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Disease modeling of bone marrow failure syndromes using iPSC-derived hematopoietic stem progenitor cells

  • Mahmoud I. Elbadry
    Affiliations
    Hematology/Respiratory Medicine, Faculty of Medicine, Institute of Medical Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan

    Department of Internal Medicine, Division of Hematology, Faculty of Medicine, Sohag University, Egypt
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  • J. Luis Espinoza
    Affiliations
    Department of Hematology and Rheumatology, Faculty of Medicine, Kindai University, Osaka, Japan
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  • Shinji Nakao
    Correspondence
    Offprint requests to: Shinji Nakao, MD, PhD, Hematology/Respiratory Medicine, Faculty of Medicine, Institute of Medical Pharmaceutical and Health Sciences, Kanazawa University, Takara-machi 13-1, 920-8641 Kanazawa, Japan
    Affiliations
    Hematology/Respiratory Medicine, Faculty of Medicine, Institute of Medical Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan
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Open ArchivePublished:January 18, 2019DOI:https://doi.org/10.1016/j.exphem.2019.01.006

      Highlights

      • Induced pluripotent stem cells (iPSCs) can be generated from patients with bone marrow failure syndromes (BMFSs).
      • Patient-derived iPSCs can generate hematopoietic progenitors.
      • Disease models of BMFSs can be tailored using iPSCs.
      The plasticity of induced pluripotent stem cells (iPSCs) with the potential to differentiate into virtually any type of cells and the feasibility of generating hematopoietic stem progenitor cells (HSPCs) from patient-derived iPSCs (iPSC-HSPCs) has many potential applications in hematology. For example, iPSC-HSPCs are being used for leukemogenesis studies and their application in various cell replacement therapies is being evaluated. The use of iPSC-HSPCs can now provide an invaluable resource for the study of diseases associated with the destruction of HSPCs, such as bone marrow failure syndromes (BMFSs). Recent studies have shown that generating iPSC-HSPCs from patients with acquired aplastic anemia and other BMFSs is not only feasible, but is also a powerful tool for understanding the pathogenesis of these disorders. In this article, we highlight recent advances in the application of iPSCs for disease modeling of BMFSs and discuss the discoveries of these studies that provide new insights in the pathophysiology of these conditions.
      Aplastic anemia (AA) is a life-threatening bone marrow failure (BMF) disorder, resulting in bone marrow hypoplasia, infection and hemorrhage, and severe peripheral pancytopenia. Although the most cases of AA are acquired and associated with the autoimmune destruction of hematopoietic stem progenitor cells (HSPCs) in the BM, in some cases, the BMF is caused by genetic or inherited anomalies that impair hematopoiesis [
      • Young NS
      • Calado RT
      • Scheinberg P
      Current concepts in the pathophysiology and treatment of aplastic anemia.
      ]. The destruction or dysfunction of HSPCs in the BM of patients with BMF syndromes (BMFSs) limits the study of these disorders because the use of conventional in vitro HSPC culture or in vivo animal models for creating patient-specific disease modeling is technically impossible due to the unavailability of patient-derived HSPCs. With the development of induced pluripotent stem cells (iPSCs) [
      • Takahashi K
      • Tanabe K
      • Ohnuki M
      • et al.
      Induction of pluripotent stem cells from adult human fibroblasts by defined factors.
      ], a promising venue for the study of rare diseases such as BMFSs has been opened. The generation of patient-derived iPSCs and their subsequent differentiation into iPSC-HSPCs offer a unique opportunity for generating disease models to study several genetic and immune backgrounds of BMFSs, facilitating the investigation of human rare diseases based on individual patients’ phenotypes (Figure 1). Previously, we summarized some aspects of using iPSCs for understanding AA pathogenesis and the methods of establishing animal models for acquired AA (aAA) using iPSCs [
      • Elbadry MI
      • Espinoza JL
      • Nakao S
      Induced pluripotent stem cell technology: a window for studying the pathogenesis of acquired aplastic anemia and possible applications.
      ]. To achieve the goal of this review, we will focus on the previous successful trials to generate iPSCs from patients with different BMFSs. By drawing upon the broad experimental expertise of the previous published works, we will try to summarize the possible future application of this technology in understanding the pathogenesis of BMFSs and the potential challenges encountered using iPSC-based models of these disorders.
      Figure 1.
      Figure 1Disease model of BMFSs. Reprograming of somatic cells (fibroblasts or monocytes) to generate iPSCs opens new opportunities for the study of BMFSs. (A) Differentiation of iPSCs into hematopoietic stem cells coupled with genome editing and mutation correction has been tested to verify the pathogenesis of inherited BMFSs including FA, DKC, SDS, DBA, FPD, congenital megakaryocytic thrombocytopenia (CMT), and PMPS. (B) The hematopoietic differentiation of iPSCs derived from patients with acquired BMFS (AA) has been used to study in vitro and in vivo hematopoiesis with an emphasis on experiments aimed at unraveling the autoimmune mechanisms involved in the pathogenesis of this disorder. These studies may result in the establishment of various therapeutic approaches such as autologous transplantation, cell therapy, the use of blocking monoclonal antibodies, or the utilization of pharmaceuticals with inhibitory or stimulatory activities.

      Generation of iPSC clones from patients with inherited BMFSs

      Inherited BMFSs are a rare group of disorders often developing in childhood that are characterized by BMF with a marked reduction of all hematopoietic lineages or a single-cell lineage usually in association with one or more physical abnormalities [
      • Dokal I
      • Vulliamy T
      Inherited bone marrow failure syndromes.
      ]. Although the genetic lesions linked with most inherited BMFSs have been identified, some of the cellular events resulting from such genetic aberrations have not been clarified [
      • Jung M
      • Dunbar CE
      • Winkler T
      Modeling human bone marrow failure syndromes using pluripotent stem cells and genome engineering.
      ]. The utilization of iPSC-derived hematopoietic cells has emerged as a promising tool for the study of the somatic and germline mutations linked to inherited BMFSs and various studies, including the use of in vitro cell culture or in vivo animal models, have been reported.

