Search

Although pluripotent stem cells are well recognized as a potential source of cell therapy, it is still needed to improve efficiency to differentiate into target cell lineages. 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) β locus and successful pre-TCR signaling. Here we defined the role of P53 on lymphopoiesis from human embryonic stem cells (ESCs).Firstly we modified P53 gene of human ESC H1 by utilizing of zinc finger nuclease targeting the P53 gene, kindly provided by Sangamo BioSciences. Sequencing analysis of the P53 knockout (KO) ES cells showed the successful deletion which induced the frame shift of the downstream sequence in both of its alleles. 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).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 × 10^6 input CD34+ cells; P53 KO: 2476 cells /1 × 10^6 input CD34+ cells; FigureFigure). 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.View Large Image | Download PowerPoint Slide

Hematopoietic stem/progenitor cells (HSPCs) derived from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have potential therapeutic applications in humans. To assess the safety and efficacy of ESC/iPSC-based therapies, reliable animal models are required prior to their clinical application. The common marmoset (CM) was recently found to be a useful nonhuman primate animal model for drug development and safety assessment. However, a method for the efficient hematopoietic differentiation of CM ESCs has not been established. In this study, we developed a novel and efficient method for differentiating CM ESCs into hematopoietic cells by transiently inhibiting the phosphoinositide 3-kinase (PI3K)-Protein kinase B (AKT) pathway, a critical pathway that maintains the undifferentiated state of CM ESCs during embryoid body (EB) formation. Compared with controls, transient inhibition of the P13K-AKT pathway resulted in a threefold increase in the proportion of enriched CD34⁺ cells (p0.001) and an increase in the number of hematopoietic colonies on day 8 of CM EB cultures. Moreover, number of blast colonies, number of hematopoietic progenitor cell populations of CD34⁺CD117⁺, CD34⁺CD45⁺, and CD43⁺CD45⁺ cells, and expression of hematopoietic genes were increased by transient inhibition of the PI3K-AKT pathway. We also demonstrated that the hematopoietic progenitor cell population was increased by inhibition of PI3K in a human system. Our novel and efficient ESC differentiation method might be useful for preclinical research on human hematopoietic disorders and may be efficiently translated to human ESC/iPSC-based regenerative medicine.

Background Antitumor immunotherapy to target tumor antigens and evade immune tolerance is considered reasonable and promising. However, no combined immunotherapy has been established. We designed a phase I trial of cancer vaccine combined with chemotherapy to investigate safety as the primary endpoint, and immune responses and clinical benefits as secondary endpoints. Methods Patients positive for HLA-A2402 who showed locally advanced, metastatic, and/or recurrent gastrointestinal, lung or cervical cancer were evaluated in a phase I clinical trial of cancer vaccine and chemotherapy. Cyclophosphamide (CPM) was administered 4 days before vaccination to eliminate regulatory T cells (Treg). 18 patients were treated in cohorts of six patients, each with escalating CPM dose (150, 300 and 600 mg/m2). Five HLA-A2402-restricted tumor-associated antigen (TAA) epitope peptides from KOC1, TTK, URLC10, DEPDC1, and MPHOSPH1 were injected once a week for 4 weeks. Patients without gastrointestinal bleeding, pleural effusion or ascites also received low dose IL-2 every after vaccination. Results The treatment was tolerated well without any associated adverse events above grade 3. The study included primary disease with 9 cases of colorectal cancer, 3 cholangiocellular carcinoma, 3 lung cancer, and one each of esophageal, gastric and cervical cancer. Nine of 13 patients (69%) assessed by Elispot assay showed cytotoxic T lymphocytes (CTL) specific for TAA to more than one of five antigens after vaccination. Log-rank test among these demonstrated that CTL responses induced by vaccine contributed significantly to overall survival (MST: 9.2 vs 3.9 months, P = 0.003). In addition, a significantly broader antigenic repertoire was found in longer survivors (p = 0.033). Treg number dropped from baseline with dose just after CPM administration. This seems to reflect direct inhibition of CPM on Treg. Interestingly, reduced Treg correlated significantly with longer overall survival (P = 0.024), suggesting CPM augmented, vaccine-induced CTL responses through Treg reduction. Conclusion This phase I clinical study demonstrated promising survival associated with Treg inhibition, as well as CTL responses that warrants further clinical studies. Disclosure K. Yoshida: I am a research staff of the company, OncoTherapy Science Inc. providing the peptide vaccines used in this study. T. Tsunoda: I am the President and CEO of the company, OncoTherapy Science, Inc. providing the peptide vaccines used in this study. Y. Nakamura: I am one of the major stock holders of the company, OncoTherapy Science, Inc. K. Tani: Our department obtained the partial research funding in 2007 from the comapany, OncoTherapy Science, Inc. All other authors have declared no conflicts of interest.