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To survey cancer-related mutations in human pluripotent stem cells and their derivatives, we analyzed >2,200 transcriptomes from 146 independent lines in the NCBI's Sequence Read Archive. Twenty-two per cent of samples had at least one cancer-related mutation; of these, 64% had TP53 mutations, which conferred a pronounced selective advantage, perturbed target gene expression and altered cellular differentiation. These findings underscore the need for robust surveillance of cancer-related mutations in pluripotent cells, especially in clinical applications.
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Human pluripotent stem cells (hPSCs) hold a central role in studying human development, in disease modeling and in regenerative medicine. These cells not only acquire genetic modifications when kept in culture, but they may also harbor epigenetic aberrations, mainly involving parental imprinting and X-chromosome inactivation. Here we present a detailed bioinformatic protocol for detecting such aberrations using RNA sequencing data. We provide a pipeline designed to process and analyze RNA sequencing data for the identification of abnormal biallelic expression of imprinted genes, and thus detect loss of imprinting. Furthermore, we show how to differentiate among X-chromosome inactivation, full activation and aberrant erosion of X chromosome in female hPSCs. In addition to providing bioinformatic tools, we discuss the impact of such epigenetic variations in hPSCs on their utility for various purposes. This pipeline can be used by any user with basic understanding of the Linux command line. It is available on GitHub as a software container ( https://github.com/Gal-Keshet/EpiTyping ) and produces reliable results in 1-4 d.
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Metilación de ADN , Impresión Genómica , Humanos , Femenino , RNA-Seq , Inactivación del Cromosoma X/genética , CromosomasRESUMEN
The laboratory culture of human stem cells seeks to capture a cellular state as an in vitro surrogate of a biological system. For the results and outputs from this research to be accurate, meaningful, and durable, standards that ensure reproducibility and reliability of the data should be applied. Although such standards have been previously proposed for repositories and distribution centers, no widely accepted best practices exist for laboratory research with human pluripotent and tissue stem cells. To fill that void, the International Society for Stem Cell Research has developed a set of recommendations, including reporting criteria, for scientists in basic research laboratories. These criteria are designed to be technically and financially feasible and, when implemented, enhance the reproducibility and rigor of stem cell research.
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Investigación con Células Madre , Humanos , Reproducibilidad de los ResultadosRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019, has become a global health concern. Therefore, there is an immense need to understand the network of virus-host interactions by using human disease-relevant cells. We have thus conducted a loss-of-function genome-wide screen using haploid human embryonic stem cells (hESCs) to identify genes involved in SARS-CoV-2 infection. Although the undifferentiated hESCs are resistant to SARS-CoV-2, their differentiated definitive endoderm (DE) progenies, which express high levels of ACE2, are highly sensitive to the virus. Our genetic screening was able to identify the well-established entry receptor ACE2 as a host factor, along with additional potential novel modulators of SARS-CoV-2. Two such novel screen hits, the transcription factor MAFG and the transmembrane protein TMEM86A, were further validated as conferring resistance against SARS-CoV-2 by using CRISPR-mediated mutagenesis in hESCs, followed by differentiation of mutant lines into DE cells and infection by SARS-CoV-2. Our genome-wide genetic screening investigated SARS-CoV-2 host factors in non-cancerous human cells with endogenous ACE2 expression, providing a unique platform to identify novel modulators of SARS-CoV-2 cytopathology in human cells.
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COVID-19 , SARS-CoV-2 , Humanos , Interacciones Microbiota-Huesped , Enzima Convertidora de Angiotensina 2/genética , COVID-19/genética , Diferenciación Celular/genéticaRESUMEN
Retinoblastoma (Rb) is a rare malignant disorder affecting the developing retina of children under the age of five. Chemotherapeutic agents used for treating Rb have been associated with defects of the retinal pigment epithelium (RPE), such as hyperplasia, gliosis, and mottling. Herein, we have developed two pluripotent stem cell (PSC)-RPE models to assess the cytotoxicity of known Rb chemotherapeutics such as Melphalan, Topotecan and TW-37. Our findings demonstrate that these drugs alter the RPE by decreasing the monolayer barrier's trans-epithelial resistance and affecting the cells' phagocytic activity. Transcriptional analyses demonstrate an altered expression of genes involved in melanin and retinol processing, tight junction and apical-basal polarity pathways in both models. When applied within the clinical range, none of the drug treatments caused significant cytotoxic effects, changes to the apical-basal polarity, tight junction network or cell cycle. Together, our results demonstrate that although the most commonly used Rb chemotherapeutic drugs do not cause cytotoxicity in RPE, their application in vitro leads to compromised phagocytosis and strength of the barrier function, in addition to changes in gene expression that could alter the visual cycle in vivo. Our data demonstrate that widely used Rb chemotherapeutic drugs can have a deleterious impact on RPE cells and thus great care has to be exercised with regard to their delivery so the adjacent healthy RPE is not damaged during the course of tumor eradication.
