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Several decades have passed since the generation of the first embryonic stem cell (ESC) lines both in mice and in humans. Since then, stem cell biologists have tried to understand their potential biological and clinical uses for their implementation in regenerative medicine. The hematopoietic field was a pioneer in establishing the potential use for the development of blood cell products and clinical applications; however, early expectations have been truncated by the difficulty in generating bonafide hematopoietic stem cells (HSCs). Despite some progress in understanding the origin of HSCs during embryonic development, the reproduction of this process in vitro is still not possible, but the knowledge acquired in the embryo is slowly being implemented for mouse and human pluripotent stem cells (PSCs). In contrast, ESC-derived hematopoietic cells may recapitulate some leukemic transformation processes when exposed to oncogenic drivers. This would be especially useful to model prenatal leukemia development or other leukemia-predisposing syndromes, which are difficult to study. In this review, we will review the state of the art of the use of PSCs as a model for hematopoietic and leukemia development. This work was funded by grants from PID2019-104695RB-I00 and PDC2021-120817-I00from AgenciaEstatal de Investigación (AEI) to AB, ERA PerMed GATA2-HuMo Funding Mechanism (Spain: Acció instrumental de SLT011/18/00006 and SLT011/18/00007 of the Department of Health of the Government of Catalonia to AG and A, the Spanish Ministry of Economy, Industry, and Competitiveness (MINECO PID2020-15591RB-100), La Marató de TV3 (202001-32) and CERCA Programme/Generalitat de Catalunya for institutional support to AG. LGP is a recipient of 2021 FI_B2 00188, AGAUR, Generalitat de Cataluña.

The importance of predisposition to leukaemia in clinical practice is being increasingly recognized. This is emphasized by the establishment of a novel WHO disease category in 2016 called "myeloid neoplasms with germline predisposition". A major syndrome within this group is GATA2 deficiency, a heterogeneous immunodeficiency syndrome with a very high lifetime risk to develop myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). GATA2 deficiency has been identified as the most common hereditary cause of MDS in adolescents with monosomy 7. Allogenic haematopoietic stem cell transplantation is the only curative option; however, chances of survival decrease with progression of immunodeficiency and MDS evolution. Penetrance and expressivity within families carrying GATA2 mutations is often variable, suggesting that co-operating extrinsic events are required to trigger the disease. Predictive tools are lacking, and intrafamilial heterogeneity is poorly understood; hence there is a clear unmet medical need. On behalf of the ERAPerMed GATA2 HuMo consortium, in this review we describe the genetic, clinical, and biological aspects of familial GATA2-related MDS, highlighting the importance of developing robust disease preclinical models to improve early detection and clinical decision-making of GATA2 carriers. This work was supported by ERA PerMed GATA2-HuMo Funding Mechanism (Spain: Acció instrumental de SLT011/18/00006 of the Department of Health of the Government of Catalonia, to AG and AB; Hungary: ED-18-1-2019-001 grant from the National Research, Development and Innovation Office to CB; and Germany: German Federal Ministry of Education and Research (BMBF) 2018-123/01KU1904 to MWW), ÚNKP-21-2-I-SE-21 and Hungarian National Academy of Scientist Education grant to KL, TKP2021-NVA-15, TKP2021-EGA-24 and EU's Horizon 2020 research and innovation programme under grant agreement no. 739593 and Elixir Hungary to CB, MSCA No 101029927-scGATA2track (H2020-MSCA-IF-2020) to OM-B, the Spanish Ministry of Economy, Industry, and Competitiveness (MINECO PID2020-15591RB-100), La Marató de TV3 (202001-32), FPS Grant 2018 by Fondazione Pisana per la Scienza ONLUS and CERCA Programme/Generalitat de Catalunya for institutional support to AG, Vera and Joseph Dresner Foundation, Evans MDS Foundation, American Lebanese Syrian Associated Charities, and BMBF MyPred 01GM1911A to MWW. Open access funding enabled and organized by ProjektDEAL.

Germline SAMD9 mutations are one of the most common alterations that predispose to pediatric myelodysplastic syndrome (MDS), a clonal disorder characterized by ineffective hematopoiesis, increasing the risk of developing acute myeloid leukemia (AML). Up to date, a disease model to study the role of SAMD9 mutation in MDS is still lacking. Here, we have generated a human induced pluripotent stem cell (hiPSC) line carrying SAMD9mut (p.I1567M), taking advantage of CRISPR/Cas9 system. As a result, the genetic engineered hiPSC line represent a new in vitro disease model to understand the impact of SAMD9 mutation at molecular and cellular level during hematopoiesis.

