OP9 Bone Marrow Stromal Cells Research Articles

Unraveling Tumorigenicity in iPSC-Derived Immune Cells: The Impact of Chromosomal Abnormalities and Proliferative Intermediates

Response to tumor cells by the immune system can determine the course of a cancer. This guiding principle and the dynamic, adaptable nature of our immune system underlies the drive to generate immune cells for cancer immunotherapy. Induced pluripotent stem cells (iPSCs) provide an unlimited resource for the generation of immune cells such as T cells and dendritic cells (DCs) for cancer immunotherapy. However, the use of iPSC-derived immune cells in clinical applications has been limited due to concerns of safety. In vitro passage of iPSCs can introduce selective pressures that promote the accumulation of mutations that confer a growth advantage. Over several passages, this can lead to the acquisition of chromosomal abnormalities. Moreover, differentiation protocols of varying efficiency permit the presence of immature cells, contaminating the differentiated cell population. However, the impact of chromosomal abnormalities and intermediate products with proliferation potential on tumorigenicity of iPSC-derived immune cells remains unclear. Here, we investigated the tumorigenic potential of iPSC-derived T cells and DCs in immunocompetent and immunodeficient mice. Pmel-1 T-cell receptor (TCR) transgenic mouse T cells were reprogrammed using Sendai virus vectors carrying Oct3/4, Sox2, Klf4 and cMyc. While our iPSCs were phenotypically normal, even at early passage numbers, various chromosomal abnormalities were found in the resulting cell population. These abnormalities included trisomy 8 and 11 and loss of X chromosome with high frequency. Furthermore, within a population of iPSCs determined to be karyotypically normal by G-banding, unbalanced translocations were found by spectral karyotyping. These findings are consistent with those reported in the literature and highlight the prevalence of chromosomal abnormalities in iPSC populations. As iPSCs can harbor chromosomal abnormalities that are undetectable by standard methods, we assessed the tumorigenic potential of iPSC-derived cells, similar to what may be used in clinical applications. Mouse iPSCs were differentiated into CD8αβ T cells by co-culture with murine OP9 bone marrow stromal cells expressing the Notch ligand Delta-like 1 (OP9-DL1) and were enriched by CD8 positive selection. Activated iPSC-derived T cells were adoptively transferred to immunocompetent C57BL/6 and immunodeficient NOD/SCID, IL-2 receptor γ chain knockout mice. Immunodeficient but not immunocompetent mice developed hepatic tumors. Reverse transcription (RT)-PCR identified Pme1-l TCR gene in tumor cells, confirming tumor origin. Moreover, histology of liver tumors is suggestive of lymphoma or lymphosarcoma. Within the population of CD8+ iPSC-derived cells, a small subpopulation co-expressing CD8 and SSEA-1 was present. Sorting the cells by SSEA-1 positivity eliminated tumor formation in vivo. Mouse iPSCs were also differentiated into DCs by co-culture with OP9-DL1 cells and granulocyte macrophage colony-stimulating factor. Despite similar use of iPSCs with chromosomal abnormalities and prolonged passaging in vitro, SSEA-1+ cells were not identified when they were differentiated to DCs, and iPSC-derived DCs did not form tumors in immunodeficient mice. Taken together, this study revealed the potential risk of tumorigenicity of iPSC-derived immune cells in our preclinical model. The presence of chromosomal abnormalities did not definitively determine risk of tumorigenesis upon transplantation. The relationship between SSEA-1 positivity and tumor formation suggests contaminating cells may carry tumorigenic characteristics. Further work is required to develop differentiation methods that generate pure populations, free of tumorigenic intermediates, to potentiate the clinical use of iPSC-derived immunotherapy.

Multi-Faceted Effects of ST6Gal1 Expression on Precursor B-Lineage Acute Lymphoblastic Leukemia.

Normal early human B-cell development from lymphoid progenitors in the bone marrow depends on instructions from elements in that microenvironment that include stromal cells and factors secreted by these cells including the extracellular matrix. Glycosylation is thought to play a key role in such interactions. The sialyltransferase ST6Gal1, with high expression in specific hematopoietic cell types, is the only enzyme thought to catalyze the terminal addition of sialic acids in an α2-6-linkage to galactose on N-glycans in such cells. Expression of ST6Gal1 increases as B cells undergo normal B-lineage differentiation. B-cell precursor acute lymphoblastic leukemias (BCP-ALLs) with differentiation arrest at various stages of early B-cell development have widely different expression levels of ST6GAL1 at diagnosis, with high ST6Gal1 in some but not in other relapses. We analyzed the consequences of increasing ST6Gal1 expression in a diagnosis sample using lentiviral transduction. NSG mice transplanted with these BCP-ALL cells were monitored for survival. Compared to mice transplanted with leukemia cells expressing original ST6Gal1 levels, increased ST6Gal1 expression was associated with significantly reduced survival. A cohort of mice was also treated for 7 weeks with vincristine chemotherapy to induce remission and then allowed to relapse. Upon vincristine discontinuation, relapse was detected in both groups, but mice transplanted with ST6Gal1 overexpressing BCP-ALL cells had an increased leukemia burden and shorter survival than controls. The BCP-ALL cells with higher ST6Gal1 were more resistant to long-term vincristine treatment in an ex vivo tissue co-culture model with OP9 bone marrow stromal cells. Gene expression analysis using RNA-seq showed a surprisingly large number of genes with significantly differential expression, of which approximately 60% increased mRNAs, in the ST6Gal1 overexpressing BCP-ALL cells. Pathways significantly downregulated included those involved in immune cell migration. However, ST6Gal1 knockdown cells also showed increased insensitivity to chemotherapy. Our combined results point to a context-dependent effect of ST6Gal1 expression on BCP-ALL cells, which is discussed within the framework of its activity as an enzyme with many N-linked glycoprotein substrates.

