ALBERT

Description: Various kinds of functional cells differentiated from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have recently been developed and expected for use in human regenerative medicine. However, the safety and efficacy of ESC/iPSC-based therapies must be carefully evaluated prior to clinical application, by using reliable animal models. The common marmoset (CM, Callithrix jacchus) is known to be a suitable preclinical model for clinical translation studies, and CM ESCs have already been established by us. Hematopoietic stem/progenitor cells (HSCs/HPCs) are one of very useful cells for transplantation therapy to treat various diseases including leukemia. However the shortage of their donors becomes a huge social problem and the expansion of HSCs/HPCs in vitro is known to be very difficult. We have previously demonstrated that CM ESCs showing indefinite self-renewal can be differentiated into hematopoietic lineages by the forced expression of hematopoietic transcription factor (TAL1/SCL). However the efficiency of their hematopoietic differentiation was quite low (less than 5%). Therefore the development of new method to promote hematopoietic differentiation of CM ESCs more efficiently is needed. To promote hematopoietic differentiation of CM ESCs, we focused on self-renewal pathway of CM ESCs and oxygen levels during EB formation. We have reported that self-renewal of CM ESCs is regulated by phosphoinositide 3-kinases (PI3Ks)-protein kinase B (AKT) pathway that is known to regulate cell cycle and cell proliferation as well as cell survival (Nii et al., 2014). On the other hand, the differentiation of mouse ESCs to hematopoietic precursors such as hemangioblasts, bipotential progenitors of endothelial and hematopoietic cells, can be enhanced by hypoxic condition (Ramírez-Bergeron et al., 2004). In addition, expansion of HSCs/HPCs can be increased by hypoxic condition in vitro (Danet et al., 2003). Thus, we hypothesized that the suppression of ESC self-renewal by the inhibition of PI3K-AKT pathway under hypoxic condition would improve hematopoietic differentiation of CM ESCs. To test our hypothesis that the inhibition of self-renewal pathway of CM ESCs could promote their hematopoietic differentiation, we treated CM ESCs with PI3K inhibitor (LY: LY294002) for the first 4 days of EB formation and examined the proportion of CD34+ cells by flow cytometric analysis, and found that the populations of CD34+ cells were significantly increased in the presence of LY. Moreover, the day8-EBs treated with LY gave rise to significantly more hematopoietic colonies than controls in colony forming unit (CFU) assay. These results indicated that hematopoietic differentiation was significantly enhanced by the inhibition of PI3K-AKT pathway in the process of EB formation. To further promote hematopoietic differentiation of CM ESCs, we conducted EB formation assay of CM ESCs and induced their differentiation into HPCs under hypoxic condition. We found that the hypoxic condition (5% O2) significantly increased the proportion of both CD34+ and CD34+/CD117+ cells in day8-EBs especially when PI3K-AKT pathway was inhibited by the LY treatment. These results were also obtained from human ESCs. In the present study, we demonstrated that transient treatment of PI3K inhibitor during EB formation under hypoxia condition promoted hematopoietic differentiation of human and CM ESCs, which might contribute to the development of the valuable experimental system using CM ESCs in order to test new strategies of human regenerative medicine. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2311 The human regenerative medicine by the transplantation of the functional cells differentiated from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) have great potential of contributing to the treatments for various diseases, and thus have attracted huge public attention. However, the risk of unwelcome tumor formation originated from transplanted cells in recipients remains to be solved. Therefore the safety and efficacy of ESC/iPSC-based therapies should be carefully evaluated using reliable animal disease models before their clinical application. Among experimental animal models, common marmoset (CM, Callithrix jacchus), one of NEW WORLD monkeys, has recently been recognized as a useful non-human primate because they are small, easy to handle, highly reproductive and genetically very similar to humans. We have continuously investigated the characteristics of ESCs and iPSCs derived from CM. Understanding the self-renewal pathways in ESCs/iPSCs is crucial for the development of improved technology to culture and differentiate them into functional cells of potential therapeutic use. It has been reported that the maintenance of self-renewal in human or mouse ESCs/iPSCs require basic fibroblast growth factor (bFGF) or leukemia Inhibitory factor (LIF) respectively, however the growth factors required for the culture of CM ESCs/iPSCs have not been clearly determined. To clarify whether LIF or bFGF is more appropriate to maintain self-renewal of CM ESCs in culture, we examined the proliferation rate of CM40, a CM ESC line, maintained in the presence or absence of LIF or bFGF. CM ESCs were passaged at a ratio of 1:3 every 3 to 4 days. We found that the number of OCT3/4+cells was significantly increased by the addition of bFGF but not of LIF compared to control (w/o cytokines). Similar results were obtained when Cj11, another CM ESC line, was used. These results indicate that bFGF is essential for culturing CM ESCs, but LIF is dispensable. It has been reported that bFGF and its downstream PI3K-AKT and MEK-ERK pathways are important for maintenance of ESCs in human. Thus we examined whether PI3K-AKT and MEK-ERK pathway play crucial roles in the maintenance of self-renewal in CM ESCs. CM40 was cultured in the medium containing bFGF in the presence of PI3K inhibitor (LY294002) or MEK inhibitor (PD0325901). We found that the percentage and number of OCT3/4+ cells were gradually decreased in the presence of LY294002 (10 μM or 20 μM), suggesting that PI3K-AKT pathway is essential for the self-renewal of CM ESCs. Furthermore, the percentage and number of OCT3/4+cells were gradually decreased by addition of PD0325901 (1 μM or 5 μM) in the course of 4 passages, indicating that MEK-ERK pathway also plays a role in the self-renewal of CM ESCs. Next we examined if inhibition of self-renewal pathway such as PI3K-AKT or MEK-ERK promote hematopoietic differentiation in CM ESCs. One of methods for inducing hematopoietic cells from ESCs is embryoid body (EB) formation which is a conventional technique frequently used for in vitro differentiation of ESCs. Thus to induce hematopoietic differentiation, we performed EB formation assay by plating single-cell suspension of CM ESCs (3 × 105 cells) in StemLine II supplemented with 50 ng/ml BMP4 and 50 ng/ml VEGF with or without 10 μM LY294002 or 5 μM PD0325901 for 2 days. Then we removed half the medium and added fresh medium with the same final concentrations of BMP4, VEGF, LY294002 and PD0325901, plus 25 ng/ml SCF, 25 ng/ml TPO and 25 ng/ml FLT3L to expand the hematopoietic progenitors. We found that addition of LY294002 or PD0325901 increased the population of cells positive for CD34, a marker for hematopoietic stem/progenitor and endothelial cells, in day4-EBs. These CD34+cells showed hematopoietic differentiation potential proved by colony forming unit (CFU) assay Taken together, inhibition of self-renewal pathway such as PI3K-AKT or MEK-ERK in CM ESCs is thought to promote their hematopoietic differentiation by EB formation. Our findings might be useful to develop a better technology of the culture and hematopoietic differentiation of CM ESCs as well as to test efficacy and safety of ESC-derived hematopoietic cells using CM disease models for the future ESC/iPSC-based human regenerative medicine. Disclosures: No relevant conflicts of interest to declare.