Germline heterozygous GATA2 mutations underlie a complex disorder characterized by bone marrow failure, immunodeficiency and high risk to develop myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Our understanding about GATA2 deficiency is limited due to the lack of relevant disease models. Here we generated high quality human induced pluripotent stem cell (iPSC) lines carrying two of the most recurrent germline GATA2 mutations (R389W and R396Q) associated with MDS, using CRISPR/Cas9. These hiPSCs represent an in vitro model to study the molecular and cellular mechanisms underlying GATA2 deficiency, when differentiated into blood progenitors.

Lung cancer is the leading cause of cancer deaths. Tumor heterogeneity, which hampers development of targeted therapies, was herein deconvoluted via single cell RNA sequencing in aggressive human adenocarcinomas (carrying Kras-mutations) and comparable murine model. We identified a tumor-specific, mutant-KRAS-associated subpopulation which is conserved in both human and murine lung cancer. We previously reported a key role for the oncogene BMI-1 in adenocarcinomas. We therefore investigated the effects of in vivo PTC596 treatment, which affects BMI-1 activity, in our murine model. Post-treatment, MRI analysis showed decreased tumor size, while single cell transcriptomics concomitantly detected near complete ablation of the mutant-KRAS-associated subpopulation, signifying the presence of a pharmacologically targetable, tumor-associated subpopulation. Our findings therefore hold promise for the development of a targeted therapy for KRAS-mutant adenocarcinomas., Maroni, Bassal, Krishnan et al. characterise human non-small cell lung cancer (NSCLC) carrying Kras-mutations by single-cell RNA sequencing. They identify a tumour-specific population that is conserved in mice and responds to the drug, PTC596, which is currently in clinical trials and may offer a potential avenue for treating aggressive NSCLC expressing mutated Kras.

Summary In vertebrates, GATA2 is a master regulator of hematopoiesis and is expressed throughout embryo development and in adult life. Although the essential role of GATA2 in mouse hematopoiesis is well established, its involvement during early human hematopoietic development is not clear. By combining time-controlled overexpression of GATA2 with genetic knockout experiments, we found that GATA2, at the mesoderm specification stage, promotes the generation of hemogenic endothelial progenitors and their further differentiation to hematopoietic progenitor cells, and negatively regulates cardiac differentiation. Surprisingly, genome-wide transcriptional and chromatin immunoprecipitation analysis showed that GATA2 bound to regulatory regions, and repressed the expression of cardiac development-related genes. Moreover, genes important for hematopoietic differentiation were upregulated by GATA2 in a mostly indirect manner. Collectively, our data reveal a hitherto unrecognized role of GATA2 as a repressor of cardiac fates, and highlight the importance of coordinating the specification and repression of alternative cell fates., Graphical Abstract, Highlights • GATA2 promotes hemogenic emergence during human iPSC differentiation • GATA2 enhances hematopoietic differentiation of human iPSCs • GATA2 acts as a direct repressor of cardiac fates during mesoderm specification, Giorgetti A. and colleagues demonstrate that GATA2 induction in early mesodermal cells leads to the robust generation of hemogenic endothelial and hematopoietic cells from human iPSCs. They show the ability of GATA2 to instruct mesodermal precursors toward a hematopoietic cell fate and concurrently inhibit cardiac fates. This study expands our understanding of the regulatory networks that control early human hematopoiesis.

We have generated two human induced pluripotent stem cell (iPSC) lines from CD133

