Human-induced pluripotent stem cells from blood cells of healthy donors and patients with acquired blood disorders

Abstract Human induced pluripotent stem (iPS) cells derived from somatic cells hold promise to develop novel patient-specific cell therapies and research models for inherited and acquired diseases. We and others previously reprogrammed human adherent cells, such as postnatal fibroblasts to iPS cells, which resemble adherent embryonic stem cells. Here we report derivation of iPS cells from postnatal human blood cells and the potential of these pluripotent cells for disease modeling. Multiple human iPS cell lines were generated from previously frozen cord blood or adult CD34+ cells of healthy donors, and could be redirected to hematopoietic differentiation. Multiple iPS cell lines were also generated from peripheral blood CD34+ cells of 2 patients with myeloproliferative disorders (MPDs) who acquired the JAK2-V617F somatic mutation in their blood cells. The MPD-derived iPS cells containing the mutation appeared normal in phenotypes, karyotype, and pluripotency. After directed hematopoietic differentiation, the MPD-iPS cell-derived hematopoietic progenitor (CD34+CD45+) cells showed the increased erythropoiesis and gene expression of specific genes, recapitulating features of the primary CD34+ cells of the corresponding patient from whom the iPS cells were derived. These iPS cells provide a renewable cell source and a prospective hematopoiesis model for investigating MPD pathogenesis.

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Hematopoiesis of Human Induced Pluripotent Stem Cells Derived From Blood Cells of Healthy Donors and Patients with the JAK2-V617F Somatic Mutation.

Blood ◽

10.1182/blood.v114.22.812.812 ◽

2009 ◽

Vol 114 (22) ◽

pp. 812-812

Author(s):

Zhaohui Ye ◽

Huichun Zhan ◽

Prashant Mali ◽

Sarah Dowey ◽

Donna Williams ◽

...

Keyword(s):

Cell Lines ◽

Somatic Mutation ◽

Blood Cells ◽

Jak2 V617f ◽

Myeloproliferative Disorders ◽

Ips Cells ◽

Hematopoietic Differentiation ◽

Ips Cell ◽

Cd34 Cells ◽

Hes Cells

Abstract Abstract 812 Human induced pluripotent stem (iPS) cells derived from somatic cells hold promise to develop novel patient-specific cell therapies and research models for inherited and acquired diseases. We and others previously reprogrammed human adherent cells such as postnatal fibroblasts to iPS cells that resemble adherent human embryonic stem (hES) cells. It is also highly desirable to reprogram blood cells that are easily accessible and less exposed to environmental mutagens. The large numbers of umbilical cord blood (CB) cells that are collected and stored in multiple cell banks are examples that could be used as a source of either autologous or allogeneic but histo-compatible iPS cell lines. As in vitro expansion of hematopoietic stem/progenitor cells from CB and adult sources remains a challenge, unlimited expansion of derived iPS cells in combination with further optimized hematopoietic differentiation methods should provide a vital alternative to amplify histo-compatible blood stem cells for blood/BM transplantation purposes. More critically, the ability to reprogram blood cells is essential if one wishes to generate iPS cells containing somatic mutations that are restricted to the blood cells and found in acquired hematological disorders, such as myeloproliferative disorders (MPDs), in order to investigate their pathogenesis. The BCR/ABL-negative MPDs, which include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), are a heterogeneous group of diseases characterized by increased proliferation of erythroid, megakaryocytic and myeloid lineages alone or in combination. The acquired common somatic mutation JAK2-V617F is present in >95% of PV, and ∼50% of ET and PMF patients. The discovery of this mutation in 2005 has significantly improved our understanding of MPD mechanisms and has intensified the search for drug(s) that may effectively inhibit this aberrant kinase activation. However, new disease models are needed to answer questions it raised such as how gene dosages of JAK2-V617F and other pre-disposing mutations affect the MPD pathogenesis. Here we report the derivation of iPS cells from postnatal human blood cells and the potential of these pluripotent cells for hematopoietic differentiation and disease modeling. Multiple human iPS cell lines were generated from previously frozen cord blood, adult peripheral blood and marrow CD34+ cells of healthy donors. The hematopoietic differentiation potential of these human iPS cells was examined by an improved method of EB formation and differentiation under a feeder- and serum-free condition. After two weeks of treatment, the cells were harvested and analyzed by both hematopoietic colony-forming assays and FACS for the presence of hematopoietic markers. Both myeloid and erythroid colonies were detected as we have observed using hES cells. By FACS analysis, CD45+ (27%-64%) and CD43+ (36%-60%) hematopoietic cells co-expressing undetectable to intermediate levels of CD34 marker were also observed. Multiple iPS cell lines were also generated from peripheral blood CD34+ cells of two patients with myeloproliferative disorders (MPDs) who acquired the JAK2-V617F somatic mutation in their blood cells. The MPD-derived iPS cells containing the mutation appeared normal in phenotypes, karyotype and pluripotency. To determine if these MPD iPS cell lines could be used as a model to study abnormal human hematopoiesis, we used the same serum-free differentiation protocol to direct them into hematopoietic lineages. Similar to the increased erythropoiesis of hematopoietic progenitor (CD34+) cells isolated from PV patients, including one subject whose blood-derived iPS cells were used in this study, re-differentiated hematopoietic progenitor (CD34+CD45+) cells from the PV-iPS cells showed enhanced erythropoiesis as compared to those from the iPS cells derived from normal CD34+ cells. They also showed a gene expression pattern similar to the primary CD34+ cells from the PV patient. These iPS cells thus provide a renewable cell source and a prospective model for investigating MPD pathogenesis. In combination with the gene targeting technology we recently described, human iPS cell lines derived from patients and subsequent hematopoietic differentiation technologies provide a novel model for investigations of various blood diseases with either acquired or inherited mutations. Disclosures: No relevant conflicts of interest to declare.