      Fanconi anemia

      Fanconi anemia (FA) is the most common type of BMFS and is characterized by a spectrum of congenital physical abnormalities, coupled with progressive fatal BMF, chromosomal instability, and cancer predisposition [
      • Bagby G
      Recent advances in understanding hematopoiesis in Fanconi anemia.
      ]. At a molecular level, 21 FA genes encoding proteins involved in multiple DNA damage repair pathway essential for maintaining genomic stability have been identified [
      • Bagby G
      Recent advances in understanding hematopoiesis in Fanconi anemia.
      ,
      • Knies K
      • Inano S
      • Ramirez MJ
      • et al.
      Biallelic mutations in the ubiquitin ligase RFWD3 cause Fanconi anemia.
      ]. The complete functional characterization of FA genes is crucial for understanding the molecular pathogenesis of this disease; however, this has been hindered by the scarcity of FA patient samples and the difficulty of generating bona fide disease models. Due to the inherent defects in the DNA repair pathway, the effective reprogramming and maintenance of pluripotency of FA-derived somatic cells has been challenging. Not surprisingly, the first attempts to generate iPSCs from patients with FA reported very low efficiency in iPSC induction [
      • Raya A
      • Rodriguez-Piza I
      • Guenechea G
      • et al.
      Disease-corrected haematopoietic progenitors from Fanconi anaemia induced pluripotent stem cells.
      ,
      • Muller LU
      • Milsom MD
      • Harris CE
      • et al.
      Overcoming reprogramming resistance of Fanconi anemia cells.
      ,
      • Yung SK
      • Tilgner K
      • Ledran MH
      • et al.
      Brief report: human pluripotent stem cell models of fanconi anemia deficiency reveal an important role for fanconi anemia proteins in cellular reprogramming and survival of hematopoietic progenitors.
      ]. In addition, FA-derived iPSCs (FA-iPSCs) showed reduced potential to differentiate into the hematopoietic cells, producing fewer HSPCs and impaired erythroid and megakaryocytic differentiation capacity than normal iPSCs [
      • Liu GH
      • Suzuki K
      • Li M
      • et al.
      Modelling Fanconi anemia pathogenesis and therapeutics using integration-free patient-derived iPSCs.
      ,
      • Suzuki NM
      • Niwa A
      • Yabe M
      • et al.
      Pluripotent cell models of fanconi anemia identify the early pathological defect in human hemoangiogenic progenitors.
      ]. Liu et al. [
      • Liu GH
      • Suzuki K
      • Li M
      • et al.
      Modelling Fanconi anemia pathogenesis and therapeutics using integration-free patient-derived iPSCs.
      ] generated iPSCs from a patient with FA and succeeded in generating isogenic control iPSC lines using helper-dependent adenoviral vector (HDAdV)–mediated in situ targeted correction of the FANCA mutation. This study confirmed that genome stability was preserved in FA-iPSCs (normal karyotype at passage 13) and their differentiated progeny. Compared with control iPSCs, FA-iPSCs generated a significantly lower percentage of hematopoietic progenitor cells (HPCs) defined as a CD34high/CD43low population and those FA-HPCs were restricted to colony-forming unit-granulocyte macrophage and failed to generate erythroblast or megakaryocyte colonies. This phenotype was completely rescued by in situ FANCA gene correction [
      • Liu GH
      • Suzuki K
      • Li M
      • et al.
      Modelling Fanconi anemia pathogenesis and therapeutics using integration-free patient-derived iPSCs.
      ]. A higher reprogramming efficiency of FA fibroblasts was reported in a model of conditional reprogramming combined with the introduction of a FANCA transgene. In this study, the inhibition of CHK1 effectively restored the growth of FA-deficient iPSCs at a level comparable to that of normal iPSCs by bypassing the cell cycle at the G2-M checkpoint [
      • Chlon TM
      • Wells SI
      • Ruiz-Torres S
      • Kuhar M
      • Wells JM
      Models of pluripotent and somatic stem cells to study tissue-specific sensitivities in Fanconi anemia.
      ]. The findings that targeted gene correction rescued the phenotypic abnormalities in FA-iPSCs [
      • Liu GH
      • Suzuki K
      • Li M
      • et al.
      Modelling Fanconi anemia pathogenesis and therapeutics using integration-free patient-derived iPSCs.
      ] indicate that iPSC technology has a therapeutic potential for this disease; however, given the defects in the DNA repair pathway, further studies will be required not only to find ways to enhance the reprogramming efficiency of FA somatic cells and their differentiation into different lineages, but also to exclude the possible emergence of mutations that may occur during cell reprogramming.

      Dyskeratosis congenita

      Dyskeratosis congenita (DKC) is a rare progressive congenital disorder with a highly variable phenotype. Clinically, DKC has been characterized by the triad of abnormal nails, reticular skin pigmentation, and oral leukoplakia, with some manifestations that resemble premature aging (similar to progeria), although this phenotype is not always present [
      • Calado RT
      • Young NS
      Telomere diseases.
      ]. In addition to developing BMFS, which occurs in more than 80% of cases, DKC patients have an increased risk of malignant transformation. At the molecular level, DKC is a disorder of poor telomere maintenance, in which one or more mutations directly or indirectly affect the vertebrate telomerase RNA component (TERC), giving rise to an abnormal ribosome function.
      To study the disease mechanisms in humans, Agarwal et al. [
      • Agarwal S
      • Loh YH
      • McLoughlin EM
      • et al.
      Telomere elongation in induced pluripotent stem cells from dyskeratosis congenita patients.
      ] generated iPSCs from DKC patients harboring DKC1 mutations and observed that the reprogrammed DKC somatic cells were able to overcome TERC levels, restoring telomere maintenance and cell self-renewal despite the underlying genetic lesions affecting telomerase. In contrast, other studies reported that iPSC clones derived from fibroblasts of five DKC patients [
      • Batista LF
      • Pech MF
      • Zhong FL
      • et al.
      Telomere shortening and loss of self-renewal in dyskeratosis congenita induced pluripotent stem cells.
      ] and four patients with heterozygous mutations in either TERT or TERC and hypocellular BM [
      • Winkler T
      • Hong SG
      • Decker JE
      • et al.
      Defective telomere elongation and hematopoiesis from telomerase–mutant aplastic anemia iPSCs.
      ] exhibited some telomere abnormalities, including impaired telomere elongation or progressive telomere attrition with markedly reduced accumulation of TERC, dyskerin, and TCAB1 protein levels in Cajal bodies compared with wild-type (WT) iPSCs reprogrammed in parallel, which could be related to mislocalization of the telomerase complex. These observations substantiate the therapeutic potential of methods aimed at increasing TERC expression in DKC. At the same time, the ability to generate DKC-iPSCs provide limitless cells with which to study functional roles of telomeres in maintaining the pluripotency and to investigate the phenotypic variability among individuals with telomerase mutations; however, the dynamic role of telomere and telomerase during the process of cell reprogramming needs to be clarified by further investigations.

