efficiently.. hES cells are typically maintained on mouse embryonic fibroblast (MEF) feeders or with MEF-conditioned medium. However, these xenosupport systems greatly limit the therapeutic applications of hES cells because of the risk of cross-transfer of animal pathogens. Recently, successful attempts have been made to use feeder free culture system to maintain hES cells. However, feeder-free cultures usually display a higher degree of spontaneous differentiation than conventional culture, requiring higher concentration of exogenous basic fibroblast growth factor (bFGF). So, for hES cells-based cell therapy it is very important to find a new method to expand hES cells in vitro.In order to avoid the animal pathogens, we had derived hFLSCs from the fetal liver of 14 weeks human embryo, which could be used as new feeder cells for hES cells culture. Our results suggested that hES cells could be maintained on hFLSCs feeder cells in prolonged culture for 10-15 passages, with the similar pluripotency, karyotypes and proliferation of hEScells MEFs.It is known that all the hESCs culture system need to supplement bFGF, but the underlying mechanisms are not fully understood. We analyzed the IGF 1 receptor (IGF1R) and FGF receptor 1 (FGFR1) expression on hFLSCs and hES cells by immunochemistryand found that IGF1R expression was exclusive to the human ES cells, whereas FGFR1 expression was restricted to surrounding hES cells derived fibroblasts and hFLSCs. Our results suggested that eventhough FGFR1 were not dominantly expressed by the pluripotent hES cells, they were very important for maintenance of hES cells.bFGF is the only cytokine that need be supplemented in hES cell culture medium. So we tried to establish a transgenic hFLSCs that stably express bFGF and use it as a new feeder cells to culture hES cells without exogenous bFGF.The bFGF recombinant lentiviral plasmid was steadily transfected into hFLSCs. The low and high bFGF-expression hFLSCs were sorted by fluorescence-activated cell sorting (FACS) according to weak and strong GFP expression. The low and high bFGF-expression hFLSCs were named"Low-bFGF-hFLSCs"and"bFGF-hFLSCs", respectively. The expression and secretion of bFGF was confirmed by RT-PCR, Western blot, and ELISA. We then used the"bFGF-hFLSCs"as feeder cells to expand hES cells in vitro. This transgenic feeder cells can maintain the properties of hES cells in prolonged culture for 10-15 passages. Proliferation and pluripotency of hES cells on bFGF-hFLSCs was similar to those on mouse feeders. Furthermore, these cells could differentiate into cell types of the three germ layers in vitro.In order to understand the mechanisms that bFGF-hFLSCs maintaining hES cells without additional bFGF, we analyzed the difference of gene expression between hFLSCs and bFGF-hFLSCs by RT-PCR and Western blot. We found that bFGF-hFLSCs expressed IGF2 more highly than hFLSCs. And some other genes such as TGF-β, FGFR1, IGF1R were also unregulated by bFGF-hFLSCs. These results suggested that that IGF2 secrected by bFGF-hFLSCs maintains the hES cells via IGF2/IGF1R axis on hES cells.In summary, bFGF-hFLSCs could function as feeder cells sustain hES cells in vitro by supplying an eligible microenvirnment free of animal feeder layers. The new feeder cells could support the pluripotency, karyotypes and proliferation of hESCs without exogenous bFGF2 in prolonged cultures as efficiently as that on MEFs. bFGF-hFLSCs could establish a paracrine signaling as being required for self-renewal and pluripotency of hESCs by producing of supportive factors, including IGF2 and TGF-βfactors.2. In vitro differentiation of hES cells to hematopoietic stem/progenitors and large scale production of red blood cells from hematopoietic stem cellsHuman erythropoiesis is a complex multistep process that involves the differentiation of early erythroid progenitors to mature erythrocytes. In order to derive red blood cells from hES cells, we separate the differentiation process into two independent parts in our experiments. We first differentiate hES cells to hematopoietic stem/progenitors cells, and then differentiate the hematopoietic progenitors to full mature red blood cells in large scale.hES cells were cultured in low cell-attachment dishes to form embryoid body (EB) which were induced into hematopoietic