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Hematopoietic Differentiation of Human Induced Pluripotent Stem Cells

Blood ◽

10.1182/blood.v112.11.731.731 ◽

2008 ◽

Vol 112 (11) ◽

pp. 731-731

Author(s):

Kyung-Dal Choi ◽

Junying Yu ◽

Kimberly Smuga-Otto ◽

Jessica Dias ◽

Giorgia Salvagiotto ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Differential Expression ◽

Pluripotent Stem Cells ◽

Blood Cells ◽

Hematopoietic Cells ◽

Patient Specific ◽

Hematopoietic Differentiation ◽

Differentiation Potential ◽

Induced Pluripotent

Abstract Induced pluripotent stem cells (iPSCs) provide an unprecedented opportunity for modeling of human diseases in vitro as well as for developing novel approaches for regenerative therapy based on immunologically compatible cells. In the present study, we employed an OP9 differentiation system to characterize the hematopoietic differentiation potential of seven human iPSC lines obtained from human fetal, neonatal, and adult fibroblasts through reprogramming with POU5F1, SOX2, NANOG, and LIN28 and compared it with the differentiation potential of five human embryonic stem cell lines (hESC; H1, H7, H9, H13, and H14). Similar to hESCs, all iPSCs in coculture with OP9 generated all types of colony forming cells (CFCs) as well as CD34+ cells that can be separated into distinct subsets based on differential expression of CD43 and CD31. CD34+CD31+CD43− cells obtained from all iPSCs expressed molecules present on endothelial cells and readily formed a monolayer when placed in endothelial conditions, while hematopoietic CFC potential was restricted to CD43+ cells. iPSC-derived CD43+ cells could be separated into three major subsets based on differential expression of CD235a/CD41a and CD45: CD235a+CD41a+/− (erythro-megakaryocytic progenitors), and lin-CD34+CD43+CD45− (multipotent), and lin-CD34+CD43+CD45+ (myeloid-skewed) primitive hematopoietic cells. Both subsets of primitive hematopoietic cells expressed genes associated with myeloid and lymphoid development, although myeloid genes were upregulated in CD45+ cells, which are skewed toward myeloid differentiation. Cytogenetic analysis demonstrated that iPSCs and derived from them CD43+ cells maintained normal karyotype. In addition short tandem repeat analysis of CFCs generated from IMR90-1 cells has been performed to confirm that blood cells are in fact derived from reprogrammed IMR90 cells, and not from contaminating hESCs. While we observed some variations in the efficiency of hematopoietic differentiation between different iPSCs, the pattern of differentiation was very similar in all seven tested iPSC and five hESC lines. Using different cytokine combinations and culture conditions we were able to expand iPSC-derived myeloid progenitors and induce their differentiation toward red blood cells, neutrophils, eosinophils, macrophages, ostoeclasts, dendritic and Langerhans cells. Although several issues remain to be resolved before iPSC-derived blood cells can be administered to humans for therapeutic purposes, patient-specific iPSCs can already be used for characterization of mechanisms of blood diseases and to identify molecules that can correct affected genetic networks.