      Shwachman–Diamond syndrome

      Shwachman–Diamond syndrome (SDS) is a rare, autosomal-recessive congenital disorder characterized by BMF, exocrine pancreatic insufficiency, skeletal abnormalities, and short stature [
      • Shimamura A
      Shwachman–Diamond syndrome.
      ]. At the molecular level, the genetic defect in this syndrome lies on the long arm of 7 position 7q11. The Shwachman–Bodian–Diamond syndrome (SBDS) gene is expressed in all tissues and encodes a protein of 250 amino acid residues [
      • Shimamura A
      Shwachman–Diamond syndrome.
      ]. However, the function of this protein is not known, so many aspects of SDS pathogenesis are not completely understood. The generation of iPSCs derived from SDS patients showed a significant heterogeneity among iPSC clones because one patient's iPSC lines could not differentiate into the hematopoietic lineage at all, whereas another patient's iPSC lines showed impaired hematopoietic differentiation. Although, under the appropriate culture conditions, these iPSC lines differentiated into pancreatic progenitor cells, they showed impaired ductal formations and deficient synthetic functions. Intracellular granules containing proteases were observed in iPSC-derived myeloid and pancreatic cells, suggesting that adding protease inhibitors during hematopoietic and pancreatic differentiation could be helpful to investigate the pathogenesis of SDS and may contribute to identify new drug targets for SDS [
      • Tulpule A
      • Kelley JM
      • Lensch MW
      • et al.
      Pluripotent stem cell models of Shwachman–Diamond syndrome reveal a common mechanism for pancreatic and hematopoietic dysfunction.
      ]. Recently, Ruiz-Gutierrez et al. [
      • Ruiz–Gutierrez M
      • Vargel Bolukbasi O
      • Vo L
      • et al.
      Modeling bone marrow failure and MDS in Shwachman–Diamond syndrome using induced pluripotent stem cells.
      ] established SDS-iPSCs with homozygous IVS2+2 T>C SBDS mutations that expressed low levels of SBDS protein with reduced production of HSPCs, similar to levels noted in the primary patient samples. Interestingly, they also modeled SDS with IVS2+2 T>C SBDS mutations and deletion of 7q at locus (11.2) by generating SDS (del7q) iPSCs using a modified Cre-Lox approach. The SDS-iPSCs with del(7q) demonstrated a marked reduction in proliferation without an increase in cell death in comparison with isogenic SDS-iPSCs. These isogenic SDS-iPSCs with or without del(7q) models revealed a novel strategy to determine the effects of del 7q on hematopoiesis and new drug targets for this cryptic clinical disorder.

      Diamond–Blackfan anemia

      Diamond–Blackfan anemia (DBA) is an inherited BMFS characterized by severely decreased erythroid precursors coupled with progress to overt BMF and leukemia predisposition. The genetic aberrations observed in this disease mainly affect different ribosomal gene loci, leading to altered ribosomal functions [
      • Giri N
      • Kang E
      • Tisdale JF
      • et al.
      Clinical and laboratory evidence for a trilineage haematopoietic defect in patients with refractory Diamond–Blackfan anaemia.
      ]. However, the exact mechanism by which haploinsufficiency results in erythroid failure and other clinical manifestations remains uncertain. The generation of iPSC lines from fibroblasts of DBA patients showed reprogramming inefficiently, although stable clones similar to those generated from healthy individuals were obtained. The mutant clones exhibited globally defective hematopoiesis upon multipotent hematopoietic progenitor differentiation and erythroid potential with ability to be rescued using zinc finger nuclease (ZFN) by site-directed gene correction [
      • Garcon L
      • Ge J
      • Manjunath SH
      • et al.
      Ribosomal and hematopoietic defects in induced pluripotent stem cells derived from Diamond–Blackfan anemia patients.
      ]. Recently, Doulatov et al. [
      • Doulatov S
      • Vo LT
      • Macari ER
      • et al.
      Drug discovery for Diamond–Blackfan anemia using reprogrammed hematopoietic progenitors.
      ] succeeded in generating DBA-iPSC clones recapitulating the defects in erythroid differentiation that are typically observed in the disease. Interestingly, they also identified small-molecule enhancers of rapamycin 28 (SMER28), a small-molecule inducer of autophagy, which rescued the erythropoiesis in in vitro models as well as in vivo models of DBA. These findings suggest that autophagy may be dysregulated in DBA and provide an explanation for enhancing erythropoiesis by upregulating the expression of globin genes using SMER28, which acted through autophagy factor ATG5 in RPS19-deficient cells. These findings demonstrate the power of iPSC models in drug screening strategies for hematological diseases.