Description: Tumor suppressor P53 regulates multiple signaling pathways triggered by diverse cellular stresses including DNA damages, oncogenic stimulations, and hypoxic stress, resulting in cell-cycle arrest, apoptosis, and senescence. P53 signaling is also important for double-stranded DNA breaks (DSBs) induced during physiologic events, i.e., rearrangement of antigen-specific receptors. It has been reported that P53-mediated DSB checkpoint contribute to normal murine T lymphopoiesis, especially at the double-negative (DN) stage which is defined as CD4-CD8- fraction in thymus and requires rearrangements of the T cell receptor (TCR) b locus and successful pre-TCR signaling (Guidos CJ et al., Genes Dev, 1996; Jiang D et al., J Exp Med 2006; HaksMC et al., Immunity, 1999). Here we defined the role of P53 on hematopoietic development, especially lymphopoiesis, from human embryonic stem cells (ESCs). Firstly we modified P53 gene of human ESC H1 by utilizing genome editing tool of zinc finger nuclease (ZFN) targeting the 5th exon of the P53 gene, kindly provided by Sangamo BioSciences. Sequencing analysis of the P53 knockout (KO) ES cells showed the successful deletion at the 5th exon which induced the frame shift of the downstream sequence in both of its alleles. qRT-PCR showed no stable expression of full length P53 mRNA and western blot analysis of P53 phosphorylation status in P53 KO ESCs showed undetectable levels of phosphorylated or non-phosphorylated P53 proteins when cultured in the presence or absence of apoptotic signal triggered by mitomycin C (MMC). In consistent with this, P53 KO ESCs showed significant resistance to MMC-induced cell death. In addition, P53 KO ESCs lacked apoptotic stimulation-induced upregulation of P53 downstream target genes including P53 up-regulated modulator of apoptosis (PUMA). On the other hand induction of P53 target gene P21 was not observed both in H1 and P53 KO ESCs, as reported previously by other groups (Ginis I et al., Dev Biol, 2004; Barta T et al., Stem Cells, 2010; Garc'a CP et al., Stem Cell Res, 2014; World J et al., Stem Cells, 2014). We then induced hematopoietic differentiation of P53 KO ESCs through embryoid body formation. Erythroid lineage cells developed from human ESCs were significantly suppressed in the absence of P53 signaling during embryoid body maturation. Pharmacological inhibition of P53 had the same effect as genetic disruption of P53 gene. CD34+ hematopoietic precursors were isolated from embryoid bodies originated from H1 and P53 KO ECSs, plated on OP9-DL1 stromal cells, and cultured in the presence of stem cell factor (SCF), FLT3 ligand, and interleukin (IL)-7. After 3-4 weeks of culture, CD45+CD3+ T lineage cells were induced from both H1 and P53 KO ECSs-derived CD34+ cells. Among these cells, most of the cells were in CD4+CD8+ double-positive (DP) stage, with increase in the yield of DP cells in the absence of P53 signaling (H1: 343 cells/1 x 106 input CD34+ cells; P53 KO: 2476 cells/1 x 106 input CD34+ cells; Figure). Whether pharmacological inhibition of P53 had the similar effect on T lymphopoiesis as genetic disruption of P53 gene needs to be investigated furthermore. Our data indicate that P53 mediated signaling regulate in vitro early T lymphopoiesis from human pluripotent stem cells, especially at the transition from double negative into DP stage. These observations promoted us to perform high throughput transcriptome analysis including cDNA microarray analysis between early T lineage cells derived from H1 and P53 KO ESCs. Genes associated with the early T lymphopoiesis from human ESCs were identified and currently under further characterization. Disclosures No relevant conflicts of interest to declare.