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Neoplasias de la Retina , Retinoblastoma , Niño , Humanos , Epitelio Pigmentado de la Retina/metabolismo , Retinoblastoma/tratamiento farmacológico , Retinoblastoma/genética , Retinoblastoma/metabolismo , Retina , Neoplasias de la Retina/tratamiento farmacológico , Neoplasias de la Retina/genética , Neoplasias de la Retina/metabolismo , Expresión Génica , Diferenciación CelularRESUMEN
ATM depletion is associated with the multisystemic neurodegenerative syndrome ataxia-telangiectasia (A-T). The exact linkage between neurodegeneration and ATM deficiency has not been established yet, and no treatment is currently available. In this study, we aimed to identify synthetic viable genes in ATM deficiency to highlight potential targets for the treatment of neurodegeneration in A-T. We inhibited ATM kinase activity using the background of a genome-wide haploid pluripotent CRISPR/Cas9 loss-of-function library and examined which mutations confer a growth advantage on ATM-deficient cells specifically. Pathway enrichment analysis of the results revealed the Hippo signaling pathway as a major negative regulator of cellular growth upon ATM inhibition. Indeed, genetic perturbation of the Hippo pathway genes SAV1 and NF2, as well as chemical inhibition of this pathway, specifically promoted the growth of ATM-knockout cells. This effect was demonstrated in both human embryonic stem cells and neural progenitor cells. Therefore, we suggest the Hippo pathway as a candidate target for the treatment of the devastating cerebellar atrophy associated with A-T. In addition to the Hippo pathway, our work points out additional genes, such as the apoptotic regulator BAG6, as synthetic viable with ATM-deficiency. These genes may help to develop drugs for the treatment of A-T patients as well as to define biomarkers for resistance to ATM inhibition-based chemotherapies and to gain new insights into the ATM genetic network.
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Ataxia Telangiectasia , Células Madre Embrionarias Humanas , Humanos , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Vía de Señalización Hippo , Células Madre Embrionarias Humanas/metabolismo , Redes Reguladoras de Genes , Ataxia Telangiectasia/tratamiento farmacológico , Chaperonas Moleculares/metabolismoRESUMEN
Anticancer drugs are at the frontline of cancer therapy. However, innate resistance to these drugs occurs in one-third to one-half of patients, exposing them to the side effects of these drugs with no meaningful benefit. To identify the genes and pathways that confer resistance to such therapies, we performed a genome-wide screen in haploid human embryonic stem cells (hESCs). These cells possess the advantage of having only one copy of each gene, harbour a normal karyotype, and lack any underlying point mutations. We initially show a close correlation between the potency of anticancer drugs in cancer cell lines to those in hESCs. We then exposed a genome-wide loss-of-function library of mutations in all protein-coding genes to 10 selected anticancer drugs, which represent five different mechanisms of drug therapies. The genetic screening enabled us to identify genes and pathways which can confer resistance to these drugs, demonstrating several common pathways. We validated a few of the resistance-conferring genes, demonstrating a significant shift in the effective drug concentrations to indicate a drug-specific effect to these genes. Strikingly, the p53 signalling pathway seems to induce resistance to a large array of anticancer drugs. The data shows dramatic effects of loss of p53 on resistance to many but not all drugs, calling for clinical evaluation of mutations in this gene prior to anticancer therapy.
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Antineoplásicos , Células Madre Embrionarias Humanas , Humanos , Células Madre Embrionarias Humanas/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Haploidia , Antineoplásicos/farmacología , Antineoplásicos/metabolismo , MutaciónRESUMEN
Humans are diploid organisms, and triploidy in human embryos is responsible for â¼10% of spontaneous miscarriages. Surprisingly, some pregnancies proceed to triploid newborns that suffer from many neuro-developmental disorders. To investigate the impact of triploidy on human development, we generate triploid human embryonic stem cells (hESCs) by fusing isogenic haploid and diploid hESCs. Comparison of the transcriptome, methylome, and genome-wide replication timing shows general similarity between diploid and triploid hESCs. However, triploid cells have a larger volume than diploid cells, demonstrating decreased surface-area-to-volume ratio. This leads to a significant downregulation of cell surface ion channel genes, which are more essential in neural progenitors than in undifferentiated cells, leading to inhibition of differentiation, and it affects the neuronal differentiation ability of triploid hESCs, both in vitro and in vivo. Notably, our research establishes a platform to study triploidy in humans and points to their pathology as observed in triploid embryos.