      Congenital amegakaryocytic thrombocytopenia

      Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare, inherited, autosomal-recessive disorder that presents with BMF characterized by an isolated and severe decrease in the number of platelets and megakaryocytes during the first years of life that can progress to pancytopenia later in childhood [
      • Ballmaier M
      • Germeshausen M
      Congenital amegakaryocytic thrombocytopenia: clinical presentation, diagnosis, and treatment.
      ]. The cause of this disorder appears to be a mutation in the gene for the thrombopoietin (TPO) receptor, c-Mpl. Hirata et al. [
      • Hirata S
      • Takayama N
      • Jono-Ohnishi R
      • et al.
      Congenital amegakaryocytic thrombocytopenia iPS cells exhibit defective MPL-mediated signaling.
      ] succeeded in establishing iPSCs from a patient with CAMT due to compound heterozygous mutations in the myeloproliferative leukemia (MPL) virus oncogene 1499delT from the father and MPL Q186X from the mother. The CAMT-iPSCs lines showed fewer megakaryocytes upon hematopoietic differentiation and this defect was not rescued by TPO treatment. Interestingly, retrovirus-mediated MPL gene transfer rescued the hematopoietic phenotype and restored MPL expression [
      • Hirata S
      • Takayama N
      • Jono-Ohnishi R
      • et al.
      Congenital amegakaryocytic thrombocytopenia iPS cells exhibit defective MPL-mediated signaling.
      ]. This CAMT-iPSC model suggested a novel role for MPL signaling in erythropoiesis because it showed that increased expression of MPL in MPLlow CAMT iPSCs led to comparable erythroid and megakaryocyte output. In contrast, the CAMT iPSC with MPLhigh expression showed increased megakaryocytic differentiation but displayed blocked erythropoiesis. TPO and MPL signaling controls megakaryocytic and erythroid bifurcation through the cross-antagonism of the transcriptional factors Friend leukemia virus integration 1 (FLI1) toward megakaryopoiesis and Kruppel-like factor 1 (KLF1) toward erythropoiesis [
      • Dore LC
      • Crispino JD
      Transcription factor networks in erythroid cell and megakaryocyte development.
      ]. Conversely, Kuvardina et al. [
      • Kuvardina ON
      • Herglotz J
      • Kolodziej S
      • et al.
      RUNX1 represses the erythroid gene expression program during megakaryocytic differentiation.
      ] found that, during megakaryopoiesis, runt-related transcription factor 1 (RUNX1) inhibited erythroid differentiation of primary human CD34+ progenitor cells and murine megakaryocytic and erythroid progenitors through its blockage of the KLF1-dependent erythroid gene expression. These findings with CAMT-iPSCs models could lead to the identification of new roles for TPO and MPL signaling in megakaryocytic and erythroid bifurcation.

      Familial platelet disorder

      Familial platelet disorder (FPD) with predisposition to acute myelogenous leukemia (AML) is a very rare disorder characterized by mild to moderate thrombocytopenia, an abnormal platelet function, and an increased risk of developing blood malignancies especially myelodysplastic syndrome (MDS) and AML [
      • Michaud J
      • Wu F
      • Osato M
      • et al.
      In vitro analyses of known and novel RUNX1/AML1 mutations in dominant familial platelet disorder with predisposition to acute myelogenous leukemia: implications for mechanisms of pathogenesis.
      ]. The disease is an inherited, autosomal-dominant disorder caused by heterozygous loss-of-function mutations in RUNX1 [
      • Osato M
      Point mutations in the RUNX1/AML1 gene: another actor in RUNX leukemia.
      ]. Impaired hematopoiesis and defects in megakaryocytic differentiation were reported in iPSC lines derived from patients with FPD. In these studies, FPD-iPSCs displayed normal early erythroid–megakaryocyte progenitor formation, but the megakaryocytic differentiation and maturation from FPD-iPSCs were profoundly impaired. The FPD-HSPC phenotype was rescued by overexpression of WT RUNX1 in FPD-iPSCs with megakaryocyte differentiation [
      • Sakurai M
      • Kunimoto H
      • Watanabe N
      • et al.
      Impaired hematopoietic differentiation of RUNX1–mutated induced pluripotent stem cells derived from FPD/AML patients.
      ,
      • Connelly JP
      • Kwon EM
      • Gao Y
      • et al.
      Targeted correction of RUNX1 mutation in FPD patient-specific induced pluripotent stem cells rescues megakaryopoietic defects.
      ,
      • Antony-Debre I
      • Manchev VT
      • Balayn N
      • et al.
      Level of RUNX1 activity is critical for leukemic predisposition but not for thrombocytopenia.
      ], supporting the notion that RUNX1 mutations are responsible for defects in megakaryocytic differentiation in FPD patients. Furthermore, the facts that genomic instability was consistently observed in the RUNX1-mutated iPSCs and that the RUNX1-iPSC phenotype depended on the regulation of NR4A3 in RUNX1-mutated cells [
      • Bluteau D
      • Gilles L
      • Hilpert M
      • et al.
      Down-regulation of the RUNX1-target gene NR4A3 contributes to hematopoiesis deregulation in familial platelet disorder/acute myelogenous leukemia.
      ] suggest that a marked loss of RUNX1 expression increases the risk of malignant transformation observed in this disease

      Pearson marrow pancreas syndrome

      Pearson marrow pancreas syndrome (PMPS) is a fatal BMFS caused by heteroplasmic deletions in mitochondrial DNA (mtDNA) that translate to pancreatic insufficiency and other systemic organ dysfunction, transfusion-dependent sideroblastic anemia, and other cytopenias [
      • Rotig A
      • Cormier V
      • Blanche S
      • et al.
      Pearson's marrow-pancreas syndrome. A multisystem mitochondrial disorder in infancy.
      ,
      • Farruggia P
      • Di Marco F
      • Dufour C
      Pearson syndrome.
      ]. The cause of the hematopoietic failure in PMPS is unknown and there are no experimental models to reproduce the specific defects in this disorders [
      • Farruggia P
      • Di Marco F
      • Dufour C
      Pearson syndrome.
      ]. A distinctive characteristic of PMPS is the varying levels of mutant mtDNA in different cells, which ultimately determines the severity of the clinical manifestations in individuals with the disease. Cherry et al. [
      • Cherry AB
      • Gagne KE
      • McLoughlin EM
      • et al.
      Induced pluripotent stem cells with a mitochondrial DNA deletion.
      ] generated iPSCs from three patients with PMPS (PMPS-iPSCs) and, despite technical challenges, were able to isolate PMPS-iPSCs that had no mutant mtDNA. All PMPS-iPS lines were capable of forming functional HSPCs; however, compared with isogenic PMPS-iPSC lines without mitochondrial mutations, samples carrying deleted mtDNA showed a trend toward reduced numbers of colonies. In addition, colonies carrying a high burden of mutant mtDNA, yielded high numbers of erythroid precursors with pathologic iron granule deposition compared with PMPS-iPSCs, with less mutant mtDNA [
      • Cherry AB
      • Gagne KE
      • McLoughlin EM
      • et al.
      Induced pluripotent stem cells with a mitochondrial DNA deletion.
      ]. These data demonstrate clonal variation in changes in mtDNA heteroplasmy during culture and recapitulate a tissue-specific phenotype by directed differentiation of iPSCs carrying mutant mtDNA. Although the pathogenic mechanisms underlying this disorder remain largely unknown, iPSC-derived models for PMPS and other disorders with mtDNA mutations open up new ways to use patient cells in research. This is particularly important considering the fact that targeted modification of the mitochondrial genome is technically challenging [
      • Hämäläinen RH
      Induced pluripotent stem cell-derived models for mtDNA diseases.
      ].