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Targeted Gene Correction of Glanzmann Thrombasthenia Induced Pluripotent Stem Cells Restores Surface Expression and Fibrinogen Binding of Integrin αIIbβ3,

Blood ◽

10.1182/blood.v118.21.4173.4173 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4173-4173

Author(s):

Spencer Sullivan ◽

Jason A. Mills ◽

Li Zhai ◽

Prasuna Paluru ◽

Guohua Zhao ◽

...

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Cell Lines ◽

Surface Expression ◽

Ips Cells ◽

Ips Cell ◽

Glanzmann Thrombasthenia ◽

Integrin Αiibβ3 ◽

Fibrinogen Binding ◽

Induced Pluripotent

Abstract Abstract 4173 Glanzmann Thrombasthenia (GT) is a rare, autosomal recessive disorder resulting from an absence of functional platelet integrin αIIbβ3, leading to impaired platelet aggregation and clinically presenting with severe bleeding. It is a model of an inherited platelet disorder that might benefit from corrective gene therapy. Treatment options for GT are limited and largely supportive. They include anti-fibrinolytics, activated factor VII, platelet transfusions, and bone marrow transplantation. Recent gene therapy research in a canine model for GT demonstrated that lentiviral transduction of mobilized hematopoietic stem cells could restore 6% αIIbβ3 receptors in thrombasthenic canine platelets relative to wild type (WT) canine platelets. As an alternative gene therapy strategy, we generated induced pluripotent stem (iPS) cell lines from the peripheral blood of two patients with GT and examined whether a megakaryocyte-specific promoter driving αIIb cDNA expression within the AAVS1 safe harbor locus could ameliorate the GT phenotype in iPS cell-derived megakaryocytes. Patient 1 is a compound heterozygote for αIIb with the following two missense mutations: exon 2 c.331T>C (p.L100P) and exon 5 c.607G>A (p.S192N). Patient 2 is homozygous for a c.818G>A (p.G273D) mutation adjacent to the first calcium-binding domain of αIIb, leading to impaired intracellular transport of αIIbβ3. Both patients express <5% αIIbβ3 on the surface of their platelets. Peripheral blood mononuclear cells from both GT patients and WT controls were efficiently reprogrammed to pluripotency using a doxycycline-inducible polycistronic lentivirus containing OCT4, KLF4, SOX2, and CMYC. Transgene constructs using a murine GPIbα promoter driving either a green fluorescent protein (GFP) reporter or αIIb cDNA were inserted into a gene-targeting vector specific for the first intron of AAVS1, a locus amenable to gene targeting and resistant to transgene silencing in human iPS cells. The GPIbα-driven GFP transgene was efficiently targeted into AAVS1 in WT iPS cells using zinc finger nuclease-mediated homologous recombination, as was the αIIb construct into GT iPS cell lines. PCR and Southern blot analyses confirmed single, non-random, transgene integrations. The iPS cells were differentiated into megakaryocytes using a feeder-free/serum-free adherent monolayer protocol and analyzed by flow cytometry. GFP, along with endogenous CD41 (αIIb), was initially expressed in primitive WT hematopoietic progenitor cells. GFP expression was lost in erythrocytes and myeloid cells, but maintained in CD41+/CD42+ megakaryocytes, demonstrating that this transgenic construct mirrors endogenous CD41 expression. The GT phenotype was confirmed in megakaryocytes derived from patient iPS cells, showing loss of αIIbβ3 expression. When compared to WT iPS cell-derived megakaryocytes, gene-corrected GT iPS cell-derived megakaryocytes showed >50% and >70% αIIbβ3 surface expression for patients 1 and 2, respectively. Both patients' iPS cell-derived megakaryocytes also demonstrated fibrinogen binding upon thrombin activation. This is the first report of the generation and genetic correction of iPS cell lines from patients with a disease affecting platelet function. These findings suggest that this GPIbα-promoter construct targeted to the AAVS1 locus drives megakaryocyte-specific expression at a therapeutically significant level, which offers the possibility of correcting severe inherited platelet disorders beginning with iPS cells derived from these affected individuals. Disclosures: Lambert: Cangene: Honoraria.