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Células Madre Embrionarias Humanas , Triploidía , Recién Nacido , Embarazo , Femenino , Humanos , Diferenciación Celular/genética , Genoma , Genómica , DiploidiaRESUMEN
Genomic imprinting underlies the mammalian requirement for sexual reproduction. Nonetheless, the relative contribution of the two parental genomes during human development is not fully understood. Specifically, a fascinating question is whether the formation of the gonad, which holds the ability to reproduce, depends on equal contribution from both parental genomes. Here, we differentiated androgenetic and parthenogenetic human pluripotent stem cells (hPSCs) into ovarian granulosa-like cells (GLCs). We show that in contrast to biparental and androgenetic cells, parthenogenetic hPSCs present a reduced capacity to differentiate into GLCs. We further identify the paternally expressed gene IGF2 as the most upregulated imprinted gene upon differentiation. Remarkably, while IGF2 knockout androgenetic cells fail to differentiate into GLCs, the differentiation of parthenogenetic cells supplemented with IGF2 is partly rescued. Thus, our findings unravel a surprising essentiality of genes that are only expressed from the paternal genome to the development of the female reproductive system.
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Células Madre Embrionarias Humanas , Células Madre Pluripotentes , Animales , Humanos , Femenino , Impresión Genómica , Diferenciación Celular/genética , Partenogénesis/genética , Células de la Granulosa , MamíferosRESUMEN
It is well established that human pluripotent stem cells (hPSCs) can acquire genetic and epigenetic changes during culture in vitro. Given the increasing use of hPSCs in research and therapy and the vast expansion in the number of hPSC lines available for researchers, the International Society for Stem Cell Research has recognized the need to reassess quality control standards for ensuring the genetic integrity of hPSCs. Here, we summarize current knowledge of the nature of recurrent genetic and epigenetic variants in hPSC culture, the methods for their detection, and what is known concerning their effects on cell behavior in vitro or in vivo. We argue that the potential consequences of low-level contamination of cell therapy products with cells bearing oncogenic variants are essentially unknown at present. We highlight the key challenges facing the field with particular reference to safety assessment of hPSC-derived cellular therapeutics.
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Epigenómica , Células Madre Pluripotentes , Humanos , Investigación con Células Madre , Oncogenes , Epigénesis GenéticaRESUMEN
Human pluripotent stem cells (hPSCs) are currently evaluated for clinical applications due to their proliferation and differentiation capacities, raising the need to both assess and enhance, the safety of hPSC-based treatments. Distinct molecular features contribute to the tumorigenicity of hPSCs, manifested in the formation of teratoma tumors upon transplantation in vivo. Prolonged in vitro culturing of hPSCs can enhance selection for specific genetic aberrations, either at the chromosome or gene level. Some of these aberrations are tightly linked to human tumor pathology and increase the tumorigenic aggressiveness of the abnormal cells. In this perspective, we describe major tumor-associated risk factors entailed in hPSC-based therapy, and present precautionary and safety measures relevant for the development and application of such therapies.
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Células Madre Pluripotentes , Teratoma , Carcinogénesis , Diferenciación Celular , Humanos , Teratoma/patologíaRESUMEN
X chromosome inactivation is a mammalian dosage compensation mechanism, where one of two X chromosomes is randomly inactivated in female cells. Previous studies have suggested that primed human embryonic stem cells (hESCs) maintain an eroded state of the X chromosome and do not express XIST, while in naïve transition, both XIST and the eroded X chromosome are reactivated. However, the pattern of chromosome X reactivation in naïve hESCs remains mainly unknown. In this study, we examine the variations in the status of X chromosome between primed and naïve hESCs by analyzing RNA sequencing samples from different studies. We show that most samples of naïve hESCs indeed reactivate XIST and there is an increase in gene expression levels on chromosome X. However, most of the naïve samples do not fully activate chromosome X in a uniform manner and present a distinct eroded pattern, probably as a result of XIST reactivation and initiation of re-inactivation of chromosome X. This large-scale analysis provides a higher-resolution description of the changes occurring in chromosome X during primed-to-naïve transition and emphasizes the importance of taking these variations into consideration when studying X inactivation in embryonic development.