Murine Oncostatin M Has Opposing Effects on the Proliferation of OP9 Bone Marrow Stromal Cells and NIH/3T3 Fibroblasts Signaling through the OSMR.

The IL-6 family cytokine Oncostatin M (OSM) is involved in cell development, growth, hematopoiesis, inflammation, and cancer. Intriguingly, OSM has proliferative and antiproliferative effects depending on the target cell. The molecular mechanisms underlying these opposing effects are not fully understood. Previously, we found OSM upregulation in different myeloproliferative syndromes. However, OSM receptor (OSMR) expression was detected on stromal cells but not the malignant cells themselves. In the present study, we, therefore, investigated the effect of murine OSM (mOSM) on proliferation in stromal and fibroblast cell lines. We found that mOSM impairs the proliferation of bone marrow (BM) stromal cells, whereas fibroblasts responded to mOSM with increased proliferation. When we set out to reveal the mechanisms underlying these opposing effects, we detected increased expression of the OSM receptors OSMR and LIFR in stromal cells. Interestingly, Osmr knockdown and Lifr overexpression attenuated the OSM-mediated effect on proliferation in both cell lines indicating that mOSM affected the proliferation signaling mainly through the OSMR. Furthermore, mOSM induced activation of the JAK-STAT, PI3K-AKT, and MAPK-ERK pathways in OP9 and NIH/3T3 cells with differences in total protein levels between the two cell lines. Our findings offer new insights into the regulation of proliferation by mOSM.

FGF7 Supports Hematopoietic Stem and Progenitor Cells and Niche-Dependent Myeloblastoma Cells Via Autocrine Action On Bone Marrow Stromal Cells In Vitro

Some fibroblast growth factor (FGF)/FGF receptor (FGFR) systems, namely FGF1 and FGF2 which are produced mainly by megakaryocytes, and their cognate receptors expressed on either hematopoietic stem or progenitor cells (HSPCs) or bone marrow (BM) stromal cells, are reported to support HSPCs under stressed conditions. In this study, we show that mesenchymal cell-derived FGF7 (also called keratinocyte growth factor or KGF) may be a novel niche factor for HSPC support and niche-dependent leukemia-initiating cell growth. We previously reported that mouse embryonic fibroblasts (MEFs) have characteristics that mimic BM stromal cells and possess an ability of HSPCs support in vitro, and that this ability is profoundly attenuated in MEFs deficient for the MED1 subunit of the Mediator transcriptional master coregulator complex, subcomplex of RNA polymerase II holoenzyme. We found through the microarray analyses that the FGF7 expression (both at mRNA and protein levels) was specifically and profoundly attenuated in Med1-/- MEFs compared with Med1+/+ MEFs. When normal mouse BM cells were cocultured with mitomycin C-treated Med1+/+ MEFs or BM stromal cells (OP-9 or MS-5) in the presence of anti-FGF7 antibody, the DNA synthesis and resultant growth of BM cells, and the number of long-term culture-initiating cells (LTC-ICs), were significantly suppressed. However, the addition of anti-FGF7 antibody did not enhance cell death in BM cells. In contrast, the addition of recombinant FGF7 to the coculture of BM cells with mitomycin C-treated Med1-/- MEFs robustly upregulated the DNA synthesis and growth of BM cells, and the number of LTC-ICs. The Ph+ myeloblastoma cells MB-1, originally isolated from a patient with blast crisis chronic myeloid leukemia, is niche-dependent myeloblastic cells that require mesenchymal stromal cells for survival and growth. During coculture typically with OP-9 BM stromal cells, these MB-1 cells form cobblestone areas, which most probably reconstitute a stochastic model of leukemic stem or initiating cells in vitro. We next analyzed the role of FGF7 in support of MB-1 cells for growth and survival during coculture. Indeed, the addition of anti-FGF7 antibody to the coculture with mitomycin C-treated BM stromal cells (either OP-9 or MS-5) substantially attenuated DNA synthesis and resultant proliferation of stromal cell-dependent myeloblastoma cells MB1. Notably, cobblestone formation (i.e., signature of stem cell phenotype) of MB-1 cells was significantly attenuated when anti-FGF7 antibody was added to the coculture with OP-9 cells. However, the addition of anti-FGF7 antibody did not elicit cell death of MB-1 cells. Thus, FGF7 appears to promote both proliferation and maintenance of leukemia stem or initiating cell feature of MB-1 cells. While neither FGF7 nor its cognate receptor FGFR2IIIb mRNAs were detectable in BM cells, the mesenchymal cells including Med1+/+ MEFs and (OP-9 and MS-5) BM stromal cells expressed them. The expression levels of FGFR2IIIb were comparable between Med1+/+ and Med1-/- MEFs, and BM stromal cells. Therefore, we hypothesized that FGFR2IIIb on these mesenchymal stromal cells might be functional and possibly elicit secretion of niche signal(s) secondarily in response to the downstream target gene(s) of FGFR2IIIb, followed by subsequent support of HSPCs and leukemic cells. To prove that FGF7 is functional in these cells, we examined response of the downstream targets of FGFR2IIIb in these cells, including phosphorylation of FRS2 (the immediate downstream target of FGFR2IIIb tyrosine kinase), phosphorylation of MAP kinases (the intermediate hub of various intracellular signals), and mRNA expression of immediate early response genes including c-Jun, c-Fos and c-Myc. Indeed, exogenously added recombinant FGF7 elicited phosphorylation of both FRS2 and MAP kinases within 10 min, and full induction of these immediate early response genes within 30 min, similarly in both Med1+/+ and Med1-/- MEFs. The OP-9 and MS-5 BM stromal cells also exhibited similar responses to exogenously added FGF7. These results indicate that FGFR2IIIb expressed on mesenchymal stromal cells are functional, and that FGF7 and FGFR2IIIb produced by these cells constitute an autocrine (or paracrine) loop. Taken together, we propose that FGF7 produced by BM stromal cells supports HSPCs and leukemic progenitor cells indirectly via FGFR2IIIb expressed on the BM stromal cells. Disclosures:No relevant conflicts of interest to declare.