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UM171 Regulates the Hematopoietic Differentiation of Human Acquired Aplastic Anemia-Derived Induced Pluripotent Stem Cells

Blood ◽

10.1182/blood-2019-127466 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2500-2500

Author(s):

Tellechea Maria Florencia ◽

Flavia S. Donaires ◽

Tiago C. Silva ◽

Lilian F. Moreira ◽

Yordanka Armenteros ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Pluripotent Stem Cells ◽

Patient Specific ◽

Hematopoietic Differentiation ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Induced Pluripotent

Aplastic anemia (AA) is characterized by a hypoplastic bone marrow associated with low peripheral blood counts. In acquired cases, the immune system promotes hematopoietic stem and progenitor cell (HSPC) depletion by the action of several pro-inflammatory Th1 cytokines. The current treatment options for severe cases consist of sibling-matched allogeneic hematopoietic stem cell transplantation (HSCT) and immunosuppressive therapy (IST) with anti-thymocyte globulin, cyclosporine, and eltrombopag. However, most patients are not eligible for HSCT and, although about 85% of patients respond to IST with eltrombopag, a proportion of patients eventually relapse, requiring further therapies. Failure to respond adequately to immunosuppression may be attributed to the scarcity of HSPCs at the time of diagnosis. Induced pluripotent stem cells (iPSCs) are potentially an alternative source of patient-specific hematopoietic cells. Patient-specific HSPCs derived from in vitro iPSC differentiation may serve as a tool to study the disease as well as a source of hematopoietic tissue for cell therapies. The pyrimidoindole molecule UM171 induces ex vivo expansion of HSCs of human cord and peripheral blood and bone marrow, but the pathways modulated by this molecule are not well understood. Here we evaluated the hematopoietic differentiation potential of iPSCs obtained from patients with acquired AA. We further determined the effects of UM171 on this differentiation process. First, we derived iPSCs from 3 patients with acquired AA after treatment (1 female; average age, 31 years; 2 partial responders, 1 complete responder) and 3 healthy subjects (3 females; average age, 61 years) and induced differentiation in vitro through the embryoid body system in cell feeder and serum-free medium supplemented with cytokines. The hematopoietic differentiation of healthy-iPSCs yielded 19% ± 8.1% (mean ± SEM) of CD34+cells after 16 days in culture, in contrast with 11% ± 4.9% of CD34+cells obtained from the differentiation of AA-iPSCs, which corresponds to a 1.7-fold reduction in CD34+cell yield. The total number of erythroid and myeloid CFUs was lower in the AA-iPSC group as compared to healthy-iPSCs (12±4.2 vs.24±7.2; respectively; p<0.03). These findings suggest that erythroid-derived AA-iPSC have an intrinsic defect in hematopoietic differentiation. Next, we tested whether UM171 modulated hematopoietic differentiation of AA-iPSCs. We found that UM171 significantly stimulated the differentiation of both healthy and AA-iPSCs. In the healthy-iPSC group, the percentage of CD34+cells was 1.9-fold higher when treated with UM171 compared to controls treated with DMSO (37% ± 7.8% vs.19% ± 8.1%; respectively; p<0.03) and in AA-iPSCs the increase was 3.9-fold (45% ± 11% vs. 11% ± 4.9%; p<0.07). The clonogenic capacity of progenitors to produce erythroid and myeloid colonies also was augmented in both groups in comparison to DMSO (28±11 vs. 23±7.2) for healthy-iPSCs and for AA-iPSCs (23±8.5 vs. 12±4.2, p<0.06). We then investigated the molecular pathways influenced by UM171. The transcriptional profile of differentiated CD34+cells showed that UM171 up-regulated genes involved in early hematopoiesis from mesoderm (BRACHYURY and MIXL1) and primitive streak specification (APELA and APLNR), to hemangioblasts and primitive hematopoietic progenitor commitment (TDGF1, SOX17, and KLF5). We also observed the up-regulation of pro-inflammatory NF-kB activators (MAP4K1, ZAP70, and CARD11) and the anti-inflammatory gene PROCR, a marker of cultured HSCs and an NF-kB inhibitor. This balanced network has been previously suggested to be modulated by UM171 (Chagraoui et. al. Cell Stem Cell 2019). Taken together, our results showed that acquired AA-iPSCs may have intrinsic defects that impair hematopoietic differentiation in vitro. This defect may be atavic to the cell or, alternatively, the consequence of epigenetic changes in erythroid precursors provoked by the immune attack. In addition, our findings demonstrate that UM171 significantly stimulate the hematopoietic differentiation of AA-iPSCs and identified a novel molecular mechanism for UM171 as an enhancer of early hematopoietic development programs. These observations may be valuable for improving the achievement of de novo hematopoietic cells. Disclosures No relevant conflicts of interest to declare.