      Generation of iPSC clones from patients with acquired BMFS

      Paroxysmal nocturnal hemoglobinuria

      Human phosphatidylinositol glycan class A (PIGA)-null iPSCs were successfully engineered using plasmids expressing ZFN-mediated homologous recombination [
      • Zou J
      • Maeder ML
      • Mali P
      • et al.
      Gene targeting of a disease-related gene in human induced pluripotent stem and embryonic stem cells.
      ] or a knockout model [
      • Yuan X
      • Braunstein EM
      • Ye Z
      • et al.
      Generation of glycosylphosphatidylinositol anchor protein-deficient blood cells from human induced pluripotent stem cells.
      ], allowing for the production of glycosylphosphatidylinositol-anchored protein (GPI-AP)-deficient iPSCs for testing the importance of GPI-APs in hematopoiesis. First, Yuan et al. [
      • Yuan X
      • Braunstein EM
      • Ye Z
      • et al.
      Generation of glycosylphosphatidylinositol anchor protein-deficient blood cells from human induced pluripotent stem cells.
      ] examined the potential GPI-AP role in the generation of hematopoietic cells by using human iPSC lines derived from adult male dermal fibroblasts (hFib2-iPS5), a PIGA gene knockout model of the hFib2-iPS5 cells (PIGA-null iPSCs), and PIGA-null iPSCs reconstituted with PIGA transgene expression (iPSCs-PIGA). These PIGA-null iPSCs were unable to form embryonic bodies or to generate HSPCs or any cells expressing the CD59, CD34, and CD45 markers and were defective in generating mesodermal cells expressing kinase insert domain receptor [
      • Yuan X
      • Braunstein EM
      • Ye Z
      • et al.
      Generation of glycosylphosphatidylinositol anchor protein-deficient blood cells from human induced pluripotent stem cells.
      ]. Their biological and phenotypic defects were rescued by PIGA transgene expression. Interestingly, the investigators succeeded in establishing a paroxysmal nocturnal hemoglobinuria (PNH) disease model that could provide a limitless source of GPI-AP-deficient blood cells by transducing the PIGA-null iPSCs with a doxycycline (Dox)-inducible PIGA expression system. Culturing these CD59+ iPSCs in medium with Dox for up to 14 days followed by further culturing without Dox in medium supplemented with myeloid or erythroid-inducing cytokines allowed for the generation of both myeloid and erythroid lineages that were CD59 deficient [
      • Yuan X
      • Braunstein EM
      • Ye Z
      • et al.
      Generation of glycosylphosphatidylinositol anchor protein-deficient blood cells from human induced pluripotent stem cells.
      ]. These results confirmed that GPI-APs are critical for primitive hematopoiesis. Phondeechareon et al. [
      • Phondeechareon T
      • Wattanapanitch M
      • U-Pratya Y
      • et al.
      Generation of induced pluripotent stem cells as a potential source of hematopoietic stem cells for transplant in PNH patients.
      ] succeeded in establishing iPSCs derived from a PNH patient's dermal fibroblasts that capable of producing normal autologous HSPCs that could be used to treat PNH patients by autologous transplantation.

      Acquired aplastic anemia

      Recently, three iPSC lines were successfully generated from three severe AA (SAA) patients by Melguizo-Sanchis et al. [
      • Melguizo-Sanchis D
      • Xu Y
      • Taheem D
      • et al.
      iPSC modeling of severe aplastic anemia reveals impaired differentiation and telomere shortening in blood progenitors.
      ]. The iPSCs showed failure to elongate their telomeres during the reprogramming process and reduction in hematopoietic differentiation to erythroid and myeloid cells, which was not rescued by eltrombopag. This study suggests that some (not all) SAA cases could be related to an underlying genetic predisposition with negative effects on the proliferation or differentiation of myeloid and erythroid and cells.
      We previously reported that leukocytes lacking HLA class I alleles are frequently detectable in patients with aAA due to copy number neutral loss of heterozygosity of the short arm of chromosome 6 (6pLOH) or other allelic mutations [
      • Katagiri T
      • Sato-Otsubo A
      • Kashiwase K
      • et al.
      Frequent loss of HLA alleles associated with copy number-neutral 6pLOH in acquired aplastic anemia.
      ]. Whereas pediatric SAA can often be attributable to genetic causes, the HLA-lacking cells support the widely accepted immunological nature of aAA pathogenesis in both children and adults. Recently, we succeeded in reprogramming monocytes from a patient with aAA in remission to generate three different iPSC lines with unique HLA-B*40:02 mutations, including the start–loss mutation, in addition to WT and 6pLOH(+) iPSC clones. Monocytes from another aAA patient (non-SAA) were also successfully reprogrammed to generate three different iPSC clones with unique HLA-B*54:01 mutations, including nonsense and start codon mutations, in addition to WT and 6pLOH(+) iPSC clones [
      • Espinoza JL
      • Elbadry MI
      • Chonabayashi K
      • et al.
      Hematopoiesis by iPSC-derived hematopoietic stem cells of aplastic anemia that escape cytotoxic T-cell attack.
      ,
      • Nakagawa N
      • Elbadry MI
      • Akatsuka Y
      • et al.
      Identification of T cell receptors specific to antigens presented by HLA-B5401 on IPS cell-derived hematopoietic stem cells in a patient with acquired aplastic anemia carrying B5401-lacking leukocytes.
      ]. The generation of iPSC clones from monocytes lacking specific HLA alleles that are produced by the patients’ HSPCs during the development or the progression of this disease is consistent with the notion that a strong immune pressure directed toward HSPCs is involved in the pathogenesis of this disease.
      HSPCs derived from HLA-lacking iPSCs showed a unique HLA-gene expression pattern and different mutation types similar to the primary monocytes from the aAA patients. We observed an apparent higher expression of the retained HLA-A alleles in 6pLOH clones (probably as a compensatory response to the missing alleles) and no expression of HLA-A alleles in iPSC-HSPCs with 6pLOH. When these iPSC-HSPCs were transplanted to immunodeficient mice, the engraftment and blood-repopulating capacity were similar, regardless of the HLA expression, to WT, 6pLOH, and HLA-A(+)B(–) clones. Importantly, hematopoietic cells generated in the transplanted mice retained the phenotype of the original iPSC clones, indicating that this method could be an excellent tool for the study of aAA in vivo. Table 1 summarizes the successful reports of iPSC generation from patients with different BMFSs and the effects of the gene defect correction on the proliferation and hematopoietic differentiation of their iPSCs.
      Table 1Induction of iPSC-HSPCs from patients with inherited and acquired BMFSs
      BMFS typeCells usedGenes affectedReprogramming efficiencyFunctionality assayPathogenesisBlood repopulating capacityGenome-editing methodGene correction effectReference
      FAFA patients’ fibroblastsFANCA, and D2DecreasedIn vitro and in vivoDefect in DNA damage repairNo reduction in E/MLentiviruses vectorsMaintain a fully functional FA pathway
      • Raya A
      • Rodriguez-Piza I
      • Guenechea G
      • et al.
      Disease-corrected haematopoietic progenitors from Fanconi anaemia induced pluripotent stem cells.
      FA patients’ fibroblasts