MGP Interacts with BMP-4 and BMP-2 and Supports Normal and Malignant Hematopoietic Stem/Progenitor Cells

The general transcriptional coregulatory complex Mediator, subcomplex of the RNA polymerase II holoenzyme, is the endpoint convergence of a variety of intracellular signals, and initiates transcription through recruitment of general initiation factors and formation of a functional preinitiation complex. Among the Mediator subunits, MED1 is crucial for hematopoiesis: it is known to be employed in transcription of genes involved in hematopoietic niche function, as well as in differentiation of hematopoietic precursor cells (e.g., RARα- and VDR-mediated myelomonopoiesis, GATA1-mediated erythromegakaryopoiesis, and iNKT cell development). Among the attenuated genes in Med1-/- mesenchymal stromal cells was matrix Gla protein (MGP), modulator of the BMP-SMAD signals. As MGP is abundantly expressed in bone tissues and reportedly interacts with niche factors BMP-4 and BMP-2, we hypothesized that MGP might modulate niche function in hematopoiesis through the interaction with these BMPs. We tested this possibility by an in vitro niche model composed of a coculture of bone marrow (BM) mesenchymal stromal cells and BM hematopoietic cells. When mouse BM hematopoietic cells were cocultured with OP-9 or MS-5 mouse BM stromal cells in the presence of the blocking antibody against MGP, the growth and DNA synthesis of BM hematopoietic cells were attenuated and the number of long-term culture-initiating cells (LTC-ICs), which corresponded to the supported hematopoietic stem/progenitor cells (HSPCs), decreased. We next asked if MGP also functioned as a niche factor for malignant HSPCs, To this end, we utilized MB-1 cells, blast crisis CML-derived myeloblastic leukemia cells. MB-1 cells are dependent on cocultured BM stromal cells and possess characteristics of leukemia stem cells (LSCs). When MB-1 cells were cocultured with OP-9 or MS-5 BM stromal cells in the presence of the anti-MGP antibody, the number of MB-1 cells, as well as the number of cobblestone formation, i.e., LSC nature, of MB-1 cells, was profoundly attenuated. Therefore, MGP appears to have a trophic effect on hematopoietic cells and to support normal and malignant HSPCs in our niche model. MGP was, both at mRNA and protein levels, expressed abundantly in BM stromal cells but scarcely in normal hematopoietic cells. However, MB-1 leukemia cells (presumably ectopically) expressed a meaningfully high level of MGP. During the coculture of normal BM hematopoietic cells with OP-9 or MS-5 BM stromal cells, MGP was induced prominently and transiently after one day. MGP, expressed in MS-5 or OP-9 BM stromal cells, was likewise transiently induced after 1 day of culture with BM hematopoietic cells that were physically dissociated from these BM stromal cells by the transwell apparatus, indicating that secreted humoral factor(s) induced the MGP expression in the BM stromal cells. As for a malignant situation, the expressions of MGP in both MB-1 leukemia cells and OP-9 or MS-5 BM stromal cells were fluctuated in an oscillated days period. Since the support of HSPCs required their association with BM stromal cells, the induced MGP expression was apparently insufficient for HSPCs support, and the action of induced MGP may be indirect. Therefore, we next asked if BMP-4 and BMP-2, which reportedly associate with MGP, were also induced by the coculture and were employed in HSPCs support jointly with MGP. Indeed, during the coculture, BMP-4 was rapidly and transiently induced within a few days, simultaneously with MGP, followed by a subsequent and sustained induction of BMP-2 that lasted for over a week. However, neither BMP-4 nor BMP-2 was produced by BM stromal cells when cultured with dissociated BM hematopoietic cells using the transwell apparatus. Therefore, the induction of BMP2/BMP4 was dependent on physical association of hematopoietic cells and stromal cells. GST-pulldown assays and mammalian two-hybrid assays confirmed that MGP specifically interacted with both BMP-4 and BMP-2. To elucidate the mechanism of action of the blocking antibody against MGP, we tested if the antibody inhibited the interaction between MGP and BMP-4. Indeed, serial GST pulldown assays disclosed that the addition of the antibody specifically abolished the interaction between GST-MGP and BMP-4. These results indicate that MGP might act as a niche factor for normal and malignant HSPCs, at least in part, through interacting with, and modulating the action of, BMP-4 and BMP-2. DisclosuresNo relevant conflicts of interest to declare.