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Reduced Production of Mature Neutrophils From Induced Pluripotent Stem Cells Derived From a Severe Congenital Neutropenia Patient with HAX1 Gene Deficiency

Blood ◽

10.1182/blood.v118.21.2402.2402 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2402-2402 ◽

Cited By ~ 1

Author(s):

Tatsuya Morishima ◽

Ken-ichiro Watanabe ◽

Akira Niwa ◽

Takayuki Tanaka ◽

Katsutsugu Umeda ◽

...

Keyword(s):

Cell Lines ◽

Normal Control ◽

Annexin V ◽

Ips Cells ◽

Patient Specific ◽

Es Cell ◽

Severe Congenital Neutropenia ◽

Congenital Neutropenia ◽

Ips Cell ◽

Induced Pluripotent

Abstract Abstract 2402 Induced pluripotent stem (iPS) cells are reprogrammed somatic cells with embryonic stem (ES) cell-like characteristics. As iPS cells can be generated from somatic cells of patients with a certain disease, they are expected to be a novel model to study pathogenesis of various diseases. Recently, we established a neutrophil differentiation system from human iPS cells (Morishima T, et al. J Cell Physiol. 2011). In an attempt to apply the system to investigate pathophysiology of neutrophil-affected disorders, we generated iPS cells from a severe congenital neutropenia (SCN) patient with HAX1 gene deficiency. The patient was an 11-year-old boy with severe congenital neutropenia as well as developmental delay and epilepsy. DNA sequence analysis revealed HAX1 gene mutation in exon 2 (Matsubara K, et al. Haematologica. 2007). Four iPS cell lines were generated from skin fibroblasts of the patient by retroviral overexpression of the three or four transcription factors Oct3/4, Sox2, and Klf4, with or without c-Myc. These patient-derived iPS cell lines showed human ES cell like morphology and could be maintained under human ES cell culture condition. They also expressed typical human ES cell markers and were capable of differentiating into the cell lineages and tissues representing three germ layers by teratoma formation in vivo. These cells had normal karyotype and short tandem repeat analysis indicated that they were derived from parental skin fibroblast. DNA sequencing analysis of the iPS cell lines identified the same mutation carried in the parental skin fibroblasts, thus confirmed that we had established the HAX1 deficiency patient-specific iPS cells (HAX1-iPSCs). Next these HAX1-iPSCs and the healthy-person derived iPS cells were differentiated into neutrophils in vitro using feeder-free culture protocols established in our laboratory. In this culture system, small human iPS cell clumps were cultured on the matrigel-coated dish with recombinant cytokines and without any feeder cells and fetal calf serum. Around day 25 of culture, mature neutrophils were obtained as floating cells. Morphologically, the majority of HAX1-iPSCs-derived cells were classified into myeloblast or promyelocyte stage and there were only a few mature neutrophils. The proportion of mature neutrophils was only less than 10% in HAX1-iPSCs-derived cells whereas more than 40% in normal control. Flow cytometric analysis revealed that the percentage of immature CD34 positive cells was significantly higher and that of myeloid-committed CD11b positive cells was lower in the HAX1-iPSCs-derived cells than normal control. Immunocytochemical analysis for neutrophil specific granules showed that lactoferrin- and gelatinase-positive cells decreased in the HAX1-iPSCs-derived cells compared with normal control, confirming that HAX1-iPSCs-derived cells contained less mature neutrophils than normal control. Apoptosis assay by Annexin V staining revealed that HAX1-iPSCs-derived cells showed higher percentage of Annexin V-positive cells compared with normal control. Overall, these HAX1 deficiency patient-specific iPS cell lines recapitulate the hematological phenotype in the patient. These results indicate that patient-derived iPS cells provide us a novel disease model and make a contribution to the understanding of the pathophysiology of the diseases that affect neutrophils. Disclosures: No relevant conflicts of interest to declare.