et al., Coenzyme Q (CoQ) plays a crucial role in mitochondria as an electron carrier within the mitochondrial respiratory chain (MRC) and is an essential antioxidant. Mutations in genes responsible for CoQ biosynthesis (COQ genes) cause primary CoQ deficiency, a rare and heterogeneous mitochondrial disorder with no clear genotype–phenotype association, mainly affecting tissues with high-energy demand including brain and skeletal muscle (SkM). Here, we report a four-year-old girl diagnosed with minor mental retardation and lethal rhabdomyolysis harboring a heterozygous mutation (c.483G > C (E161D)) in COQ4. The patient's fibroblasts showed a decrease in [CoQ], CoQ biosynthesis, MRC activity affecting complexes I/II + III, and respiration defects. Bona fide induced pluripotent stem cell (iPSCs) lines carrying the COQ4 mutation (CQ4-iPSCs) were generated, characterized and genetically edited using the CRISPR-Cas9 system (CQ4-iPSCs). Extensive differentiation and metabolic assays of control-iPSCs, CQ4-iPSCs and CQ4-iPSCs demonstrated a genotype association, reproducing the disease phenotype. The COQ4 mutation in iPSC was associated with CoQ deficiency, metabolic dysfunction, and respiration defects. iPSC differentiation into SkM was compromised, and the resulting SkM also displayed respiration defects. Remarkably, iPSC differentiation in dopaminergic or motor neurons was unaffected. This study offers an unprecedented iPSC model recapitulating CoQ deficiency-associated functional and metabolic phenotypes caused by COQ4 mutation., This work was supported by the ISCIII/FEDER (E-Rare-2 Call PI12/03112 to P.M.), FIS/ISCIII/FEDER project (PI14/01962 to P.N.) and the European Research Council (ERC-2014-CoG646903 to P.M.). D.R.M. and C.P. are supported by PFIS scholarships (FI11/0511 and FI12/00468, respectively). C.B is supported by a Miguel Servet II contract (CPII13/00011). P.M. also acknowledges the financial support from The Obra Social La Caixa-Fundacio Josep Carreras and The Generalitat de Catalunya (SGR330). P.M. and J.L.-B. are investigators of the Spanish Cell Therapy cooperative network (TERCEL). A.G. is supported by Ramon y Cajal Program (RyC-2013–13221).

Recent studies in zebrafish and mice have revealed that proinflammatory signaling is a positive regulator of definitive hematopoietic development. Whether proinflammatory signaling also regulates human hematopoietic specification remains unknown. Here, we explored the impact of the proinflammatory cytokines tumor necrosis factor-α (TNFα), interferon-γ (IFNγ), and interleukin-1β (IL1β) on in vitro hematopoietic differentiation using human pluripotent stem cells. Gene expression analysis and enzyme-linked immunosorbent assay revealed the absence of a proinflammatory signature during hematopoietic development of human pluripotent stem cells. Functionally, the emergence of hemogenic endothelial progenitors (CD31+CD34+CD45- or CD34+CD43-CD73-) and hematopoietic cells (CD43+CD45+) was not affected by treatment with increasing doses of TNFα, IFNγ, and IL1β irrespective of the developmental window or the differentiation protocol used (embryoid body or OP9 co-culture based). Similarly, knockdown of endogenous NF-kB signaling had no impact on hematopoietic differentiation of human pluripotent stem cells. This study serves as a demonstration that TNFα, IFNγ, and IL1β signals do not improve hematopoietic differentiation of human pluripotent stem cells using current protocols and suggests that proinflammatory signaling is insufficient to drive definitive hematopoietic specification of human hematopoietic stem cells in vitro. This work was supported by the European Research Council (646903 to PM) and the Spanish Ministry of Economy and Competitiveness (SAF2013-43072R to AG, SAF2013-43065R to PM, and ISCIII/FEDER-PI14/01119 to CB). CB is supported by a Miguel Servet contract (CPII13/00011). AG is supported by the Ramon y Cajal Program (RyC-2013-13221). PM also acknowledges support from Obra Social La Caixa-Fundacio Josep Carreras and Generalitat de Catalunya (SGR330).

Coenzyme Q

Summary Induced pluripotent stem cells (iPSCs) are a powerful tool for disease modeling. They are routinely generated from healthy donors and patients from multiple cell types at different developmental stages. However, reprogramming leukemias is an extremely inefficient process. Few studies generated iPSCs from primary chronic myeloid leukemias, but iPSC generation from acute myeloid or lymphoid leukemias (ALL) has not been achieved. We attempted to generate iPSCs from different subtypes of B-ALL to address the developmental impact of leukemic fusion genes. OKSM(L)-expressing mono/polycistronic-, retroviral/lentiviral/episomal-, and Sendai virus vector-based reprogramming strategies failed to render iPSCs in vitro and in vivo. Addition of transcriptomic-epigenetic reprogramming “boosters” also failed to generate iPSCs from B cell blasts and B-ALL lines, and when iPSCs emerged they lacked leukemic fusion genes, demonstrating non-leukemic myeloid origin. Conversely, MLL-AF4-overexpressing hematopoietic stem cells/B progenitors were successfully reprogrammed, indicating that B cell origin and leukemic fusion gene were not reprogramming barriers. Global transcriptome/DNA methylome profiling suggested a developmental/differentiation refractoriness of MLL-rearranged B-ALL to reprogramming into pluripotency., Graphical Abstract, Highlights • Neither primary B-ALL blasts nor leukemic B cell lines can be reprogrammed to iPSCs • Global transcriptome and DNA methylome suggest a developmental refractoriness, Despite the interest in generating iPSCs from human primary acute leukemias for disease modeling, reprogramming leukemias is an extremely inefficient process. In this article, Menéndez, Bueno, and colleagues show that many reprogramming strategies reported to date are not sufficient to generate B-ALL-derived iPSCs. Global transcriptome/DNA methylome profiling suggested a developmental/differentiation refractoriness of B-ALL to reprogramming into pluripotency.