Periostin Supports Hematopoietic Stem/Progenitor Cells and Niche-Dependent Myeloblastoma Cells In Vitro

Periostin (POSTN), the fasciclin family extracellular matrix protein also known as osteoblast-specific factor 2 (OSF-2), was previously reported to be required for optimal B lymphopoiesis in vitro. Now, our study first demonstrates the proof that POSTN might be a bona fide niche factor for both normal and malignant myelopoiesis, indicating that it is a niche molecule for hematopoietic stem cells and diverse hematopoietic precursor cells. The Mediator, composed of about 31 subunits, is a master transcriptional coregulator complex that is essential for global transcription governed by RNA polymerase II. Among the Mediator subunits, MED1 acts as a specific coactivator for activators that include nuclear receptors and GATA1. We previously reported that Med1−/− mouse embryonic fibroblasts (MEFs) have a decreased capability to support hematopoietic stem/progenitor cells (HSPCs) relative to wild-type MEFs in vitro, and that the attenuated expression of full-length osteopontin and FGF7 in Med1−/− MEFs is responsible for the observed phenotype. The microarray analyses, showing that the expression of POSTN was also suppressed in Med1−/− MEFs, prompted us to study the role for POSTN in support of both normal and malignant HSPCs in our in vitro niche model. When bone marrow (BM) cells were cocultured with mitomycin C-treated Med1+/+ MEFs, or OP-9 or MS-5 BM stromal cells, in the presence of anti-POSTN blocking antibody, the mitogenicity and growth of BM cells were attenuated. The number of long-term culture-initiating cells (LTC-ICs), i.e., number of both granulo-monocytic and erythroid colonies, was also decreased. When BM cells were cocultured with Med1-/- MEFs in the presence of recombinant POSTN, the mitogenicity and growth of BM cells and the number of LTC-ICs were restored. These results suggest that POSTN mediates mitogenicity of BM cells and HSPCs support. The MB-1 myeloblastoma cell line, originally established from a patient with myeloid crisis chronic myeloid leukemia, is a mesenchymal stromal cell-dependent cell line. These cells are unique in that they grow by forming cobblestone areas in the presence of niche cells but die of apoptosis when detached from stromal cells, thus faithfully conforming to a stochastic model of leukemic stem cells in vitro. Intriguingly, antibody-mediated blockage of stromal cells-derived POSTN markedly reduced the mitogenicity and growth, as well as the cobblestone formation, a leukemic stem cell feature, of MB-1 myeloblastoma cells. Therefore, it appears that niche cell-derived POSTN supports niche-dependent MB-1 myeloblastoma cells. While POSTN was expressed both in BM cells and variably in different BM stromal cells, expression in the latter cells was markedly increased by tactile interaction with hematopoietic cells. Specifically, POSTN was robustly induced 6 hours after BM stromal cells were cocultured with BM cells or MB-1 myeloblastoma cells, and the induction sustained for as long as 24 hours. However, POSTN expression was not enhanced when BM cells were cocultured but physically separated from MS-5 or OP-9 cells using transwell culture wells. Therefore, the major source of POSTN in the coculture appears to be the BM stromal cells associated with hematopoietic cells. The receptor for POSTN, integrin αvβ3, was expressed abundantly in BM stromal cells. Although β3 mRNA was especially prominent in both BM cells and MB-1 cells, in accordance with a previous report that integrin β3/CD61 marks HSPCs, western blot analysis showed that αv and β3 expression levels were below the detection level on BM cells. Hence, integrin αvβ3 is scarce on BM cells compared to BM stromal cells, although it does not exclude the possibility that functional integrin αvβ3 might be enriched on HSPCs as suggested previously. When an excess amount of exogenous POSTN was added to MS-5 or OP-9 BM stromal cells after 24-h serum starvation, FAK (the immediate target of integrin αvβ3) and MAP kinases ERK1/ERK2 (the intermediate hub of various intracellular signals) were robustly phosphorylated as early as 10 min, and the phosphorylation was sustained for over 60 min. Thus, POSTN effectively activates integrin αvβ3 and subsequent intracellular signaling in BM stromal cells. These results suggest that stromal cell POSTN supports both normal HSPCs and leukemia-initiating cells in vitro, at least in part, indirectly by acting on stromal cells in an autocrine or paracrine manner. DisclosuresNo relevant conflicts of interest to declare.

Efficient Induction of Myleoid and Lymphoid Hematopoiesis from Nonhuman Primate Pluripotent Stem Cells Using GSK3b Inhibitor

Induced pluripotent stem cells (iPSCs) have created novel opportunities for the scalable manufacture of blood products for clinical use. Recent advances in hematopoietic differentiation from human pluripotent stem cells have brought the clinical translation of iPSC-derived blood products close to reality and evoke an urgent need for preclinical evaluation of their efficacy, safety, and immunogenicity in large animal models. Due to substantial similarities with humans, outcomes of cellular therapies in nonhuman primate (NHP) models can be readily extrapolated to clinical setting. However, the use of this model is hampered by limited availability of clinically relevant NHP-iPSCs and low efficiency of myeloid and lymphoid differentiation from them. Here, we describe generation of clinically relevant transgene-free iPSCs from different NHP species, including rhesus, Chinese and Mauritian cynomolgus monkeys, and demonstrate that GSK3β inhibition is essential to induce rapid and efficient differentiation of the NHP-iPSCs into multipotential CD34+CD45+CD90+CD38-CD45RA-hematopoietic progenitors in coculture with OP9 bone marrow stromal cells. NHP-iPSC-derived hematopoietic progenitors were capable of differentiating further into mature erythroid cells, megakaryocytes, granulocytes, monocytes/macrophages following exposure to hematopoietic cytokines promoting development corresponding lineages. In addition, iPSC-derived CD34+CD45+ cells generated CD4+CD8+ T lymphoid cells and CD159a+ NK cells when cultured on OP9 cells expressing DLL4. To confirm T cell development, we analyzed the genomic DNA of the hematopoietic cells from OP9-DLL4 cultures for the presence of T cell receptor (TCR) rearrangements. This analysis demonstrated the presence of multiple PCR products of random V-J and D-J rearrangements at the β locus and V-J rearrangements at the γ locus, indicative of a polyclonal T lineage repertoire. To evaluate feasibility of using NHP model for evaluation of safety and clinical efficacy of iPSC-derived blood products, we generated iPSCs from Mauritian cynomolgus macaque and marked them with GFP. GFP-iPSCs were used to generate CD34+CD45+ multipotential hematopoietic progenitors using multiple rounds of differentiation followed by freezing. Subsequently, iPSC donor animal was subjected to nonmyeloablative conditioning with fludarabine (50 mg/m(2)/day for 3 days) together with low-level total body irradiation (2 Gy) and transfused with 30x106/kg autologous iPSC-derived CD34+CD45+ cells. Transfusion of iPSC-derived CD34+CD45+ cells well tolerated and animal recovered without any adverse events. By flow cytometry, GFP-marked cells (up to 1%) were detected in peripheral blood on days 20-30 post-transfusion, predominately in granulocyte, monocyte and platelet compartments. Although, GFP-positive cells mostly disappeared in circulation one month after transfusion, GFP-positive cells were detected in circulation for more than 90 days by genomic PCR. During 6 month follow-up period no signs of myelo- or lympho-proliferative disorder were detected. Overall, these studies lay the foundation for advancing a NHP model for preclinical testing of iPSC-based therapies for blood diseases. DisclosuresSlukvin:Cynata: Consultancy, Equity Ownership, Research Funding.