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Células Madre Embrionarias Humanas , ARN Largo no Codificante , Animales , Compensación de Dosificación (Genética) , Femenino , Células Madre Embrionarias Humanas/metabolismo , Humanos , Mamíferos/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Cromosoma X/metabolismo , Inactivación del Cromosoma X/genéticaRESUMEN
Fragile X syndrome (FXS), the most prevalent heritable form of intellectual disability, is caused by the transcriptional silencing of the FMR1 gene. The epigenetic factors responsible for FMR1 inactivation are largely unknown. Here, we initially demonstrated the feasibility of FMR1 reactivation by targeting a single epigenetic factor, DNMT1. Next, we established a model system for FMR1 silencing using a construct containing the FXS-related mutation upstream to a reporter gene. This construct was methylated in vitro and introduced into a genome-wide loss-of-function (LOF) library established in haploid human pluripotent stem cells (PSCs), allowing the identification of genes whose functional loss reversed the methylation-induced silencing of the FMR1 reporter. Selected candidate genes were further analyzed in haploid- and FXS-patient-derived PSCs, highlighting the epigenetic and metabolic pathways involved in FMR1 regulation. Our work sheds light on the mechanisms responsible for CGG-expansion-mediated FMR1 inactivation and offers novel targets for therapeutic FMR1 reactivation.
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Síndrome del Cromosoma X Frágil , Metilación de ADN/genética , Epigénesis Genética , Epigenómica , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/genética , HumanosRESUMEN
Cellular proliferation depends on the accurate and timely replication of the genome. Several genetic diseases are caused by mutations in key DNA replication genes; however, it remains unclear whether these genes influence the normal program of DNA replication timing. Similarly, the factors that regulate DNA replication dynamics are poorly understood. To systematically identify trans-acting modulators of replication timing, we profiled replication in 184 cell lines from three cell types, encompassing 60 different gene knockouts or genetic diseases. Through a rigorous approach that considers the background variability of replication timing, we concluded that most samples displayed normal replication timing. However, mutations in two genes showed consistently abnormal replication timing. The first gene was RIF1, a known modulator of replication timing. The second was MCM10, a highly conserved member of the pre-replication complex. Cells from a single patient carrying MCM10 mutations demonstrated replication timing variability comprising 46% of the genome and at different locations than RIF1 knockouts. Replication timing alterations in the mutated MCM10 cells were predominantly comprised of replication delays and initiation site gains and losses. Taken together, this study demonstrates the remarkable robustness of the human replication timing program and reveals MCM10 as a novel candidate modulator of DNA replication timing.
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Momento de Replicación del ADN , Proteínas de Mantenimiento de Minicromosoma , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Replicación del ADN/genética , Momento de Replicación del ADN/genética , Humanos , Proteínas de Mantenimiento de Minicromosoma/genética , Origen de RéplicaRESUMEN
Haploinsufficiency describes a phenomenon where one functioning allele is insufficient for a normal phenotype, underlying several human diseases. The effect of haploinsufficiency on human embryonic stem cells (hESC) has not been thoroughly studied. To establish a genome-wide loss-of-function screening for heterozygous mutations, we fuse normal haploid hESCs with a library of mutant haploid hESCs. We identify over 600 genes with a negative effect on hESC growth in a haploinsufficient manner and characterize them as genes showing less tolerance to mutations, conservation during evolution, and depletion from telomeres and X chromosome. Interestingly, a large fraction of these genes is associated with extracellular matrix and plasma membrane and enriched for genes within WNT and TGF-ß pathways. We thus identify haploinsufficiency-related genes that show growth retardation in early embryonic cells, suggesting dosage-dependent phenotypes in hESCs. Overall, we construct a unique model for studying haploinsufficiency and identified important dosage-dependent pathways involved in hESC growth and survival.