Description: Hematopoietic stem cell (HSC) transplantation is the most successful cellular therapy for the malignant hematopoietic diseases such as leukemia, and early recovery of host’s hematopoiesis after HSC transplantation has eagerly been expected to reduce the regimen related toxicity for many years. For the establishment of the safer and more efficient cell source for allogeneic or autologous HSC transplantation, HSCs differentiated from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) that show indefinite proliferation in an undifferentiated state and pluripotency, are considered to be one of the best candidates. Unfortunately, despite many recent efforts, the HSC-specific differentiation from ESCs and iPSCs remains poor [Kaufman, DS et al., 2001][Ledran MH et al., 2008]. In this study, we developed the new method to differentiate HSC from non-human primate ESC/iPSC. It has been reported that common marmoset (CM), a non-human primate, is a suitable experimental animal for the preclinical studies of HSC therapy [Hibino H et al., 1999]. We have been investigated the hematopoietic differentiation of CM ESCs into HSCs, and previously reported that the induction of CD34+ cells having a blood colony forming capacity from CM ESCs were promoted by lentiviral transduction of TAL1 cDNA [Kurita R et al., 2006]. However, those CD34+ cells did not have a bone marrow reconstituting ability in irradiated NOG (NOD/Shi-scid/IL-2Rγnull) mice, suggesting that transduction of TAL1 gene was not sufficient to induce functional HSCs which have self-renewal capability and multipotency. Thus, we tried to find other hematopoietic genes being able to promote hematopoietic differetiation more efficiently than TAL1. We selected 6 genes (LYL1, HOXB4, BMI1, GATA2, c-MYB and LMO2) as candidates for factors that induce the differentiation of ESCs into HSCs, based on the previous study of hematopoietic differentiation from human and mouse ESCs. And CM ESCs (Cj11) lentivirally transduced with the respective candidate gene were processed for embryoid body (EB) formation to induce their differentiation into HSCs for 9 days. We found that lentiviral transduction of LYL1 (lymphoblastic leukemia 1), a basic helix-loop-helix transcription factor, in EBs markedly increased the proportion of cells positive for CD34 (approximately 20% of LYL1-transduced cells). RT-PCR showed that LYL1-transduced EBs expressed various hematopoietic genes, such as TAL1, RUNX1 and c-KIT. To examine whether these CD34+ cells have the ability to differentiate into hematopoietic cells in vitro, we performed colony-forming unit (CFU) assay, and found that CD34+ cells in LYL1-transduced EBs could form multi-lineage blood colonies. Furthermore the number of blood colonies originated from CD34+CD45+ cells in LYL1-transduced EBs was almost the same as that from CD34+CD45+ cells derived from CM bone marrow. These results suggested that enforced expression of LYL1 in CM ESCs promoted the emergence of HSCs by EB formation in vitro. The LYL1 was originally identified as the factor of a chromosomal translocation, resulting in T cell acute lymphoblastic leukemia [Mellentin JD et al., 1989]. The Lyl1-deficient mice display the reduction of B cells and impaired long-term hematopoietic reconstitution capacity [Capron C et al., 2006]. And, transduction of Lyl1 in mouse bone marrow cells induced the increase of HSCs and lymphocytes in vitro and in vivo [Lukov GL et al., 2011]. Therefore we hypothesized that LYL1 may play essential roles in bone marrow reconstitution by HSCs differentiated from CM ESCs. To examine this, we transplanted CD34+ cells derived from LYL1-transduced CM ESCs into bone marrow of sublethally irradiated NOG mice, and found that about 7% of CD45+ cells derived from CM ESCs were detected in peripheral blood (PB) of recipient mice at 8 weeks after transplant (n=4). Although CM CD45+ cells disappeared at 12 weeks after transplant, CD34+ cells (about 3%) were still found in bone marrow at the same time point. Given that TAL1-transduced EBs derived from CM ESCs could not reconstitute bone marrow of irradiated mice at all, LYL1 rather than TAL1 might be a more appropriate transcription factor that can give rise to CD34+ HSCs having the enhanced capability of bone marrow reconstitution from CM ESCs. We are planning to do in vivo study to prove this hypothesis in CM. Disclosures: No relevant conflicts of interest to declare.