Differentiation of in vitro induced human pluripotent stem cells into hematopoietic stem/progenitor cells

This study was aimed to explore the differentiation of in vitro induced human pluripotent stem cells (iPSC) into hematopoietic stem progenitor cells. The human iPSC were induced to differentiate into hematopoietic stem/progenitor cell by co-culturing with OP9 bone marrow stromal cells. The expression of hematopoietic stem/progenitor cell surface markers were detected by flow cytometry. The regulation gene expressions of iPSC and hematopoietic stem/progenitor cells were measured by real-time PCR. The CD34(+) hematopoietic stem/progenitor cells were isolated by using immunomagnetic beads, and were used for colony formation assay. The results showed that after iPSC were co-cultured with OP9 cells for 4 days, the morphological changes of iPSC could be observed. Hematopoietic stem/progenitor cell surface markers CD34 and CD43 could be detected by flow cytometry after differentiation. The pluripotent marker gene OCT4 expression gradually decreased and blood-related transcription factor Gata-2 expression gradually increased, while Runx-1 expression was wavily changed, CD34 expression gradually increased. The erythroid colony(CFU-E), granulocyte colony(CFU-G), megakaryocytic colony(CFU-M), granulocyte-megakaryocytic colony(CFU-GM), and mixed colony(CFU-GEMM) were obtained after cultures for 14 d. It is concluded that the human iPSC cells can be induced to differentiate into hematopoietic stem/progenitor cells in vitro by co-culture with OP9 cells.

Dual signals of TCR and Notch promote antigen-specific T cell development from pluripotent stem cells (P4370)

Abstract The pivot in conducting a successful adoptive T-cell immunotherapy for cancer is the obtaining of a large number of naïve phenotyped, stem-cell like tumor antigen specific T cells. Previously we have reported that both conventional and antigen-specific T cells could be generated from induced pluripotent stem (iPS) cells by different methods. Here, we observed that dual signals of TCR and Notch played a significant role in antigen-specific T lineage differentiation from pluripotent stem cells. We used a murine MHC-I restricted, tyrosinase-related protein 2 (TRP2) specific TCR and Notch ligands Delta-like (DLL) 1 and 4 as a model system for inducing iPS cells to differentiate into melanoma-specific CD8+ T cells. Murine iPS cells were retrovirally transduced with TRP2-specific TCR (iPS-TRP2), and then co-cultured on OP9 bone marrow stromal cells overexpressing both DLL1 and DLL4 ligands (OP9-DL1/DL4) in the presence of Flt3L and IL-7. This approach has a better effect in terms of directing an in vitro iPS-TRP2 differentiation towards TRP2-specific CTLs. In addition, adoptive transfer of in vitro developed iPS-TRP2-derived precursor T cells into C57BL/6J mice bearing xenografted B16 melanoma significantly increased anti-tumor immunosurveillance and protected animal from tumor challenge. These data suggest that signals from TCR and Notch can cooperatively promote antigen-specific T cell development from pluripotent stem cells.

Differentiation Efficiency of Induced Pluripotent Stem Cells Depends on the Number of Reprogramming Factors

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by retroviral overexpression of the transcription factors Oct4, Sox2, Klf4, and c-Myc holds great promise for the development of personalized cell replacement therapies. In an attempt to minimize the risk for chromosomal disruption and to simplify reprogramming, several studies demonstrated that a reduced set of reprogramming factors is sufficient to generate iPSC, albeit at lower efficiency. To elucidate the influence of factor reduction on subsequent differentiation, we compared the efficiency of neuronal differentiation in iPSC generated from postnatal murine neural stem cells with either one (Oct4; iPSC(1F-NSC) ), two (Oct4, Klf4; iPSC(2F-NSC) ), or all four factors (iPSC(4F-NSC) ) with those of embryonic stem cells (ESCs) and iPSC produced from fibroblasts with all four factors (iPSC(4F-MEF) ). After 2 weeks of coculture with PA6 stromal cells, neuronal differentiation of iPSC(1F-NSC) and iPSC(2F-NSC) was less efficient compared with iPSC(4F-NSC) and ESC, yielding lower proportions of colonies that stained positive for early and late neuronal markers. Electrophysiological analyses after 4 weeks of differentiation identified functional maturity in neurons differentiated from ESC, iPSC(2F-NSC) , iPSC(4F-NSC) , and iPSC(4F-MEF) but not in those from iPSC(1F-NSC) . Similar results were obtained after hematoendothelial differentiation on OP9 bone marrow stromal cells, where factor-reduced iPSC generated lower proportions of colonies with hematoendothelial progenitors than colonies of ESC, iPSC(4F-NSC) , and iPSC(4F-MEF) . We conclude that a reduction of reprogramming factors does not only reduce reprogramming efficiency but may also worsen subsequent differentiation and hinder future application of iPSC in cell replacement therapies.