Induced pluripotent stem cells (iPSCs) represent an invaluable tool in a chromosomal instability syndrome such as Fanconi anemia (FA), as they can allow to study of the molecular defects underlying this disease. Many other applications, such as its use as a platform to test different methods or compounds, could also be of interest. But the greatest impact of iPSCs may be in bone marrow failure diseases, as iPSCs could represent an unlimited source of autologous cells to apply in advanced treatments such as gene therapy. At the same time, genome editing constitutes the next generation of technology to further develop a safer, personalized, targeted gene therapy. Despite the promising advantages that these two technologies would present in a disease such as FA, the specific characteristics of the disease make both of these processes especially challenging. Efficient and safer FA-hiPSC (human induced pluripotent stem cell) generation methods, robust and reliable differentiation protocols for iPSCs, as well as really efficient delivery methods to perform targeted gene correction should be developed.

Summary Neurons obtained directly from human somatic cells hold great promise for disease modeling and drug screening. Available protocols rely on overexpression of transcription factors using integrative vectors and are often slow, complex, and inefficient. We report a fast and efficient approach for generating induced neural cells (iNCs) directly from human hematopoietic cells using Sendai virus. Upon SOX2 and c-MYC expression, CD133-positive cord blood cells rapidly adopt a neuroepithelial morphology and exhibit high expansion capacity. Under defined neurogenic culture conditions, they express mature neuronal markers and fire spontaneous action potentials that can be modulated with neurotransmitters. SOX2 and c-MYC are also sufficient to convert peripheral blood mononuclear cells into iNCs. However, the conversion process is less efficient and resulting iNCs have limited expansion capacity and electrophysiological activity upon differentiation. Our study demonstrates rapid and efficient generation of iNCs from hematopoietic cells while underscoring the impact of target cells on conversion efficiency., Graphical Abstract, Highlights • Expandable iNCs are generated from CB cells by SeV at high efficiency • CB-iNCs differentiate into functional neurons in vitro • CB stem/progenitor cells are more amenable to neural conversion than PB-MNCs, Giorgetti, Sanchez-Pernaute and colleagues report a fast and efficient approach for generating functional induced neurons from both cord blood and peripheral blood cells using SOX2 and c-MYC Sendai virus. However, the reprogramming of adult peripheral blood cells is less efficient. This study provides additional evidence that the origin and the nature of target cells may impact the outcome of the conversion process.

Access to healthy or diseased human neural tissue is a daunting task and represents a barrier for advancing our understanding about the cellular, genetic, and molecular mechanisms underlying neurogenesis and neurodegeneration. Reprogramming of somatic cells to pluripotency by transient expression of transcription factors was achieved a few years ago. Induced pluripotent stem cells (iPSC) from both healthy individuals and patients suffering from debilitating, life-threatening neurological diseases have been differentiated into several specific neuronal subtypes. An alternative emerging approach is the direct conversion of somatic cells (i.e., fibroblasts, blood cells, or glial cells) into neuron-like cells. However, to what extent neuronal direct conversion of diseased somatic cells can be achieved remains an open question. Optimization of current expansion and differentiation approaches is highly demanded to increase the differentiation efficiency of specific phenotypes of functional neurons from iPSCs or through somatic cell direct conversion. The realization of the full potential of iPSCs relies on the ability to precisely modify specific genome sequences. Genome editing technologies including zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat/CAS9 RNA-guided nucleases have progressed very fast over the last years. The combination of genome-editing strategies and patient-specific iPSC biology will offer a unique platform for in vitro generation of diseased and corrected neural derivatives for personalized therapies, disease modeling and drug screening. Stem Cells 2014;32:2811–2817