Description: Human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), have the potential to self-renew indefinitely and differentiate into various cell types. hPSCs can differentiate into various stem or progenitor cell populations used for regenerative medicine and drug development. Newly developed genome editing technology has advanced the use of hPSCs for such purposes. However, to fully utilize hPSCs to achieve this goal, more efficient gene transfer methods under defined conditions are required. Development of efficient genome editing methods, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9), for use in hPSCs holds great promise in the fields of basic and clinical research. Among these methods, TALENs are more efficient and safer for use in hPSCs to achieve specific gene editing, as ZFNs had a low gene editing efficiency and CRISPR/Cas9 was accompanied by more severe off-target effects than TALENs. Electroporation is a widely used transfection method for hPSC genome editing; however, this method results in reduced cell viability and gene editing efficiency. In the past decade, various methods were developed for gene transfer into hPSCs; however, hPSCs form tightly packed colonies, making gene transfer difficult. In this study, we established a culture method of hPSCs at a single-cell-state to reduce cell density, and investigated gene transfection efficiency followed by gene editing efficiency. hPSCs cultured in a single-cell-state were transfected using non-liposomal transfection reagents with plasmid DNA driven by the human elongation factor 1-alpha 1 (EF1α) promoter or mRNA encoding enhanced green fluorescent protein (eGFP). The proportion of eGFP+ cells considerably increased in single-cell-state cultures (DNA: 95.80 ± 2.51%, mRNA: 99.70 ± 0.10%). Moreover, most of the cells were viable (control: 93.10 ± 0.40%, DNA: 83.40 ± 2.03%, mRNA: 86.71 ± 0.19%). The mean fluorescence intensity (MFI) was approximately three-fold higher than that in cells transfected by electroporation (electroporation (EPN): 6631 ± 992; transfection (TFN): 17933 ± 1595). eGFP expression was detected by fluorescence microscopy until day seven post-transfection. Our results also demonstrate an inverse correlation between cell density and transfection efficiency. To test whether transfection using this method affected the "stemness" of hPSCs, we examined SSEA4 and NANOG expression in eGFP-transfected cells by flow cytometry analysis. The percentage of both SSEA4+ and NANOG+ cells was greater than 90%. Moreover, transplantation of eGFP-transfected cells into immunodeficient mice led to the formation of teratomas. These results strongly suggested that single-cell-state hPSC culture improved transfection efficiency without inducing differentiation or loss of pluripotency. Moreover, we used our efficient transfection method to edit the hPSC genome using TALENs. We constructed a Platinum TALEN driven by the EF1α promoter targeting the adenomatous polyposis coli (APC) gene and analyzed the efficiency of gene editing using the Cel-1 assay. Our efficient transfection method induced mutations more efficiently than electroporation (Transfection: 11.1 ± 1.38%, Electroporation: 3.2 ± 0.89). These results showed that TALENs increased gene editing efficiency in single-cell-state hPSC cultures. Overall, our efficient hPSC transfection method using single-cell-state culture provides an excellent experimental system to investigate the full potential of hPSCs. We expect that this method may contribute to the fields of hPSC-based regenerative medicine and drug discovery. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1477 Poster Board I-500 Human embryonic stem (ES) cells differentiate into three lineages in vitro and in vivo as mouse ES cells. They are therefore highly promising source of various cells/tissues in the regenerative medicine. The current protocols, however, remain to be optimized for the induction of the cells/tissues required. We have recently reported that the lentiviral transduction of TAL1/SCL gene to ES cells derived from the common marmoset, a small nonhuman primate, enables efficient differentiation into hematopoietic progenitor cells even in the absence of stromal cells (Kurita et al. Stem Cells.24:2014-22, 2006). Such culture condition without any stromal cells is considered to facilitate clinical application of ES cell-derived cell/tissues therapy in the regenerative medicine. The present study addressed whether the strategy is also effective in human ES cells. First, we determined optimal culture conditions to induce multilineage hematopoietic differentiation in a human ES cell