      FA-mouse tail-tip fibroblasts
      FANCA, C, G, and D2

      FANCA, D2 and I
      Decreased

      Decreased
      In vitro

      In vitro
      No reduction in E/MNo

      No
      No

      No
      • Muller LU
      • Milsom MD
      • Harris CE
      • et al.
      Overcoming reprogramming resistance of Fanconi anemia cells.
      FA patients’ fibroblastsFANCA, C, and D2DecreasedIn vitroReduced E/M & increased HSPC apoptosisNoNo
      • Yung SK
      • Tilgner K
      • Ledran MH
      • et al.
      Brief report: human pluripotent stem cell models of fanconi anemia deficiency reveal an important role for fanconi anemia proteins in cellular reprogramming and survival of hematopoietic progenitors.
      FA patients’ fibroblastsFANCADecreasedIn vitroReduced E/M, neural and MSC differentiationHDAdVRescued thecell cycle and clonogenicity defects
      • Liu GH
      • Suzuki K
      • Li M
      • et al.
      Modelling Fanconi anemia pathogenesis and therapeutics using integration-free patient-derived iPSCs.
      FA patient's fibroblastFANCADecreasedIn vitroReduced E/M & endothelial lineagesNoNo
      • Suzuki NM
      • Niwa A
      • Yabe M
      • et al.
      Pluripotent cell models of fanconi anemia identify the early pathological defect in human hemoangiogenic progenitors.
      FA patient's fibroblastFANCADecreasedIn vitro and in vivoGeneration of squamous epithelial rafts and intestinal organoids but increase HSPC apoptosisActivated DNA damage response signaling through ATR and CHK1. by inhibition of CHK1 1Completely restored the growth of FA-deficientiPSCs through a remarkable rapid bypassof the G2-M checkpoint
      • Chlon TM
      • Wells SI
      • Ruiz-Torres S
      • Kuhar M
      • Wells JM
      Models of pluripotent and somatic stem cells to study tissue-specific sensitivities in Fanconi anemia.
      DKCDKC patients’ fibroblastsTERC 821 bp deletionSlow reprogramming capacityIn vitroTelomere attritionImpaired hematopoietic differentiationRetroviral vector and telomerase activationIncreased pluripotency, TERT and TERC upregulation
      • Agarwal S
      • Loh YH
      • McLoughlin EM
      • et al.
      Telomere elongation in induced pluripotent stem cells from dyskeratosis congenita patients.
      DKC patients’ fibroblastsTCAB1 mutationDecreasedIn vitro and in vivoProgressive telomere attritionImpaired hematopoietic differentiationReprogramming and telomerase activationNo TERT and TERC upregulation
      • Batista LF
      • Pech MF
      • Zhong FL
      • et al.
      Telomere shortening and loss of self-renewal in dyskeratosis congenita induced pluripotent stem cells.
      DKC patients’ fibroblastsTERT and TERCDecreasedIn vitroProgressivetelomere attritionImpaired hematopoietic differentiationReprogramming and telomerase activationNo TERT and TERC upregulation
      • Winkler T
      • Hong SG
      • Decker JE
      • et al.
      Defective telomere elongation and hematopoiesis from telomerase–mutant aplastic anemia iPSCs.
      SDSSDS patients’ fibroblastsSBDSDecreasedIn vitroRibosomopathyImpaired hematopoietic, pancreatic and ductal developmentlentiviral vector expressing human SBDS cDNARestored pancreatic structures, not enhance hematopoietic development
      • Tulpule A
      • Kelley JM
      • Lensch MW
      • et al.
      Pluripotent stem cell models of Shwachman–Diamond syndrome reveal a common mechanism for pancreatic and hematopoietic dysfunction.
      SDS patients’ bone marrow mononuclear cellsIVS2+2 T>C SBDS mutations