The finding that certain somatic cells can be directly converted into cells of other lineages by the delivery of specific sets of transcription factors paves the way to novel therapeutic applications. Here we show that human cord blood (CB) CD133 + cells lose their hematopoietic signature and are converted into CB-induced neuronal-like cells (CB-iNCs) by the ectopic expression of the transcription factor Sox2, a process that is further augmented by the combination of Sox2 and c-Myc. Gene-expression analysis, immunophenotyping, and electrophysiological analysis show that CB-iNCs acquire a distinct neuronal phenotype characterized by the expression of multiple neuronal markers. CB-iNCs show the ability to fire action potentials after in vitro maturation as well as after in vivo transplantation into the mouse hippocampus. This system highlights the potential of CB cells and offers an alternative means to the study of cellular plasticity, possibly in the context of drug screening research and of future cell-replacement therapies.

Generation of human induced pluripotent stem cells (hiPSCs) by the expression of specific transcription factors depends on successful epigenetic reprogramming to a pluripotent state. Although hiPSCs and human embryonic stem cells (hESCs) display a similar epigenome, recent reports demonstrated the persistence of specific epigenetic marks from the somatic cell type of origin and aberrant methylation patterns in hiPSCs. However, it remains unknown whether the use of different somatic cell sources, encompassing variable levels of selection pressure during reprogramming, influences the level of epigenetic aberrations in hiPSCs. In this work, we characterized the epigenomic integrity of 17 hiPSC lines derived from six different cell types with varied reprogramming efficiencies. We demonstrate that epigenetic aberrations are a general feature of the hiPSC state and are independent of the somatic cell source. Interestingly, we observe that the reprogramming efficiency of somatic cell lines inversely correlates with the amount of methylation change needed to acquire pluripotency. Additionally, we determine that both shared and line-specific epigenetic aberrations in hiPSCs can directly translate into changes in gene expression in both the pluripotent and differentiated states. Significantly, our analysis of different hiPSC lines from multiple cell types of origin allow us to identify a reprogramming-specific epigenetic signature comprised of nine aberrantly methylated genes that is able to segregate hESC and hiPSC lines regardless of the somatic cell source or differentiation state.

Induced pluripotent stem cells (iPSC ) provide an invaluable resource for regenerative medicine as they allow the generation/nof patient-specific progenitors with potential value for cell therapy. However, in many instances, an off-the-shelf approach is/ndesirable, such as for cell therapy of acute conditions or when the patient’s somatic cells are altered as a consequence of a chronic/ndisease or aging. Cord blood (CB) stem cells appear ideally suited for this purpose as they are young cells expected to carry/nminimal somatic mutations and possess the immunological immaturity of newborn cells; additionally, several hundred thousand/nimmunotyped CB units are readily available through a worldwide network of CB banks. Here we present a detailed protocol for the/nderivation of CB stem cells and how they can be reprogrammed to pluripotency by retroviral transduction with only two factors/n(OCT 4 and SO X2) in 2 weeks and without the need for additional chemical compounds.

Mouse and human fibroblasts were the first cell types successfully reprog- rammed by ectopic expression of OCT4, SOX2, KLF4, and c-MYC (OSKM) (Lowry et al., 2008; Maherali et al., 2007; Park et al., 2008; Takahashi et al., 2007; Taka- hashi and Yamanaka, 2006; Yu et al., 2007). Further studies have shown that the age, origin, and cell type used have a deep impact on the reprogramming effi- ciency, eventually requiring the expres- sion of fewer factors and/or reducing the timing of the whole process. In general, stem cells are rare and difficult to access and isolate in large numbers (neural stem cells, for instance [Kim et al., 2008, 2009c]) and, therefore, represent a com- plicated target for reprogramming. How- ever, Cord Blood (CB) could represent an alternative and readily accessible source of stem cells. Here, we describe reprog- ramming of CB cells to pluripotency by retroviral transduction of four (OSKM), three (OSK), and as few as two (OS) tran- scription factors, without the need for additional chemical compounds.