Sox4 Is Essential for Initiation and Progression of Acute Lymphoblastic Leukemia Induced by P190-BCR-ABL Transformation

Abstract 564Sox4 is an Sry-related HMG box transcription factor that plays critical roles in development. Sox4-null mice die at embryonic day 14 due to cardiac malformation. Recent studies have suggested that SOX4 may contribute to tumorigenesis, based on the fact that SOX4 is upregulated in many human tumor types, including brain, bladder, prostate, colon, and lung cancers. In addition, increased expression of Sox4 induced by retroviral insertional mutagenesis in mice has been shown to be associated with leukemia and lymphoma. We also found that SOX4 is highly expressed in human acute lymphoblastic leukemia (ALL) cells. We therefore set out in this study to determine the function of Sox4 and its underlying mechanisms in ALL. To overcome embryonic lethality caused by conventional Sox4 inactivation, we used Sox4 conditional null mice (Sox4fl/fl) to establish Sox4-null ALL cell lines. First, we obtained pure B220+ pro-B cells by co-culturing Sox4fl/fl fetal liver cells and OP-9 bone marrow stromal cells in the presence of IL-7. Subsequently, pro-B cells were transformed with p190-BCR-ABL and Sox4 inactivation was induced with Cre recombinase. We found that Sox4−/− pro-B transformed cells exhibited a significantly slower proliferation rate than Sox4+/− pro-B cells. Notably, cell cycle analysis indicated a lower percentage of S phase cells in Sox4−/− pro-B transformed cells and these cells yielded much fewer colonies than transformed Sox4+/− cells in colony formation assay. We further labeled the transformed cells with a luciferase reporter and injected them intravenously into sublethally irradiated NOD/SCID mice. Bioluminescence imaging showed that mice injected with Sox4−/− cells developed leukemia significantly more slowly and survive longer than mice injected with Sox4+/− cells. In conclusion, our data show that Sox4 significantly contributes to initiation and progression of leukemia from p190-BCR-ABL-transformed ALL cells in the mouse and thereby strongly support the notion that SOX4 overexpression critically contributes to acute lymphoblastic leukemia development in humans. Disclosures:No relevant conflicts of interest to declare.

Development of thymic NK cells from double negative 1 thymocyte precursors

AbstractThe differentiation of natural killer (NK) cells and a subpopulation of NK cells which requires an intact thymus, that is, thymic NK cells, is poorly understood. Previous in vitro studies indicate that double negative (CD4−CD8−, DN) thymocytes can develop into cells with NK cell markers, but these cells have not been well characterized. Herein, we generated and characterized NK cells differentiating from thymic DN precursors. Sorted DN1 (CD44+CD25−) CD122−NK1.1− thymocytes from Rag1−/− mice were adoptively transferred into Rag1−/−Ly5.1 congenic mice. After intrathymic injection, donor-derived cells phenotypically resembling thymic NK cells were found. To further study their differentiation, we seeded sorted DN1 CD122−NK1.1− thymocytes on irradiated OP9 bone marrow stromal cells with IL-15, IL-7, Flt3L, and stem cell factor. NK1.1+ cells emerged after 7 days. In vitro differentiated NK cells acquired markers associated with immature bone marrow–derived NK cells, but also expressed CD127, which is typically found on thymic NK cells. Furthermore, we found that in vitro cells generated from thymic precursors secreted cytokines when stimulated and degranulated on target exposure. Together, these data indicate that functional thymic NK cells can develop from a DN1 progenitor cell population.

Protein kinase G type-I phosphorylates c-Src at serine-17 and promotes cell survival, proliferation and attachment in human mesothelioma and non-small cell lung cancer cells

Background Previously, we demonstrated that protein kinase G type-Ia (PKG-Ia) plays an important role in promoting cell survival in neural cells (N1E-115 neuroblastoma and NG10815 neuroblastoma-glioma hybrid cells) and significantly contributes to the serine-155 phosphorylation of BAD, an apoptosis-regulating protein [1]. We also found that PKGIa promotes cell survival and proliferation in mouse OP9 bone marrow stromal cells [2] and human ovarian cancer cells [3] [determined by using both pharmacological inhibitors (ODQ, DT-2 and DT-3) and gene knockdown (siRNA) to reduce PKG-Ia/b activity]. In the case of ovarian cancer cells, which predominantly express the PKG-Ia isoform, the pro-growth and pro-survival effects involved a novel interaction between PKG-Ia and the oncogenic protein c-Src [3]. For example, intracellular activation of PKG-Ia, assessed by VASP serine-239 phosphorylation, was dependent on c-Src-catalyzed tyrosine phosphorylation of PKG-Ia (i.e. VASP phosphorylation was blocked by Src inhibitors, SKI-1 or SU6656) and the intracellular activation of c-Src was (somehow) dependent on the kinase activity and expression levels of PKG-Ia (i.e. c-Src activation was decreased by DT-2 or siRNA-induced knockdown of PKG-Ia).