      IVS2+2 T>C SBDS mutations with deletion of 7q at locus (11.2)
      Decreased

      Markedly decreased
      In vitro

      In vitro
      Ribosomopathy

      Clonal evolution to MDS and AML
      The number of CD34+ cells were limiting

      Decreased hematopoietic differentiation and clonogenic capacity
      NoNo
      • Ruiz–Gutierrez M
      • Vargel Bolukbasi O
      • Vo L
      • et al.
      Modeling bone marrow failure and MDS in Shwachman–Diamond syndrome using induced pluripotent stem cells.
      DBADBA patients’ fibroblastsRPS19 and RPL5 mutationsInefficiently for some clones and normally for othersIn vitroRibosomopathyReduced hematopoiesisZENRestored ribosome assembly and hematopoiesis
      • Garcon L
      • Ge J
      • Manjunath SH
      • et al.
      Ribosomal and hematopoietic defects in induced pluripotent stem cells derived from Diamond–Blackfan anemia patients.
      DBA patients’ fibroblastsRPS19 and RPL5 mutationsNormal as healthy cellsIn vitro and in vivoReduced differentiate into CD71+glycophorin A erythroid cellsCRISPR/Cas9 and SMER28Restored erythroid differentiation
      • Doulatov S
      • Vo LT
      • Macari ER
      • et al.
      Drug discovery for Diamond–Blackfan anemia using reprogrammed hematopoietic progenitors.
      CAMTCAMT patients’ fibroblastsMPLDecreasedIn vitroLoss of MPL-mediated signalingFewer megakaryocytes, impaired MPP to MEP transitionTPO

      Retroviral-mediatedgene transfer
      No rescue of megakaryopoiesis

      Rescue of the hematopoietic phenotype
      • Hirata S
      • Takayama N
      • Jono-Ohnishi R
      • et al.
      Congenital amegakaryocytic thrombocytopenia iPS cells exhibit defective MPL-mediated signaling.
      FPDPeripheral FPD patients’ T cellsRUNX1Normal as healthy cellsIn vitroTranscription factorsEarly E/M was not affected with megakaryocytic differentiation was decreasedVector expressing Flag-tagged WT-RUNX1 transfectionRecovered the capacity todifferentiate into hematopoietic lineage
      • Sakurai M
      • Kunimoto H
      • Watanabe N
      • et al.
      Impaired hematopoietic differentiation of RUNX1–mutated induced pluripotent stem cells derived from FPD/AML patients.
      FPD patients’ fibroblastsRUNX1 (AML1 or (CBFA2)DecreasedIn vitroImpaired E/M progenitorZENsRescued megakaryocytes differentiation
      • Connelly JP
      • Kwon EM
      • Gao Y
      • et al.
      Targeted correction of RUNX1 mutation in FPD patient-specific induced pluripotent stem cells rescues megakaryopoietic defects.
      PMPSPMPS patients’ bone marrow–derived fibroblastsMitochondrial genetic (mtDNA) deletionDecreasedIn vitroMitochondrial dysfunctionGive rise to sideroblastic erythroid progenitorsNoNo
      • Cherry AB
      • Gagne KE
      • McLoughlin EM
      • et al.
      Induced pluripotent stem cells with a mitochondrial DNA deletion.
      PNHPIGA knockout model in human iPSCsPIGADecreasedIn vitro and in vivoCD55 and CD59 lacking leading to complement-mediated intravascular hemolysisFailure of Teratomas formation and production of CD34+ or CD45+ cells upon hematopoietic differentiationPIG-A ZEN transgene expressionRescued phenotypic and biological defects and teratomas formation containing cells representing all three embryonic germ layers
      • Yuan X
      • Braunstein EM
      • Ye Z
      • et al.
      Generation of glycosylphosphatidylinositol anchor protein-deficient blood cells from human induced pluripotent stem cells.
      AASevere AA patients’ dermal fibroblastsCN-LOH (3q11.2)

      CN-LOH (7q22.1)

      CN-LOH (11p11.12-q11)

      Deletion (15q13.3)

      Duplication (16p13.11)
      Less than normal cellsIn vitroAll iPSC-derived-HSPCs showed significantly shorter telomeres than undifferentiated iPSCsImpaired E/M differentiationTPOE/M proliferation and/or differentiation not rescued by TPO
      • Melguizo-Sanchis D
      • Xu Y
      • Taheem D
      • et al.
      iPSC modeling of severe aplastic anemia reveals impaired differentiation and telomere shortening in blood progenitors.
      aAAPeripheral very SAA patients’ monocytesStart codon mutation of HLA-B*40:02 allele

      6pLOH
      VariableIn vitro and in vivoImmune alterations (CTL capable of killing WT iCD34+ cells but not B4002(−) iCD34+ cellsE/M differentiationNoNo
      • Espinoza JL
      • Elbadry MI
      • Chonabayashi K
      • et al.
      Hematopoiesis by iPSC-derived hematopoietic stem cells of aplastic anemia that escape cytotoxic T-cell attack.
      aAA=Acquired aplastic anemia; AML=acute meloid leukemia; ATR=ataxia telangiectasia mutated and Rad3 related serine/threonine kinase; BMFS=bone marrow failure syndrome; CAMT=congenial amegakaryocytic thrombocytopenia; CHK1=checkpoint kinase 1; CN-LOH=copy number-neutral loss of heterozygosity; CRISPR=clustered regularly interspaced short palindromic repeats/Cas9 enzymes that can be used to edit genes within organisms; CTL=cytotoxic T cell; DBA=Diamond–Blackfan anemia; DKC=dyskeratosis congenital; E/M=erythroid and myeloid progenitor cells; FA=Fanconi anemia; HDAdVs=Helper-dependent adenoviral vectors; HSPCs=hematopoietic stem and progenitor cells; MDS=myelodysplastic syndrome; MPL=myeloproliferative leukemia virus oncogene; PIGA=phosphatidylinositol glycan class A; 6pLOH=loss of heterozygosity of the short arm of chromosome 6; PNH=paroxysmal nocturnal hemoglobinuria; RUNX1=Runt-related transcription factor 1; SAA=severe aplastic anemia; SBDS=Shwachman–Bodian–Diamond syndrome; SMER28=small-molecule enhancers of rapamycin; TERT=telomerase reverse transcriptase; TPO=thrombopoietin; WT=wild-type cells; ZFN=zinc finger nuclease; MSC=Mesenchymal stem cells.

