Derivation of Hematopoietic Progenitor Cells From Vector-Free Human Induced Pluripotent Stem Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4746-4746
Author(s):  
Friedrich Schuening ◽  
Michail Zaboikin ◽  
Tatiana Zaboikina ◽  
Narasimhachar Srinivasakumar
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Conflicts Of Interest ◽  
Dermal Fibroblasts ◽  
Embryoid Bodies ◽  
Chromosomal Anomalies ◽  
Hematopoietic Stem ◽  
Induced Pluripotent

Abstract Abstract 4746 Induced pluripotent stem cells (iPSCs), due to their self-renewal and differentiation capability, have tremendous potential in regenerative medicine. Differentiation of IPSCs in vitro to obtain sufficient number of hematopoietic stem cells (HSCs) and their progenitors (HPCs) from iPSCs for therapeutic purposes is a holy grail of cellular therapy. To this end, we are comparing different in vitro differentiation approaches for generation of HSCs/HPCs from IPSCs. We have generated iPSCs from human adult dermal fibroblasts using two different reprogramming methods: 1) Transduction with retroviral vectors encoding human Klf4, Oct3/4, Sox2 and cMyc or 2) Electroporation with Epstein–Barr virus (EBV) based episomal plasmid vectors encoding Klf4, Oct3/4, Sox2, L-Myc and p53 targeting shRNA. The transduced/electroporated cells were reprogrammed on SNL5 mouse feeder cells. Putative iPSC-like colonies were cloned and adapted to grow under feeder-free conditions on Matrigel (BD) in mTeSR1 (Stem Cell Technologies) medium. From over 30 individual clones isolated, six were further characterized for: 1) expression of pluripotency markers (Tra-1–60, SSEA-3, SSEA-4, Nanog and Oct3/4) by immunofluorescence; 2) endogenous and total mRNA expression by quantitative real-time reverse-transcriptase PCR (RT-qPCR) for Klf4, Oct3/4, Sox2 and cMyc to distinguish between cellular and vector derived expression of reprogramming factors; 3) RT-qPCR to determine expression of other markers of pluripotency such as Nanog and DNA methyl transferease; 4) karyotype analysis to determine chromosomal anomalies. The vector-free IPSC clones were also tested for residual integrated EBV plasmid DNA by qPCR. Trilineage differentiation ability of the clones was determined through embryoid body formation in suspension cultures, and subsequent staining of resulting embryoid bodies after adherence to gelatin coated dishes for makers of ectoderm, mesoderm and endoderm. HSCs/HPCs were obtained from IPSCs by 1) coculture with OP9 stromal cells, or 2) step-wise differentiation in feeder-free conditions on Matrigel under defined conditions in the presence of appropriate growth factors [Niwa A et al. PLoS One. (2011); 6(7):e22261.]. The resultant HSCs/HPCs were subjected to colony forming assays in semi-solid medium containing hematopoietic cytokines. Both erythroid and myelomonocytic colonies could be readily identified. The influence of ambient oxygen concentration on the HSC/HPC derivation procedure is being investigated. The results of these studies will be presented. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Generation and miRNA Characterization of Equine Induced Pluripotent Stem Cells Derived from Fetal and Adult Multipotent Tissues

2019 ◽  
Vol 2019 ◽  
pp. 1-15 ◽  
Author(s):  
Laís Vicari de Figueiredo Pessôa ◽  
Pedro Ratto Lisboa Pires ◽  
Maite del Collado ◽  
Naira Caroline Godoy Pieri ◽  
Kaiana Recchia ◽  
...  
Keyword(s):  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryoid Body ◽  
Embryoid Bodies ◽  
Mirna Profile ◽  
Patient Specific ◽  
Induced Pluripotent

