ALBERT

Description: Journal of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.7b00168

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: 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.