line, khES-1, kindly provided by Dr. Nakatsuji, Kyoto University, Japan, as assessed by the expression of Brachyury, Flk1 and CD34. We found that the addition of BMP4 and VEGF augmented hematopoietic differentiation of embryoid bodies, and determined optimal concentrations of the cytokines. We established four human ES cell lines stably expressing TAL1/SCL gene by lentiviral transduction. The TAL1/SCL transduction further increased the hematopoietic differentiation under the optimal culture condition as assessed by the expression of CD34, CD235a and CD133. We also observed increased number of hematopoietic progenitor cells derived from two of the TAL1/SCL expressing human ES cell lines by colony-forming assays. Hematopoietic differentiation of the TAL1/SCL expressing ES cells in vivo is also being investigated by transplantation into irradiated immune deficient mice. These results suggest that the combination of optimal culture conditions and lentiviral TAL1/SCL gene transduction is a highly effective strategy to obtain hematopoietic stem cells from human ES cells in the absence of any stromal cells. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2206 Recently various kinds of functional cells differentiated from embryonic stem cells and induced pluripotent stem cells (ESCs/iPSCs) are expected to be utilized for cell therapy in clinical medicine. Among the transplantable functional cells differentiated from ESCs/iPSCs, endothelial progenitor cells (EPCs) and hematopoietic stem cells (HSCs) are considered to be strong candidate cells for regenerative medicine to cure various diseases such as ischemic disease and hematopoietic malignancy. Although the transplantation of EPCs and HSCs derived from human bone marrow, mobilized peripheral blood, and umbilical cord blood is commonly conducted in clinical settings, their availability for clinical use has often been hampered by both the lack of HLA compatible donor and the insufficient number of the cells. As the in vitro expansion of EPCs and HSCs derived from above sources is very difficult using current technology, it may be easier to expand EPCs and HSCs derived from ESCs/iPSCs in vitro. Hemangioblasts have the ability to differentiate into both EPCs and HSCs. Thus the technology to differentiate hemangioblast from ESCs/iPSCs that possess indefinite proliferative capacity is strongly expected. Differentiation of ESCs/iPSCs to hemangioblasts is best exemplified in recent studies that have used two step procedures to enhance hemangioblast differentiation with embryoid body (EB) formation and blast colony forming cell (BL-CFC) assay (Lu SJ et al., Nat Methods 4: 501–509, 2007). However the efficiency of hemangioblast differentiation by this method was quite low (approximately 0.35 ± 0.01%). PI3K-AKT pathway is well known to regulate various cell functions. In ESCs, PI3K-AKT pathway plays an important role in maintaining the undifferentiated state (Armstrong L et al., Hum Mol Genet 15: 1894–1913, 2006), suggesting that inhibition of PI3K may promote the differentiation of ESCs/iPSCs. Previously, we demonstrated that common marmosets (CM) are suitable laboratory animal models for preclinical studies of hematopoietic stem cell therapies (Hibino H et al., blood 1: 2839–2848, 1999). To develop the method for the more efficient generation of hemangioblasts from ESCs/iPSCs, we promoted the hemangioblast differentiation by the inhibition of PI3K-AKT pathway with the inhibitor, LY294002. CM-ESCs (Cj11 and CM40) were differentiated by EB formation in the presence of LY294002 for 4 days, and the EBs were trypsinized, and the dissociated individual cells were processed for BL-CFC assay in the methylcellulose medium containing various cytokines without LY294002 for 7 days. The number of blast colonies found in the BL-CFC assay significantly increased (approximately 10-fold; 3.5 ± 0.3%, p 〈 0.001) with the treatment of LY294002 during EB formation compared with control. The colonies formed in the BL-CFC assay were homogeneous and looked like a tuft of grapes which is one of hemangioblast characters, and expressed hemagioblast markers (FLK1+, VE-cadherin+, CD31+ and CD45−), suggesting that the inhibition of PI3K during EB formation promoted the generation of hemangioblast-like cells from CM-ESCs. To determine endothelial potential of these hemangioblast-like cells derived from CM-ESCs, we grew them as adherent layers on gelatin-coated plates in EGM-2 medium. The adherent cells derived from hemangioblast-like cells expressed endothelial cell markers (CD31 and vWF). Next, we also examined hematopoietic potential of hemangioblast-like cells by colony