Programming of antigen-specific CTLs from induced pluripotent stem cells for tumor protection (156.1)

Abstract Generation of highly reactive antigen(Ag)-specific CTLs derived from induced pluripotent stem(iPS) cells can provide an unlimited source of functional CTLs that can be used for adoptive immunotherapy. The iPS cell-derived T cells have the advantages of averting potential immune rejection, as well as circumventing ethical and practical problems associated with other types of stem cells. Previously we demonstrated that iPS cells are able to differentiate into progenitor T cells in vitro by the stimulation of Notch ligand Delta-like 1 (DL1) over-expressed on OP9 bone marrow stromal cells. Full maturation of iPS cells was observed upon adoptive transfer into RAG1-deficient mice. Here we show that iPS cells differentiate into functional CTLs in vivo after over-expressing MHC I-restricted Ag-specific T cell receptors (TCRs). In this study, murine iPS cells are genetically modified with OVA specific, MHC-I restricted TCR (OT-I) by retroviral transduction. We demonstrate that after adoptive transfer into recipient mice, the majority of OT-I/iPS cells undergo differentiation into CD8+ CTL. In vivo developed TCR-transduced iPS cells are able to respond to ex vivo peptide stimulation and secrete IL-2 and IFN-γ. Moreover, adoptive transfer of TCR-transduced iPS cells results in the infiltration of OVA-reactive CTLs into tumor tissues and protection of animals from tumor challenge. Our results provide a novel approach for generating Ag-specific T lymphocytes for T cell-based therapies.

Hematopoietic differentiation and production of mature myeloid cells from human pluripotent stem cells.

In this paper, we describe a protocol for hematopoietic differentiation of human pluripotent stem cells (hPSCs) and generation of mature myeloid cells from hPSCs through expansion and differentiation of hPSC-derived lin(-)CD34(+)CD43(+)CD45(+) multipotent progenitors. The protocol comprises three major steps: (i) induction of hematopoietic differentiation by coculture of hPSCs with OP9 bone marrow stromal cells; (ii) short-term expansion of multipotent myeloid progenitors with a high dose of granulocyte-macrophage colony-stimulating factor; and (iii) directed differentiation of myeloid progenitors into neutrophils, eosinophils, dendritic cells, Langerhans cells, macrophages and osteoclasts. The generation of multipotent hematopoietic progenitors from hPSCs requires 9 d of culture and an additional 2 d to expand myeloid progenitors. Differentiation of myeloid progenitors into mature myeloid cells requires an additional 5-19 d of culture with cytokines, depending on the cell type.

Essential roles of the nitric oxide (no)/cGMP/protein kinase G type‐Iα (PKG‐Iα) signaling pathway and the atrial natriuretic peptide (ANP)/cGMP/PKG‐Iα autocrine loop in promoting proliferation and cell survival of OP9 bone marrow stromal cells

Inappropriate signaling conditions within bone marrow stromal cells (BMSCs) can lead to loss of BMSC survival, contributing to the loss of a proper micro-environmental niche for hematopoietic stem cells (HSCs), ultimately causing bone marrow failure. In the present study, we investigated the novel role of endogenous atrial natriuretic peptide (ANP) and the nitric oxide (NO)/cGMP/protein kinase G type-Iα (PKG-Iα) signaling pathway in regulating BMSC survival and proliferation, using the OP9 BMSC cell line commonly used for facilitating the differentiation of HSCs. Using an ANP-receptor blocker, endogenously produced ANP was found to promote cell proliferation and prevent apoptosis. NO donor SNAP (S-nitroso-N-acetylpenicillamine) at low concentrations (10 and 50 µM), which would moderately stimulate PKG activity, protected these BMSCs against spontaneous apoptosis. YC-1, a soluble guanylyl cyclase (sGC) activator, decreased the levels of apoptosis, similar to the cytoprotective effects of low-level NO. ODQ (1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one), which blocks endogenous NO-induced activation of sGC and thus lowers endogenous cGMP/PKG activity, significantly elevated apoptotic levels by 2.5- and three-fold. Pre-incubation with 8-Bromo-cGMP or ANP, which bypass the ODQ block, almost completely prevented the ODQ-induced apoptosis. A highly-specific PKG inhibitor, DT-3, at 20, and 30 µM, caused 1.5- and two-fold increases in apoptosis, respectively. ODQ and DT-3 also decreased BMSCs proliferation and colony formation. Small Interfering RNA gene knockdown of PKG-Iα increased apoptosis and decreased proliferation in BMSCs. The data suggest that basal NO/cGMP/PKG-Iα activity and autocrine ANP/cGMP/PKG-Iα are necessary for preserving OP9 cell survival and promoting cell proliferation and migration.

Hematoendothelial Differentiation of Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) represent a unique population of cells capable of self-renewal and differentiation into all types of somatic cells, including hematopoietic and endothelial cells. Since the pattern of hematopoietic and endothelial development observed in the embryo can be reproduced using ESCs differentiated in culture, hESCs can be used as a model for studies of specification and diversification of hematoendothelial progenitors. In addition, hESCs can be seen as a scalable source of hematopoietic and endothelial cells for in vitro studies. This unit describes a method for efficient differentiation of hESCs into hematopoietic progenitors and endothelial cells through coculture with mouse OP9 bone marrow stromal cells, as well as an approach for their analysis and isolation. Support protocols are provided for culture of mouse embryonic fibroblasts, evaluation of hematopoietic and endothelial differentiation by flow cytometry and colony-forming assay, removal of OP9 cells, and propagation of hESC-derived endothelial cells. Curr. Protoc.