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Human DC3 Antigen Presenting Dendritic Cells from Induced Pluripotent Stem Cells

10.1101/2021.06.22.449451 ◽

2021 ◽

Author(s):

Taiki Satoh ◽

Marcelo A Szymanski de Toledo ◽

Janik Boehnke ◽

Kathrin Olschok ◽

Niclas Flosdorf ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

Cd8 T Cells ◽

Pluripotent Stem Cells ◽

Jak2 V617f ◽

Ips Cells ◽

Patient Specific ◽

Ips Cell ◽

Induced Pluripotent ◽

Antigen Presenting

Dendritic cells (DC) are professional antigen-presenting cells that develop from hematopoietic stem cells. Different DC subsets exist based on ontogeny, location and function, including the recently identified proinflammatory DC3 subset. DC3 have the prominent activity to polarize CD8+ T cells into CD8+ CD103+ tissue resident T cells. Here we describe human DC3 differentiated from induced pluripotent stem cells (iPS cells). iPS cell-derived DC3 have the gene expression and surface marker make-up of blood DC3 and polarize CD8+ T cells into CD8+ CD103+ tissue-resident memory T cells in vitro. To test the impact of malignant JAK2 V617F mutation on DC3, we differentiated patient-specific iPS cells with JAK2 V617Fhet and JAK2 V617Fhom mutations into JAK2 V617Fhet and JAK2 V617Fhom DC3. The JAK2 V617F mutation enhanced DC3 production and caused a bias towards erythrocytes and megakaryocytes. The patient-specific iPS cell-derived DC3 are expected to allow studying DC3 in human diseases and developing novel therapeutics.

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Nonhuman Primate Induced Pluripotent Stem Cells in Regenerative Medicine

Stem Cells International ◽

10.1155/2012/767195 ◽

2012 ◽

Vol 2012 ◽

pp. 1-7 ◽

Cited By ~ 15

Author(s):

Yuehong Wu ◽

Anuja Mishra ◽

Zhifang Qiu ◽

Steven Farnsworth ◽

Suzette D. Tardif ◽

...

Keyword(s):

Stem Cells ◽

Regenerative Medicine ◽

Induced Pluripotent Stem Cells ◽

Cell Lines ◽

Pluripotent Stem Cells ◽

Patient Specific ◽

Ips Cell ◽

Disease States ◽

Autologous Cell Therapy ◽

Induced Pluripotent

Among the various species from which induced pluripotent stem cells have been derived, nonhuman primates (NHPs) have a unique role as preclinical models. Their relatedness to humans and similar physiology, including central nervous system, make them ideal for translational studies. We review here the progress made in deriving and characterizing iPS cell lines from different NHP species. We focus on iPS cell lines from the marmoset, a small NHP in which several human disease states can be modeled. The marmoset can serve as a model for the implementation of patient-specific autologous cell therapy in regenerative medicine.

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Analysis of Viral Integration Sites in Human Induced Pluripotent Stem Cells.

Blood ◽

10.1182/blood.v114.22.1485.1485 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1485-1485

Author(s):

Thomas Winkler ◽

Amy R Cantelina ◽

Jean-Yves Metais ◽

Xiuli Xu ◽

Anh-Dao Nguyen ◽

...

Keyword(s):