      HLA lacking iPSC-derived CD34+ (iCD34+) cells from patients with aAA evade attack by autologous cytotoxic T cells specific to WT HSPCs

      HLA-lacking iCD34+ cells enabled us to study the phenotype of HSPCs and the influence of the immune system by examining the ability of patient-derived cytotoxic T lymphocytes (CTLs) to kill autologous HSPCs and to discriminate between HLA-retaining [HLA(+)] and HLA-lacking [HLA(–)] iCD34+ cells in coculture experiments (Figure 2). Stimulation of the patient's CD8+ T cells with the WT iCD34+ cells generated a CTL line capable of killing WT iCD34+ cells, but not B4002(–) iCD34+ cells [
      • Espinoza JL
      • Elbadry MI
      • Chonabayashi K
      • et al.
      Hematopoiesis by iPSC-derived hematopoietic stem cells of aplastic anemia that escape cytotoxic T-cell attack.
      ]. These data suggest that B4002(–) iCD34+ cells show a repopulating ability similar to WT iCD34+ cells when autologous T cells are absent and CTL precursors capable of selectively killing WT HSPCs are present in the patient's peripheral blood. Experiments using HLA-B5401(–) iCD34+ cells confirmed the ability of these cells to escape from autologous CTLs specific to WT HSPCs when the four T-cell receptor transfectants were tested for their response to WT and B5401-lacking iCD34+ cells [
      • Nakagawa N
      • Elbadry MI
      • Akatsuka Y
      • et al.
      Identification of T cell receptors specific to antigens presented by HLA-B5401 on IPS cell-derived hematopoietic stem cells in a patient with acquired aplastic anemia carrying B5401-lacking leukocytes.
      ]. These findings strongly indicate that HSPCs that deleted HLA alleles evade CTL attack and support the patient's hematopoiesis.
      Figure 2.
      Figure 2Disease model of aAA. Reprograming of somatic cells (monocytes) from patients with aAA harboring various HLA genotypes (WT-HLA, 6pLOH (+), or HLA-Class I mutant) to generate iPSCs with the corresponding genotypes has opened new opportunities for the study of the pathogenesis of this disorder. The differentiation of iPSCs into HSPCs and the transplantation of HSPCs into immunodeficient mice has been tested to verify the impact of HLA loss in the hematopoietic potential of HSPCs. The enrichment of CTLs from patients with aAA was used to verify that pathogenic CTLs are able to kill WT HSPCs but fail to recognize the HLA-lacking counterparts in vitro and the identification of the TCR spectrotypes likely involved in the recognition and killing of HSPCs. This model can be tested in vivo by injecting pathogenic CTLs into mice harboring iPSC-HSPCs. It is expected that this disease model would lead to better understating the pathogenesis of aAA, which eventually may lead to the development of novel therapeutic approaches such as cell therapies, therapeutic monoclonal antibodies, or therapeutic drugs.

      Role of iPSCs in studying clonal hematopoiesisin BMFSs

      DNMT3A, ASXL1, and BCOR/BCORL1 mutations, as well as 6pLOH and PIGA, are genetic abnormalities found in <40% of patients with aAA and the first three mutations involve genes commonly mutated in myeloid malignancies [
      • Ogawa S
      Clonal hematopoiesis in acquired aplastic anemia.
      ]. In addition, up to 20% of patients with aAA who do not receive allogeneic hematopoietic stem cell transplantation (HSCT) eventually develop MDS or AML [
      • Afable 2nd, MG
      • RV Tiu
      • Maciejewski JP
      Clonal evolution in aplastic anemia.
      ]. This indicates that the early identification of patients who will evolve to MDS could help make decision regarding the use of immunosuppressive therapy (IST) or HSCT; however, until now, the risk factors associated with progression to MDS or acute AML remain poorly defined. The generation of iPSCs carrying the previously mentioned mutations may be helpful for identifying the factors predisposing patients to MDS and AML progression. These include the chronological profiles, clone size, and role of autoimmunity in clonal selection. Although MDS with monosomy 7/del(7q) is a frequent clonal abnormality that emerges in the context of inherited BMFSs such as SDS, a critical question remains as to whether monosomy 7/del(7q) acts as a driver cause of MDS and leukemic risk or whether it is just an marker associated with clonal progression in BMFS. Kotini et al. [
      • Kotini AG
      • Chang CJ
      • Boussaad I
      • et al.
      Functional analysis of a chromosomal deletion associated with myelodysplastic syndromes using isogenic human induced pluripotent stem cells.
      ] reported that del(7q) iPSCs derived from BM cells of two patients with del(7q) MDS showed increased apoptosis with decreased proliferation and ineffective hematopoiesis, with markedly decreased clonogenic capacity. Ruiz-Gutierrez et al. [
      • Ruiz–Gutierrez M
      • Vargel Bolukbasi O
      • Vo L
      • et al.
      Modeling bone marrow failure and MDS in Shwachman–Diamond syndrome using induced pluripotent stem cells.
      ] succeeded in generating SDS del7q+ iPSCs with del(7q) models, which also showed markedly decreased hematopoietic differentiation compared with isogenic SDS-iPSCs without an increase in cell death. The previous approach highlights the usefulness of human iPSC-based disease modeling for studying the role of 7q loss in clonal evolution from BMFSs in addition to facilitating a functional mapping of large-scale chromosomal deletions linked to BMFSs. In addition, the use of in vitro cell cultures or in vivo animal models with iPSCs carrying these mutations would also be suitable to investigate the role of those genetic abnormalities in response to IST. Therefore, these mouse models engrafted with iPSCs carrying distinct mutations have a broad range of applications for the study of BMFSs and numerous benefits could be obtained from these models in investigations about the relationship among BMFSs, clonal evolution, and leukemic risk.

      Acknowledgments

      This work was supported by grants from the Ministry of Health , Labor, and Welfare; the Ministry of Education, Culture, Sports, and Technology; and a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science .

      Conflict of interest disclosure

      The authors declare no competing financial interests.

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      • Introduction
        Experimental HematologyVol. 71
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          The biomedical field has been periodically revolutionized by major discoveries. A notable example was Evans and Kaufman's [1] observation that cells from the inner cell mass of the mouse blastocyst could be propagated indefinitely in vitro with maintenance of their totipotentiality. The enormous potential of this technology to elucidate and manipulate processes regulating the development of the mouse was thus very rapidly realized. The subsequent creation of human embryonic stem cell (ESC) lines with analogous properties [2] expanded these avenues of experimental study to previously inaccessible stages of human development.
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