Adoptive transfer of in vitro generated T cell precursors improves T cell reconstitution and mediates graft-versus-tumor activity without graft-versus-host disease in allogeneic hematopoietic stem cell transplantation recipients

Immunoincompetence after allogeneic hematopoietic stem cell transplantation (HSCT) particularly affects the T cell lineage resulting in significant morbidity and mortality from opportunistic infections. Recent studies have shown that murine T cells and their precursors can be generated from hematopoietic stem cells (HSC) in vitro using OP9 bone marrow stromal cells (H2Kk/H2Kb) expressing the Notch 1 ligand Delta-like 1 and growth factors. In this study we determined the effects of adoptively transferred in vitro generated T cell precursors on T cell reconstitution after allogeneic HSCT. We selected HSC (Lin-Sca-1hi c-kit+) from mouse bone marrow (BM) of various strains and cultured these cells on a monolayer of OP9-DL1 cells in the presence of growth factors. T lineage cell development proved to be strain independent and is therefore not MHC restricted in this culture system. C57BL/6 dervied HSC expanded 2000–5000-fold within 3–4 weeks and consisted of 90–95% CD4-CD8-double negative (DN) T cell precursors after 16–28 days of culture. We infused these cells (8 ×106) with T cell depleted (TCD) BM (5 × 106) or purified HSC into allogeneic recipients using minor antigen mismatched and MHC class I/II mismatched transplant models. Progeny of OP9-DL1 derived T cell precursors were found in the thymus and the periphery significantly improving thymic cellularity, and thymic and splenic donor T cell chimerism. Combination of T cell precursor administration and pre-conditioning with keratinocyte growth factor (KGF) further improved thymic engraftment of OP9-DL1 derived T cell precursors. T cell receptor repertoire and proliferative response to foreign antigen of OP9-DL1 derived mature T cells were intact. Moreover, Th1-type cytokine secretion of OP9-DL1 derived splenic T cells after stimulation with PMA/ionomycin was better than that of BM or HSC derived donor T cells. Progeny of OP9-DL1-derived T cell precursors remained detectable for at least 60 days after transplant with intact cytokine production. Administration of in vitro generated T cell precursors did not induce GVHD but mediated significant graft-versus-tumor (GVT) activity (determined by in vivo bioluminescence imaging) resulting in a subsequent significant survival benefit. We conclude that the adoptive transfer of OP9-DL1 derived T cell precursors significantly enhances donor T cell reconstitution in allogeneic HSCT recipients and results in GVT activity in the absence of GVHD.

Adoptive Transfer of In Vitro Generated T Cell Precursors Enhances Donor T Cell Reconstitution and Graft-Versus-Tumor Activity in Allogeneic Hematopoietic Stem Cell Transplantation Recipients.

Allogeneic hematopoietic stem cell transplantation (HSCT) is associated with a varying period of immunoincompetence that particularly affects he T cell lineage resulting in significant morbidity and mortality from opportunistic infections. Recent studies have shown that murine T cells and their precursors can be generated from hematopoietic stem cells (HSC) in vitro using a OP9-DL1 coculture system consisting of OP9 bone marrow stromal cells expressing the Notch 1 ligand Delta-like 1 and growth factors (interleukin 7 and fms-like tyrosine kinase-3 ligand). In this study we determined the effects of adoptively transferred in vitro generated T cell precursors on T cell reconstitution after allogeneic HSCT. We selected HSC (Lin- Sca-1hi c-kithi) from bone marrow (BM) of C57BL/6 mice and cultured these cells on a monolayer of OP9-DL1 cells in the presence of growth factors. These HSC expanded 2,000–5,000-fold within 3–4 weeks and consisted of >95% CD4-CD8-double negative (DN) T cell precursors after 16–28 days of culture. We infused these cells (8x106) with T cell depleted (TCD) BM (5x106) or purified HSC into allogeneic recipients using minor antigen mismatched and MHC class I/II mismatched transplant models. Control mice received TCD BM or purified HSC only. Progeny of OP9-DL1 derived T cell precursors were found in thymus and spleen increasing thymic cellularity and significantly improving thymic and splenic donor T cell chimerism. This effect was even more pronounced when purified HSC instead of whole BM were used as allograft. T cell receptor repertoire and proliferative response to foreign antigen (determined by third party MLR) of in vivo differentiated OP9-DL1 derived mature T cells were intact. Administration of in vitro generated T cell precursors did not induce graft-versus-host disease (GVHD) but mediated significant graft-versus-tumor (GVT) activity (determined by in vivo bioluminescence imaging) resulting in a subsequent significant survival benefit. This advantage was associated with better cytokine responses (IL-2, INF-g, TNF-a) in T cells originating from OP9-DL1 derived T cell precursors compared to BM donor derived T cells. We conclude that the adoptive transfer of OP9-DL1 derived T cell precursors significantly enhances post-transplant T cell reconstitution and GVT activity in the absence GVHD.

Human embryonic stem cell–derived CD34+ cells: efficient production in the coculture with OP9 stromal cells and analysis of lymphohematopoietic potential

AbstractEmbryonic stem (ES) cells have the potential to serve as an alternative source of hematopoietic precursors for transplantation and for the study of hematopoietic cell development. Using coculture of human ES (hES) cells with OP9 bone marrow stromal cells, we were able to obtain up to 20% of CD34+ cells and isolate up to 107 CD34+ cells with more than 95% purity from a similar number of initially plated hES cells after 8 to 9 days of culture. The hES cell–derived CD34+ cells were highly enriched in colony-forming cells, cells expressing hematopoiesis-associated genes GATA-1, GATA-2, SCL/TAL1, and Flk-1, and retained clonogenic potential after in vitro expansion. CD34+ cells displayed the phenotype of primitive hematopoietic progenitors as defined by co-expression of CD90, CD117, and CD164, along with a lack of CD38 expression and contained aldehyde dehydrogenase–positive cells as well as cells with verapamil-sensitive ability to efflux rhodamine 123. When cultured on MS-5 stromal cells in the presence of stem cell factor, Flt3-L, interleukin 7 (IL-7), and IL-3, isolated CD34+ cells differentiated into lymphoid (B and natural killer cells) as well as myeloid (macrophages and granulocytes) lineages. These data indicate that CD34+ cells generated through hES/OP9 coculture display several features of definitive hematopoietic stem cells.