Introduction. Pluripotent stem cells are believed to have greater clinical potential than mesenchymal stem cells due to their ability to differentiate into almost any cell type of an organism, and since 2006, the generation of patient-specific induced pluripotent stem cells (iPSCs) has become possible in multiple species. Objectives. We hypothesize that different cell types respond differently to the reprogramming process; thus, the goals of this study were to isolate and characterize equine adult and fetal cells and induce these cells to pluripotency for future regenerative and translational purposes. Methods. Adult equine fibroblasts (eFibros) and mesenchymal cells derived from the bone marrow (eBMmsc), adipose tissue (eADmsc), and umbilical cord tissue (eUCmsc) were isolated, their multipotency was characterized, and the cells were induced in vitro into pluripotency (eiPSCs). eiPSCs were generated through a lentiviral system using the factors OCT4, SOX2, c-MYC, and KLF4. The morphology and in vitro pluripotency maintenance potential (alkaline phosphatase detection, embryoid body formation, in vitro spontaneous differentiation, and expression of pluripotency markers) of the eiPSCs were characterized. Additionally, a miRNA profile analysis of the mesenchymal and eiPSCs was performed. Results. Multipotent cells were successfully isolated, but the eBMmsc failed to generate eiPSCs. The eADmsc-, eUCmsc-, and eFibros-derived iPSCs were positive for alkaline phosphatase, OCT4 and NANOG, were exclusively dependent on bFGF, and formed embryoid bodies. The miRNA profile revealed a segregated pattern between the eiPSCs and multipotent controls: the levels of miR-302/367 and the miR-92 family were increased in the eiPSCs, while the levels of miR-23, miR-27, and miR-30, as well as the let-7 family were increased in the nonpluripotent cells. Conclusions. We were able to generate bFGF-dependent iPSCs from eADmsc, eUCmsc, and eFibros with human OSKM, and the miRNA profile revealed that clonal lines may respond differently to the reprogramming process.

Modeling Congenital Amegakaryocytic Thrombocytopenia Using Patient-Specific Induced Pluripotent Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 703-703
Author(s):  
Naoya Takayama ◽  
Shinji Hirata ◽  
Ryoko Jono-Ohnishi ◽  
Sou Nakamura ◽  
Sho-ichi Hirose ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Conflicts Of Interest ◽  
Receptor Gene ◽  
Patient Specific ◽  
Gene Correction ◽  
Donor Skin ◽  
Induced Pluripotent

Abstract Abstract 703 Patient-specific, induced pluripotent stem cells (iPSCs) enable us to study disease mechanisms and drug screening. To clarify the phenotypic alterations caused by the loss of c-MPL, the thrombopoietin (TPO) receptor, we established iPSCs derived from skin fibroblasts of a patient who received curative bone marrow transplantation for congenital amegakarycytic thrombocytopenia (CAMT) caused by the loss of the TPO receptor gene, MPL. The resultant CAMT-iPSCs exhibited mutations corresponding to the original donor skin. Then using an in vitro culture system yielding hematopoietic progenitor cells (HPCs), we evaluated the role of MPL on the early and late phases of human hematopoiesis. Although CAMT-iPSCs generated CD34+ HPCs, per se, their colony formation capability was impaired, as compared to control CD34+ HPCs. Intriguingly, both Glycophorin A (GPA)+ erythrocyte development and CD41+ megakaryocyte yields from CAMT-iPSCs were also impaired, suggesting that MPL is indispensable for MEP (megakaryocyte erythrocyte progenitors) development. Prospective analysis along with the hematopoietic hierarchy revealed that, in CAMT-iPSCs but not control iPSCs expressing MPL, mRNA expression and phosphorylation of putative signaling molecules downstream of MPL are severely impaired, as is the transition from CD34+CD43+CD41-GPA- MPP (multipotent progenitors) to CD41+GPA+ MEP. Additional analysis also indicated that c-MPL is required for maintenance of a consistent supply of megakaryocytes and erythrocytes from MEPs. Conversely, complimentary transduction of MPL into CAMT-iPSCs using a retroviral vector restored the defective erythropoiesis and megakaryopoiesis; however, excessive MPL signaling appears to promote aberrant megakaryopoiesis with CD42b (GPIba)-null platelet generation and impaired erythrocyte production. Taken together, our findings demonstrate the usefulness of CAMT-iPSCs for validation of functionality in the human hematopoiesis system. For example, it appears that MPL is not indispensable for the emergence of HPCs, but is indispensible for their maintenance, and for subsequent MEP development. Our results also strongly indicate that an appropriate expression level of an administered gene is necessary to achieve curative gene correction / therapy using patient-derived iPSCs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Exogenous LIN28 Is Required for the Maintenance of Self-Renewal and Pluripotency in Presumptive Porcine-Induced Pluripotent Stem Cells