Transcription Factors ◽

Gene Transfer ◽

Cell Lines ◽

Chromosomal Location ◽

Somatic Cells ◽

Ips Cells ◽

Ips Cell ◽

Integration Sites ◽

Equity Ownership ◽

Induced Pluripotent

Abstract Abstract 1485 Poster Board I-508 The recently discovered approach for the direct reprogramming of somatic cells into induced pluripotent stem (IPS) cells by expression of defined transcription factors may provide new approaches for regenerative medicine, gene therapy and drug screening. Successful reprogramming currently requires at least temporary expression of one to four different transcription factors (among Oct3/4, Sox2, Klf4, c-Myc, Nanog and Lin28) in the targeted cells. Non-viral based reprogramming technologies have been reported, but expression of the reprogramming factors after γ-retroviral or lentiviral gene transfer remains the most efficient and commonly used approach. Since the reprogramming frequency is consistently low in these studies, it has been speculated that gene activation or disruption via proviral integration sites (IS) may play a role in obtaining the pluripotent phenotype. Here we present for the first time an extensive analysis of the lentiviral integration profile in human IPS-cells. We analysed the IS of 8 IPS cell lines derived from either human fetal fibroblasts (IMR90) or newborn foreskin fibroblasts (FS) after lentiviral gene transfer of Oct4, Sox2, Nanog, and Lin28, using linear amplification-mediated PCR (LAM-PCR). With 5 to15 IS per individual IPS clone we identified a total of 78 independent IS. Finally we assigned 75 IS to a unique chromosomal location. In addition to LAM-PCR, we confirmed the total number of IS via Southern blot. Interestingly, in 6 of 8 IPS clones some of these IS were found in pairs, integrated into the same chromosomal location within 4 base pairs of each other. This integration pattern has not been detected in our previous analysis of 702 IS in rhesus macaques transplanted with CD34+ cells transduced with retroviral vectors. Of the 75 valid IS 53 (70.7%) could be mapped to a gene-coding region, 52 located in introns and 1 in an exon, annotated in a human reference sequence in the UCSC Genome Browser RefSeq Genes track. The different IPS-clones had no integration site in common. To investigate the impact of integration on the regulation of vector targeted genes we analyzed the mRNA expression profiles using available microarray data from these clones. Out of 46 evaluable genes only two (WDR66 and MYST2 in clone IMR90-2, p<0.0001) were significantly over-expressed. The expression of two genes in clone FS-1 (ACVR2A p=0.01, RAF1 p=0.02) and one in FS-2 (KIAA0528, p=0.03) was decreased compared to the expression data of all other clones combined. In summary our data suggest that efficient reprogramming of human somatic cells is not dependent on insertional activation or deactivation of specific genes or gene classes. Furthermore, identification of the insertion profile of the IPS cell clones IMR90-1 and -4 as well as FS-1 will be useful to other researchers using these cell lines distributed by the Wisconsin International Stem Cell (WISC) bank. Disclosures: Antosiewicz-Bourget: Cellular Dynamics International: Consultancy, Equity Ownership. Thomson: Cellular Dynamics International: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Dunbar: ASH: Honoraria.

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Human induced pluripotent stem cells are capable of B-cell lymphopoiesis

Blood ◽

10.1182/blood-2010-08-299941 ◽

2011 ◽

Vol 117 (15) ◽

pp. 4008-4011 ◽

Cited By ~ 50

Author(s):

Lee Carpenter ◽

Ram Malladi ◽

Cheng-Tao Yang ◽

Anna French ◽

Katherine J. Pilkington ◽

...

Keyword(s):

Stem Cells ◽

B Cell ◽

Embryonic Stem ◽

Ips Cells ◽

Ips Cell ◽

Lymphoid Lineage ◽

Human Ips Cells ◽

Induced Pluripotent ◽

First Time ◽

Unique Potential

Abstract Induced pluripotent stem (iPS) cells offer a unique potential for understanding the molecular basis of disease and development. Here we have generated several human iPS cell lines, and we describe their pluripotent phenotype and ability to differentiate into erythroid cells, monocytes, and endothelial cells. More significantly, however, when these iPS cells were differentiated under conditions that promote lympho-hematopoiesis from human embryonic stem cells, we observed the formation of pre-B cells. These cells were CD45+CD19+CD10+ and were positive for transcripts Pax5, IL7αR, λ-like, and VpreB receptor. Although they were negative for surface IgM and CD5 expression, iPS-derived CD45+CD19+ cells also exhibited multiple genomic D-JH rearrangements, which supports a pre–B-cell identity. We therefore have been able to demonstrate, for the first time, that human iPS cells are able to undergo hematopoiesis that contributes to the B-cell lymphoid lineage.