cells by treated by BMP4, bFGF, VEGF and SCF. Then, the expression of CD34 was examined by flow cytometry. We were able to obtain up to 10% of CD34 cells by days 14 EBs, which had the ability to form the different hematopoietic clusters in Colony-forming assays. And more important, we have demonstrated that a comparable hemangioblast population exists in the day 4 EB, which could be identified by their capacity to generate blast colonies displaying both hematopoietic and vascular potential in vitro.After derivation of hematopoietic stem/progenitors cells from hES cells, we tried to induce red blood cells from these progenitors in a large scale. We used cord blood HSCs as seed cells to derive erythroid progenitors in a high efficiency. Under optimal conditions, sustained proliferation of erythroid progenitors resulted in a more than 108-fold expansion within 50 days. And in the present methods, the erythroid progenitors were exclusively expanded, and other lineage hematopoietic cells almost disappear in the culture medium. And the induced erythroid progenitors were characterized by morphology, Wright-Giemsa stain, surface marker expression and colony-forming assays.Coculturing with stromal cell lines now have been used to encourage the differentiation of erythroid progenitors to enucleated red blood cells. As fetal liver is an important hematopoietic organ in human embryo and may have a microenvironment for terminal maturation of erythroid cells. We had derived hFLSCs from the fetal liver of 24 weeks human embryo. Then we cocultured the erythroid progenitors with the hFLSCs to mimick the marrow microenvironment in vitro to induce terminal differentiation of erythroid progenitors into fully mature. This process resulted in extrusion of the pycnotic nuclei in up to over 80% of the cells. And the cells underwent multiple maturation events, including a progressive decrease in size, increase in glycophorin A expression, and chromatin and nuclear condensation.More impotantly, almost all the induced erythroid cells expressed the adult definitiveβ-globin chain at the mRNA level, suggesting that the cells undergo definitive hematopoiesis.Since combinatorial control of transcription factors is believed to determine the molecular basis of erythroid lineage progression and globin synthesis, we further evaluated the expression of GATA-1, SCL/TAL-1, and PU-1 hematopoietic transcription factors in different time points by RT-PCR. We found that in the whole differentiation process erythroid cells expressed GATA-1 mRNA but were devoid of detectable expression of SCL/Tal-1, and PU-1 mRNAs only expressed a little in some stages. The expression profile of the erythroid hinted that the present culture system could mimick the normal erythroid cells terminal maturation to some extent, and could be used to investigate the mechanisms of erythroiesis.In summary, we first induced the hES cells to hematopoietic stem/progenitors and HA in vitro, which could supplied abundant hematopoietic stem/progenitors cells for further induction to red blood cells. And then we succeeded to produce human erythroid progenitors from cord blood HSCs in large scale. Finally, we cocultured the in vitro derived erythroid progenitor cells with hFLSCs for 10 days to induce enucleation efficiently.SummaryOur purpose in the present study was to resolve two issues which limit the derivation of human red blood cells from hES cells. For the expansion of hES cells more safe and lower cost, we established bFGF-hFLSCs as feeder cells for sustainment of hES cells in vitro free of aniamal feeder layers and exogenous bFGF. For the large scale production of erythroid progenitors and inducing it to full mature red blood cells, we established a method for ex vivo production of human erythroid progenitors in a large-scale, and by coculturing the erythroid progenitor cells with hFLSCs we could derive enucleated red blood cells in a high efficiency. By resolving these two issues, it may be possible to produce large quantities of mature red blood cells from human ES cells much more safely and efficiently. Large-scale production of cultured RBCs would have implications for gene therapy, blood transfusion. And it also could be used to analyze erythropoiesis and study important human viral or parasitic infections that target erythroid cells.