2021 ◽  
Vol 9 ◽  
Author(s):  
Warunya Chakritbudsabong ◽  
Somjit Chaiwattanarungruengpaisan ◽  
Ladawan Sariya ◽  
Sirikron Pamonsupornvichit ◽  
Joao N. Ferreira ◽  
...  
Keyword(s):  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryoid Bodies ◽  
Self Renewal ◽  
Fetal Fibroblasts ◽  
Induced Pluripotent

Porcine species have been used in preclinical transplantation models for assessing the efficiency and safety of transplants before their application in human trials. Porcine-induced pluripotent stem cells (piPSCs) are traditionally established using four transcription factors (4TF): OCT4, SOX2, KLF4, and C-MYC. However, the inefficiencies in the reprogramming of piPSCs and the maintenance of their self-renewal and pluripotency remain challenges to be resolved. LIN28 was demonstrated to play a vital role in the induction of pluripotency in humans. To investigate whether this factor is similarly required by piPSCs, the effects of adding LIN28 to the 4TF induction method (5F approach) on the efficiency of piPSC reprogramming and maintenance of self-renewal and pluripotency were examined. Using a retroviral vector, porcine fetal fibroblasts were transfected with human OCT4, SOX2, KLF4, and C-MYC with or without LIN28. The colony morphology and chromosomal stability of these piPSC lines were examined and their pluripotency properties were characterized by investigating both their expression of pluripotency-associated genes and proteins and in vitro and in vivo differentiation capabilities. Alkaline phosphatase assay revealed the reprogramming efficiencies to be 0.33 and 0.17% for the 4TF and 5TF approaches, respectively, but the maintenance of self-renewal and pluripotency until passage 40 was 6.67 and 100%, respectively. Most of the 4TF-piPSC colonies were flat in shape, showed weak positivity for alkaline phosphatase, and expressed a significantly high level of SSEA-4 protein, except for one cell line (VSMUi001-A) whose properties were similar to those of the 5TF-piPSCs; that is, tightly packed and dome-like in shape, markedly positive for alkaline phosphatase, and expressing endogenous pluripotency genes (pOCT4, pSOX2, pNANOG, and pLIN28), significantly high levels of pluripotent proteins (OCT4, SOX2, NANOG, LIN28, and SSEA-1), and a significantly low level of SSEA-4 protein. VSMUi001-A and all 5F-piPSC lines formed embryoid bodies, underwent spontaneous cardiogenic differentiation with cardiac beating, expressed cardiomyocyte markers, and developed teratomas. In conclusion, in addition to the 4TF, LIN28 is required for the effective induction of piPSCs and the maintenance of their long-term self-renewal and pluripotency toward the development of all germ layers. These piPSCs have the potential applicability for veterinary science.

scholarly journals Differentiation of human induced pluripotent stem cells into erythroid cells

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Mohsen Ebrahimi ◽  
Mehdi Forouzesh ◽  
Setareh Raoufi ◽  
Mohammad Ramazii ◽  
Farhoodeh Ghaedrahmati ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Promising Alternative ◽  
Human Ipscs ◽  
Induced Pluripotent ◽  
Human Ipsc

AbstractDuring the last years, several strategies have been made to obtain mature erythrocytes or red blood cells (RBC) from the bone marrow or umbilical cord blood (UCB). However, UCB-derived hematopoietic stem cells (HSC) are a limited source and in vitro large-scale expansion of RBC from HSC remains problematic. One promising alternative can be human pluripotent stem cells (PSCs) that provide an unlimited source of cells. Human PSCs, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are self-renewing progenitors that can be differentiated to lineages of ectoderm, mesoderm, and endoderm. Several previous studies have revealed that human ESCs can differentiate into functional oxygen-carrying erythrocytes; however, the ex vivo expansion of human ESC-derived RBC is subjected to ethical concerns. Human iPSCs can be a suitable therapeutic choice for the in vitro/ex vivo manufacture of RBCs. Reprogramming of human somatic cells through the ectopic expression of the transcription factors (OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG) has provided a new avenue for disease modeling and regenerative medicine. Various techniques have been developed to generate enucleated RBCs from human iPSCs. The in vitro production of human iPSC-derived RBCs can be an alternative treatment option for patients with blood disorders. In this review, we focused on the generation of human iPSC-derived erythrocytes to present an overview of the current status and applications of this field.