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Células Madre Embrionarias Humanas , Alelos , Haploidia , Haploinsuficiencia/genética , Heterocigoto , HumanosRESUMEN
Retinoblastoma (Rb) is a childhood cancer of the developing retina, accounting for up to 17% of all tumors in infancy. To gain insights into the transcriptional events of cell state transitions during Rb development, we established 2 disease models via retinal organoid differentiation of a pRB (retinoblastoma protein)-depleted human embryonic stem cell line (RB1-null hESCs) and a pRB patient-specific induced pluripotent (iPSC) line harboring a RB1 biallelic mutation (c.2082delC). Both models were characterized by pRB depletion and accumulation of retinal progenitor cells at the expense of amacrine, horizontal and retinal ganglion cells, which suggests an important role for pRB in differentiation of these cell lineages. Importantly, a significant increase in the fraction of proliferating cone precursors (RXRγ+Ki67+) was observed in both pRB-depleted organoid models, which were defined as Rb-like clusters by single-cell RNA-Seq analysis. The pRB-depleted retinal organoids displayed similar features to Rb tumors, including mitochondrial cristae aberrations and rosette-like structures, and were able to undergo cell growth in an anchorage-independent manner, indicative of cell transformation in vitro. In both models, the Rb cones expressed retinal ganglion and horizontal cell markers, a novel finding, which could help to better characterize these tumors with possible therapeutic implications. Application of Melphalan, Topotecan, and TW-37 led to a significant reduction in the fraction of Rb proliferating cone precursors, validating the suitability of these in vitro models for testing novel therapeutics for Rb.
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Células Madre Pluripotentes , Neoplasias de la Retina , Retinoblastoma , Diferenciación Celular , Niño , Humanos , Organoides/metabolismo , Células Madre Pluripotentes/metabolismo , Retina/metabolismo , Neoplasias de la Retina/genética , Neoplasias de la Retina/metabolismo , Neoplasias de la Retina/patología , Retinoblastoma/genética , Retinoblastoma/metabolismo , Retinoblastoma/patología , Proteína de Retinoblastoma/genéticaRESUMEN
In mammals, imprinted genes are regulated by differentially methylated regions (DMRs) that are inherited from germ cells, leading to monoallelic expression in accordance with parent-of-origin. Yet, it is largely unknown how imprinted DMRs are maintained in human embryos despite global DNA demethylation following fertilization. Here, we explored the mechanisms involved in imprinting regulation by employing human parthenogenetic embryonic stem cells (hpESCs), which lack paternal alleles. We show that although global loss of DNA methylation in hpESCs affects most imprinted DMRs, many paternally-expressed genes (PEGs) remain repressed. To search for factors regulating PEGs, we performed a genome-wide CRISPR/Cas9 screen in haploid hpESCs. This revealed ATF7IP as an essential repressor of a set of PEGs, which we further show is also required for silencing sperm-specific genes. Our study reinforces an important role for histone modifications in regulating imprinted genes and suggests a link between parental imprinting and germ cell identity.
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Sistemas CRISPR-Cas , Regulación de la Expresión Génica , Impresión Genómica , Haploidia , Células Madre Embrionarias Humanas/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Células Cultivadas , Secuenciación de Inmunoprecipitación de Cromatina/métodos , ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Metilación de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células HeLa , Células Madre Embrionarias Humanas/citología , Humanos , Sistema de Señalización de MAP Quinasas/genética , Masculino , Partenogénesis/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Espermatogénesis/genéticaRESUMEN
Haploid human embryonic stem cells (ESCs) provide a powerful genetic system but diploidize at high rates. We hypothesized that diploidization results from aberrant DNA replication. To test this, we profiled DNA replication timing in isogenic haploid and diploid ESCs. The greatest difference was the earlier replication of the X Chromosome in haploids, consistent with the lack of X-Chromosome inactivation. We also identified 21 autosomal regions that had delayed replication in haploids, extending beyond the normal S phase and into G2/M. Haploid-delays comprised a unique set of quiescent genomic regions that are also underreplicated in polyploid placental cells. The same delays were observed in female ESCs with two active X Chromosomes, suggesting that increased X-Chromosome dosage may cause delayed autosomal replication. We propose that incomplete replication at the onset of mitosis could prevent cell division and result in re-entry into the cell cycle and whole genome duplication.
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Genomic imprinting is a parent-of-origin dependent monoallelic expression of genes. Previous studies showed that conversion of primed human pluripotent stem cells (hPSCs) into naive pluripotency is accompanied by genome-wide loss of methylation that includes imprinted loci. However, the extent of aberrant biallelic expression of imprinted genes is still unknown. Here, we analyze loss of imprinting (LOI) in a large cohort of both bulk and single-cell RNA sequencing samples of naive and primed hPSCs. We show that naive hPSCs exhibit high levels of non-random LOI, with bias toward paternally methylated imprinting control regions. Importantly, we show that different protocols used for the primed to naive conversion led to different extents of LOI, tightly correlated to FGF signaling. This analysis sheds light on the process of LOI occurring during the conversion to naive pluripotency and highlights the importance of these events when modeling disease and development or when utilizing the cells for therapy.