forming unit (CFU) assay. Unexpectedly no colonies were formed regardless of whether LY294002 was added or not during EB formation, indicating that hemangioblast-like cells derived from CM-ESC might be endothelial progenitors rather than hemangioblasts. Our novel technology is 10-fold more efficient in inducing endothelial differentiation from ESCs than previously reported methods. It should be emphasized that these endothelial progenitors are morphologically homogenous and expressed endothelial cell markers in a defined adherent cell culture condition, suggesting that our novel technology will be useful for an efficient generation of homogeneous EPCs for future regenerative medicine against ischemic diseases. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2348 Since the successful establishment of human embryonic stem cells (ESCs) in 1998, transplantation of functional cells differentiated from ESCs to the specific impaired organ has been expected to cure its defective function [Thomson JA et al., Science 282:1145–47, 1998]. For the establishment of the regenerative medicine using ESCs, the preclinical studies utilizing animal model systems including non-human primates are essential. We have demonstrated that non-human primate of common marmoset (CM) is a suitable experimental animal for the preclinical studies of hematopoietic stem cells (HSCs) therapy [Hibino H et al., Blood 93:2839–48, 1999]. Since then we have continuously investigated the in vitro and in vivo differentiation of CM ESCs to hematopoietic cells by the exogenous hematopoietic gene transfer. In earlier study, we showed that the induction of CD34+ cells having a blood colony forming capacity from CM ESCs is promoted by lentiviral transduction of TAL1 cDNA [Kurita R et al., Stem Cells 24:2014-22,2006]. However those CD34+ cells did not have a bone marrow reconstituting ability in irradiated NOG (NOD/Shi-scid/IL-2Rγnull) mice, suggesting that transduction of TAL1 gene is not enough to induce functional HSCs which have self-renewal capability and multipotency. Thus we tried to find other hematopoietic genes being able to promote hematopoietic differetiation more efficiently than TAL1. We selected 6 genes (LYL1, HOXB4, BMI1, GATA2, c-MYB and LMO2) as candidates for factors that induce the differentiation from ESCs to HSCs, based on the comparison of gene expression level between human ESCs and HSCs by Digital Differential Display from the Uni-Gene database at the NCBI web site (http://www.ncbi.nlm.nih.gov/UniGene/). Then, we transduced the respective candidate gene in CM ESCs (Cj11), and performed embryoid body (EB) formation assay to induce their differentiation to HSCs for 9 days. We found that lentiviral transduction of LYL1, a basic helix-loop-helix transcription factor, in EBs derived from Cj11, one of CM ESC lines, markedly increased the number of cells positive for CD34, a marker for hematopoietic stem/progenitors. The lymphoblastic leukemia 1 (LYL1) was originally identified as the factor of a chromosomal translocation, resulting in T cell acute lymphoblastic leukemia [Mellentin JD et al., Cell 58:77-83.1989]. These class II bHLH transcription factors regulate gene expression by binding to target gene sequences as heterodimers with E-proteins, in association with Gata1 and Gata2 [Goldfarb AN et al., Blood 85:465-71.1995][Hofmann T et al., Oncogene 13:617-24.1996][Hsu HL et al., Proc Natl Acad Sci USA 91:5947-51.1994]. The Lyl1-deficient mice display the reduction of B cells and impaired long-term hematopoietic reconstitution capacity [Capron C et al., Blood 107:4678-4686. 2006]. And, overexpression of Lyl1 in mouse bone marrow cells induced the increase of HSCs, HPCs and lymphocytes in vitro and in vivo [Lukov GL et al., Leuk Res 35:405-12. 2011]. These information indicate that LYL1 plays important roles in hematopoietic differentiation in primate animals including human and common marmoset. To examine whether overexpression of LYL1 in EBs can promote hematopoietic differentiation in vitro we performed colony-forming unit (CFU) assay, and found that LYL1-overexpressing EBs showed the formation of multi-lineage blood cells consisting of erythroid cells, granulocytes and macrophages. Next, we analyzed gene expression level by RT-PCR, and found that the transduction of LYL1 induced the expression of various hematopoietic genes. These results suggested that the overexpression of LYL1 can promote the differentiation of CM ESCs to HSCs in vitro. Furthermore we found that the combined overexpression of TAL1 and LYL1 could enhance the differentiation of CD34+ cells from CM ESCs than the respective overexrpession of TAL1 or LYL1. Collectively, our novel technology to differentiate hematopoietic cells from ESCs by the transduction of specific transcription factors is novel, and might be applicable to expand human hematopoietic stem/progenitor cells in vitro for future regenerative medicine to cure human hematopoietic cell dyscrasias. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3122 Pioneering work by Yamanaka's group has demonstrated that mammalian somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by the ectopic expression of Oct3/4 (also called Pou5f1), Klf4, Sox2 with or without c-Myc (OKSM) [Takahashi, K., and Yamanaka, S. (2006). Cell 126, 663–676, Takahashi et al. (2007). Cell 131, 861–872, Nakagawa et al., (2008) Nat Biotechnol 26, 739–740.]. The iPSCs are similar to embryonic stem cells (ESCs) in their morphology, and their abilities of differentiation to three germ layers in vitro and in vivo. Comparing with ESCs, iPSCs have less concerns regarding the ethically controversial limitation or immune rejection. And iPSCs have great potential of clinical application for personalized stem cell-based therapies. However, the efficiency of iPSC generation is quite low (0.01∼1%), which is considered to be an impediment of the clinical utility of iPSCs. Although the molecular mechanisms of reprogramming process during the generation of iPSCs have not been fully understood, senescence and apoptosis induced by the ectopic expression of OKSM have been considered to be the major road block in the process of iPSC generation. Therefore the new strategy of iPSC generation which can alleviate such hurdles during the reprogramming process should be developed. The signaling pathway mediated by the activation of phosphoinositide 3-kinase (PI3K) has been shown to play crucial roles for various aspects of cell biology including cell survival, proliferation, migration, metabolism and vesicular trafficking in variety of cell types. In addition previous studies have shown that PI3K pathway is an important regulator of ES cell cycle by promoting G0 to G1 transition and ES cell proliferation. In this study we hypothesized that activation of PI3K pathway might facilitate the reprogramming process for iPSC generation. To test this hypothesis we first induced the expression of OKSM in mouse embryonic fibroblasts (MEFs) lacking Pten which is a negative regulator of PI3K pathway. About 7 days after the retroviral transduction of OKSM into Pten−/− MEFs, ES like round-shaped colonies were found (Figure 1). Twenty-two days after the retroviral transduction, the efficiency of iPSC generation was examined by the number of colonies having alkaline phosphatase (AP) activity. We found that significantly higher number of AP+ colonies were formed from Pten−/− MEFs (74 ± 15) compared to Pten+/− or wild-type MEFs (17 ± 5 and 16 ± 2, respectively). Moreover the number of SSEA1+ colonies induced by the expression of OKSM significantly increased from Pten−/− MEFs (103 ± 2) in comparison to Pten+/− or wild-type MEFs (40 ± 9 and 21 ± 9, respectively). Similar results were obtained when OKS were transduced without c-Myc. Next we tested the hematopoietic differentiation in vitro with the reprogrammed MEFs by using embryoid bodies (EBs) formation assay. After the following colony forming unit (CFU) assay, we observed the higher percentage of hematopoietic cells from reprogrammed Pten−/− MEF group comparing with controls, indicating that reprogrammed Pten−/− MEFs had the higher potential to differentiate into hematopoietic cells. Continuous activation of PI3K pathway may cause the transformation of the cells; therefore to efficiently and safely generate iPSCs transient activation of the pathway combined with the transduction of OKSM would be desirable. To establish transient activation of the PI3K pathway we used Pten inhibitor, Dipotassium Bisperoxo(5-hydroxypyridine-2-carboxyl) oxovanadate (V) (bpV), during the generation of iPSCs. The efficiency of AP+ cell-generation from MEFs by transducing Yamanaka factors with bpV was approximately 9%. This was 2 times higher than that by transducing Yamanaka factors without bpV in our hand. These AP+ cells showed a normal karyotype and an ability of differentiation into three germ layers in immunodeficient mice. Therefore ectopic expression of OKSM in the presence of Pten inhibitor can establish the efficient generation of iPSCs. Overall, transient activation of PI3K pathway by Pten inhibition offers a highly efficient method generating iPSCs, and might contribute to the establishment of human regenerative medicine with iPSCs. Disclosures: No relevant conflicts of interest to declare.