Generation of HIV Resistant and Functional Macrophages From Hematopoietic Stem Cell Derived Induced Pluripotent Stem Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 562-562
Author(s):  
Amal Kambal ◽  
Gaela Mitchell ◽  
Whitney Cary ◽  
William Gruenloh ◽  
Yunjoon Jung ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Conflicts Of Interest ◽  
Lentiviral Vector ◽  
Patient Specific ◽  
Hematopoietic Stem ◽  
Induced Pluripotent ◽  
Anti Hiv ◽  
Hiv 1

Abstract Abstract 562 Induced pluripotent stem cells (iPSCs) have radically advanced the field of regenerative medicine by making possible the production of patient-specific pluripotent stem cells from adult individuals. By developing iPSCs to treat HIV, there is the potential for generating a continuous supply of therapeutic cells for transplantation into HIV infected patients. In this study, we have utilized human hematopoietic stem cells (HSCs) to generate anti-HIV gene expressing iPSCs for HIV gene therapy. HSCs were de-differentiated into continuously growing iPSC lines with four reprogramming factors and a combination anti-HIV lentiviral vector containing a CCR5 shRNA and a human/rhesus chimeric TRIM5α gene. Upon directed differentiation of the anti-HIV iPSCs towards the hematopoietic lineage, a robust quantity (>35%) of colony forming CD133+ HSCs were obtained. These cells were further differentiated into functional end-stage macrophages which displayed a normal phenotypic profile. Upon viral challenge, the anti-HIV iPSC derived macrophages exhibited strong protection (>3 logs) from HIV-1 infection. Here we demonstrate the ability of iPSCs to develop into HIV-1 resistant immune cells and highlight the potential use of iPSCs for HIV gene and cellular therapies. Disclosures: No relevant conflicts of interest to declare.

Dysregulation of the TGF β Pathway in Induced Pluripotent Stem Cells (iPSCs) Generated from Patients with Diamond Blackfan Anemia (DBA)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 254-254
Author(s):  
Jingping Ge ◽  
Marisa Apicella ◽  
Jason A. Mills ◽  
Loic Garcon ◽  
Deborah L. French ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Ribosome Biogenesis ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Ectopic Expression ◽  
Canonical Pathway ◽  
Hematopoietic Stem ◽  
Induced Pluripotent

Abstract DBA is an inherited bone marrow failure syndrome that usually develops in the first year of life with clinical features of red cell aplasia and sometimes variable developmental abnormalities. Most affected patients have heterozygous loss of function mutations of one of the 11 ribosomal proteins (RPs) or mutations in the GATA1 gene which encodes an erythroid specific transcription factor. We have previously demonstrated that induced pluripotent stem cells (iPSCs) from fibroblast of DBA patients with RPS19 or RPL5 mutations recapitulate the pathogenesis of DBA, with the mutant lines showing abnormal ribosome biogenesis and altered erythropoiesis. The mechanism whereby haploinsufficiency for RPs causes failure of erythropoiesis and the other DBA features is still unknown. We investigated the pathways that are affected in these DBA iPSCs using an Affymetrix human exon array, and we observed the striking dysregulation of the TGF β pathway in DBA lines. The TGF β downstream target genes, such as DKK1, BAMBI, FN1, COL3A1, COLA1A1 and PAI-1 significantly increased in the DBA iPSCs. The TGF β signaling is complex and can occur via a canonical pathway or by a number of non-canonical pathways. We measured levels of a number of intermediates in these pathways by western blot, and observed a significant increase in the levels of p-JNK, a mediator of a non-canonical pathway, in the DBA iPSCs. Moreover, when the mutant cells were corrected by ectopic expression of WT RPS19 or RPL5, levels of p-JNK returned to normal. We also investigated the SMAD family, which are mediators of the TGF β canonical pathway and are known to negatively regulate the regeneration of hematopoietic stem cells. We observed a drastic decrease in SMAD4, but no change in p-SMAD2. Again corrected lines showed normal expression levels of SMAD4. Our data suggests that the activation of a non-canonical TGF β pathway in the DBA iPSCs may lead increased expression of the downstream genes; and the decrease of anti-proliferative factor SMAD4 may explain how DBA iPSCs maintain their growth. We conclude that the mutations of RPS19 or RPL5 both affect ribosome biogenesis and TGF β signaling, which can cause the failure of erythropoiesis at the stem cell stage. We further suggest that the suppression of SMAD4 may be used as a therapeutic target for DBA treatment. Disclosures No relevant conflicts of interest to declare.