The majority of clinical studies on endothelial progenitor cells (EPCs) focuses on the role of these cells in cardiovascular diseases and no systematic studies exist regarding their variations in healthy subjects. In order to define the burden of angiogenesis in physiological conditions we assessed the frequency of peripheral blood endothelial colonies (PB-ECs) and their relation with other factors possibly involved in their function such as high-sensitivity C-reactive protein (hs-CRP), endothelial cell-specific mitogen factor (VEGF) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in a highly selected healthy population. A PB sample was obtained from 37/47 healthy subjects (age 40.2+/-15.0yrs; M/F 15/22) without known cardiovascular risk factors. The serum level of hs-CRP, VEGF, TIMP-1, the frequency of PB-ECs by clonogenic assay, and the number of early EPCs and late EPCs by flow cytometry analysis were evaluated. PB-ECs were formed by 40.5% of studied subjects with a mean of 0.40+/-0.82 colonies/10(6) cells. The differences in the frequency of colony formation between genders were not statistically significant. The subjects with PB-ECs were characterized by higher values of hs-CRP, when compared with those not forming colonies, 0.276+/-0.230 vs 0.095+/-0.077 mg/l (p=0.003) respectively, and of VEGF, 328.3+/-162.9 vs 202.68+/-118.53 pg/ml (p=0.02). No significant differences were found in TIMP-1 values. The EPC clonogenic potential seems to be related to hs-CRP and VEGF levels even in healthy population supporting the concept that these mediators are involved in physiological ECs function.

Circulating endothelial progenitor cells (EPCs) are believed to contribute to vascular homeostasis; unfortunately, the response of EPCs in physiological conditions remains largely unknown. Herein we report our observations of a 44-year-old healthy subject after a trek in the Himalayas that support high-altitude hypoxia and exercise oxygen demands are strong stimuli for clonogenic endothelial cell activation and activity, as shown by the increase in the number of mature EPCs and in the endothelial colony-forming unit capacity. Both of these effects were completely reverted at sea level, 45 days after the subject's trek. Am. J. Hematol. 79:76-78, 2005. 2005 Wiley-Liss, In

Traditionally, somatic tissue-derived stem cells of mammalian adults have been viewed as pluripotent precursors capable of lifelong maintenance of cellular compartments typical of the tissue in which they reside. However in recent years, in vitro cultures and in vivo transplantation assays have indicated that adult somatic stem cell of various species are capable of adopting multiple fates. Bone marrow cells can give rise to a wide array of phenotypes, including blood, endothelial, bone, cartilage, fat, tendon, lung, liver, muscle, marrow stroma, and even brain cells. Conversely, neural stem cells as well as progenitors present in the muscle may contribute to blood cell production, indicating that adult stem cells present in numerous tissues may generate multiple cell types even of different dermal origin. Therefore, the developmental potential of adult somatic stem cells might be reassessed, although the mechanisms that ultimately lead to determination of cell fate are not completely defined. The successful long-term culturing and expansion of somatic adult stem cells together with their intrinsic versatility leads to future hope of stem cell therapeutic use in a wide spectrum of diseases and disorders of several, even not easily accessible, tissues.

Recent studies indicate that human-induced pluripotent stem cells contain genomic structural variations and point mutations in coding regions. However, these studies have focused on fibroblast-derived human induced pluripotent stem cells, and it is currently unknown whether the use of alternative somatic cell sources with varying reprogramming efficiencies would result in different levels of genetic alterations. Here we characterize the genomic integrity of eight human induced pluripotent stem cell lines derived from five different non-fibroblast somatic cell types. We show that protein-coding mutations are a general feature of the human induced pluripotent stem cell state and are independent of somatic cell source. Furthermore, we analyse a total of 17 point mutations found in human induced pluripotent stem cells and demonstrate that they do not generally facilitate the acquisition of pluripotency and thus are not likely to provide a selective advantage for reprogramming.

Gamete failure-derived infertility affects millions of people worldwide; for many patients, gamete donation by unrelated donors is the only available treatment. Embryonic stem cells (ESCs) can differentiate in vitro into germ-like cells, but they are genetically unrelated to the patient. Using an in vitro protocol that aims at recapitulating development, we have achieved, for the first time, complete differentiation of human induced pluripotent stem cells (hiPSCs) to postmeiotic cells. Unlike previous reports using human ESCs, postmeiotic cells arose without the over-expression of germline related transcription factors. Moreover, we consistently obtained haploid cells from hiPSCs of different origin (keratinocytes and cord blood), produced with a different number of transcription factors, and of both genetic sexes, suggesting the independence of our approach from the epigenetic memory of the reprogrammed somatic cells. Our work brings us closer to the production of personalized human gametes in vitro.