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Micro-RNA Profiling Reveals the Key Role of miR-206 in the Hematopoietic Potential of Human Embryonic and Induced Pluripotent Stem Cells

Blood ◽

10.1182/blood-2018-99-110729 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1275-1275

Author(s):

Stephane Flamant ◽

Jean-Claude Chomel ◽

Christophe Desterke ◽

Olivier Feraud ◽

Emilie Gobbo ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Line ◽

Cell Lines ◽

Pluripotent Stem Cells ◽

Target Genes ◽

Hematopoietic Differentiation ◽

Induced Pluripotent

Abstract Although human pluripotent stem cells (hPSCs) can theoretically be differentiated into any cell type, their ability to generate hematopoietic cells shows a major variability from one cell line to another. The reasons of this variable differentiation potential, which is constant and reproducible in a given hPSC line, are not clearly established. In order to study this phenomenon, we comparatively studied 4 human embryonic stem cell lines (hESC) and 11 human induced pluripotent stem cell (hiPSC) lines using transcriptome assays. These cell lines exhibited a significant variability to generate in vitro hematopoiesis as evaluated by day-16 embryoid body (EB) formation followed by clonogenic (CFC) assays. Four out of 11 iPSC lines (PB6, PB9, PB12.1, and PB14.3) were found to lack any hematopoietic differentiation ability whereas 7 cell lines showed variable hematopoietic potential. Among hESC lines, H9 and CL0 had low H1 and SA01 exhibited high hematopoietic potential using the above assays. Among hESC and hIPSC displaying hematopoietic potential, two sub-groups were further defined based on their hematopoietic CFC efficiency: a group of poor (generation of less than 100 CFC/105 cells, PB4 / PB10 /H9 /CL01), and high hematopoietic competency (more than 120 CFC/105 cells, PB3/ PB6.1 /PB7 /PB13 /PB17 /SA01/H1). Using global miRNome analysis performed at the pluripotency stage, the expression of 754 individual miRNAs was analyzed from 15 hPSC lines in order to explore a potential predictive marker between both sub-groups of pluripotent cells according to their hematopoietic potency. Using this approach, 27 miRNAs out of 754 appeared differentially expressed allowing the identification of a miRNA signature associated with hematopoietic-competency. The hematopoietic competency was associated with down-regulation of miR-206, miR-135b, miR-105, miR-492, miR-622 and upregulation of miR-520a, miR-296, miR-122, miR-515, miR-335. Amongst these, miR-206 harbored the most significant variation (0.04-Fold change). To explore the role of miRNA-206 in this phenomenon, we have generated a miR-206-eFGP-Puro lentiviral vector which was transfected in hESC line H1 followed by puromycin selection. As a control, H1 cell line was transfected with a Arabidopsis thaliana microRNA sequence (ath-miR-159a), which has no specific targets in mammalian cells. The correct expression of the transgenes were evaluated by flow cytometry (using GFP) and q-RT-PCR for miR-206 expression. The hematopoietic potential of H1 cell line and its miR-206-overexpressing counterpart was then tested using standard in vitro assays via d16-EB generation. We found that both CFC numbers and percentage of CD34+ were significantly lower in H1-mir-206-derived day-16 EB cells than in H1-ath- derived day-16 EB cells (p < 0.05). Thus, over-expression of miR-206 in this blood-competent hESC appeared to repress its hematopoietic potential at very early stage, since a similar lower CFC efficiency was observed in day-3 EB cells derived from miR-206 overexpressing H1 cell line. We then conducted an integrative bioinformatics analysis on miR-206 predicted target genes. To this end, 773 mRNA target transcripts of the broadly conserved (across vertebrates) miR-1-3p/206 family were identified in the TargetScan database and were integrated into the global transcriptomic analysis performed by microarray on day-16 EB cells. Using supervised ranking product analysis, 62 predicted gene targets of the miR-1-3p/206 family were found to be significantly up-regulated in hematopoietic-competent EB samples including the transcription factors RUNX1 and TAL1. Hierarchical unsupervised clustering, based on this subset of 62 predicted mir-206 target genes, fully discriminated hematopoietic-deficient from hematopoietic-competent cells. In conclusion, miRNA profiling performed at pluripotency stage could be useful to predict the ability to human iPSC to give rise to blood cell progenitors. This work emphasizes for the first time the critical role of the muscle-specific miR-206 in hematopoietic differentiation. Finally, these results suggest that genetic manipulation of hESC/iPSC could be used to enhance their hematopoietic potential and to design protocols for generation of hPSC-derived hematopoietic stem cells with long-term reconstitution ability. Disclosures No relevant conflicts of interest to declare.

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