scholarly journals Analysis of Embryoid Bodies Derived from Human Induced Pluripotent Stem Cells as a Means to Assess Pluripotency

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Steven D. Sheridan ◽  
Vasudha Surampudi ◽  
Raj R. Rao
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryoid Body ◽  
Embryoid Bodies ◽  
Functional Assay ◽  
Differentiation Potential ◽  
Disease Etiology ◽  
Induced Pluripotent

Human induced pluripotent stem cells (hiPSCs) have core properties of unlimited self-renewal and differentiation potential and have emerged as exciting cell sources for applications in regenerative medicine, drug discovery, understanding of development, and disease etiology. Key among numerous criteria to assess pluripotency includes thein vivoteratoma assay that has been widely proposed as a standard functional assay to demonstrate the pluripotency of hiPSCs. Yet, the lack of reliability across methodologies, lack of definitive clinical significance, and associated expenses bring into question use of the teratoma assay as the “gold standard” for determining pluripotency. We propose use of thein vitroembryoid body (EB) assay as an important alternative to the teratoma assay. This paper summarizes the methodologies for creating EBs from hiPSCs and the subsequent analyses to assess pluripotency and proposes its use as a cost-effective, controlled, and reproducible approach that can easily be adopted to determine pluripotency of generated hiPSCs.

Cancellation of c-MYC Silencing in Human Induced Pluripotent Stem Cells Contributes to the Efficient in Vitro Production of Platelets with the Ability of Hemostasis In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1488-1488 ◽  
Author(s):  
Naoya Takayama ◽  
Sou Nakamura ◽  
Satoshi Nishimura ◽  
Ryoko Ohnishi ◽  
Kazutoshi Takahashi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Blood Platelets ◽  
Dermal Fibroblasts ◽  
Efficient Production ◽  
Nog Mice ◽  
Induced Pluripotent

Abstract Abstract 1488 Poster Board I-511 Human induced pluripotent stem cells (hiPSCs) generated from somatic cells by introduction of OCT3/4, SOX2, KLF4 and c-MYC represent a potential source of hematopoietic cells for transfusion without the risk of immune rejection. We recently established an in vitro culture system with which hiPSCs could be differentiated into the unique structure of an “in vitro hematopoietic niche” containing hematopoietic progenitors. Upon further cultivation under appropriate conditions, these hematopoietic progenitor cells differentiated into megakaryocytes, which could then generate platelets with morphologies indistinguishable from peripheral blood platelets regardless of either 4-factor iPSCs (n=8, 4-factor hiPSC clones generated from adult dermal fibroblasts through induction with c-MYC) or 3-factor iPSC clones (without c-MYC, n=3). It is well known that iPSC differentiation yields a heterogeneous population of clones. To select the best hiPSC clone for platelet production, we quantified thrombopoiesis with 11 independent hiPSC clones by comparison with human embryonic stem cells (hESCs) evaluated previously (Takayama et al., Blood, 2008) as a reference. Particularly noteworthy is our finding that 4-factor iPSCs have an advantage over 3-factor iPSCs or hESCs (P<0.01) that is mediated through cancellation of c-MYC silencing (re-activation) over the course of differentiation evidenced by RT-PCR studies. Indeed, ectopic expression of c-MYC, but not OCT3/4, SOX2 or KLF4, using a retroviral vector in hESC-derived progenitors accelerated both megakaryopoiesis and thrombopoiesis. By contrast, the platelet activation statuses (i.e., PAC-1 ligation with activated integrin αIIbβ3 following agonist stimulation) were comparable for platelets obtained from 4-factor hiPSCs and hESCs, though levels of c-MYC clearly differed, indicating that at least integrin activation is independent of c-MYC. To further estimate the in vivo functionality of iPSC-derived platelets, we developed a mouse model for transfusion. Irradiation (2.0 Gy, 9 days beforehand) induced thrombocytopenia in NOG (nod-scid/IL-2 γc-null) mice. Subsequent flow cytometry showed that 2 hrs after transfusion (1.0∼1.2×107 platelets per a mouse) of NOG mice via the tail vein, the circulating levels of selected 4-factor iPSC-derived platelets were similar to those of human adult platelets (platelet chimerism of human CD41/mouse CD41; 4∼10%). Moreover, by using our recently established in vivo imaging system, which enables observation of single platelet behavior, we observed that 4-factor iPSC-derived platelets circulate in NOG mice and contribute to the development thrombi within their vessels, suggesting the in vivo functionality of iPSC-derived platelets is intact. A number of studies have suggested that c-MYC can have deleterious effects leading to in vivo oncogeneity after transplantation in vivo. By contrast, our data strongly indicate the importance of c-MYC for platelet generation from hiPSCs and hESCs. Given that anucleate platelets are routinely irradiated before transfusion in clinical settings, use of c-MYC for hiPSCs generation may contribute to the efficient production of HLA-matched platelet concentrates for those requiring repeated transfusion. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Modeling Fanconi Anemia Using Human Induced Pluripotent Stem Cells By Reversible Complementation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3856-3856
Author(s):  
R. Grant Rowe ◽  
William Marion ◽  
Sonya Ruiz-Torres ◽  
Edroaldo Lummertz da Rocha ◽  
Yosra Girvan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Fanconi Anemia ◽  
Pluripotent Stem Cells ◽  
Erythroid Differentiation ◽  
Embryoid Bodies ◽  
Directed Differentiation ◽  
Sequencing Analysis ◽  
Hematopoietic Stem ◽  
Induced Pluripotent

Abstract Modeling of Fanconi anemia (FA) using human induced pluripotent stem cells (iPSCs) has been hindered by the requirement for an intact FA DNA repair pathway for effective reprogramming and maintenance of pluripotency in patient-derived iPSCs. Temporary complementation of FA pathway defects could permit effective reprogramming, with removal of complementation upon directed differentiation to hematopoietic stem and progenitor cells (HSPCs) to model the hematopoietic phenotypes of FA. In this study, we used three FA patient-derived iPSC lines engineered with doxycycline inducible complementation of disease-causing FANCA mutations. We maintained complementation with doxycycline exposure in the pluripotent state and during morphogen directed differentiation of hemogenic endothelium (HE) within embryoid bodies, which possesses the capacity to differentiate to HSPCs. Upon initiation of the endothelial-to-hematopoietic transition (EHT) in purified iPSC-derived HE, doxycycline was either removed or maintained in culture to either inactivate or sustain FANCA expression. Morphologic EHT and emergence of CD34+CD45+ immunophenotypic human HSPCs were not affected by the status of FANCA expression, which allowed for the isolation of otherwise isogenic FANCA-expressing and FANCA-deficient HSPCs for disease modeling. FANCA-deficient HSPCs were unable to form FANCD2/γH2AX foci relative to FANCA-expressing HSPCs in response to genotoxic stress, confirming impairment of the function of the FA pathway. We found that uncomplimented, FANCA-deficient HSPCs showed impaired cell cycle progression, increased apoptosis, and markedly decreased colony forming activity in methylcellulose compared to complemented, FANCA-expressing HSPCs. Unexpectedly, we also found that FANCA-deficient HSPCs showed accelerated erythroid differentiation compared to cells with an intact FA pathway. RNA sequencing analysis showed enrichment of signatures of normal HSPCs in complemented HSPCs, while uncomplimented HSPCs showed signatures of more mature hematopoietic cells and cells undergoing erythroid differentiation. Together, these findings demonstrate the utility of reversible complementation in modeling FA with patient-derived iPSCs, and provide an inexhaustible source of otherwise isongenic complimented and uncomplimented human FA HSPCs for use in the investigation of new therapies. Disclosures No relevant conflicts of interest to declare.

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