Pluripotent stem cell-derived gametes: truth and (potential) consequences.
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ABSTRACT:
SUBMITTER: Mathews DJ
PROVIDER: S-EPMC5226919 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Recently, platelet-derived growth factors present in lysates became an extreme interest in the field of regenerative medicine such as in wound healing and as substitutes to foetal bovine serum in xeno-free cell culture systems. However, the generation of such platelet lysates completely depends on the availability of platelet donors. In this study, the possibility to use in vitro-generated megakaryocytes derived from induced pluripotent stem cells (iPSCs) as a cell source for typical platelet growth factors was investigated. Therefore, the presence and levels of those factors were characterized in in vitro-produced megakaryocytes. In comparison with platelets, in vitro-generated megakaryocytes showed a multifold increased content in transcript and protein levels of typical platelet growth factors including platelet-derived growth factors (PDGFs), transforming growth factor (TGF)-1?, vascular endothelial cell factor (VEGF)-A, epidermal growth factor (EGF), insulin-like growth factor (IGF)-1 and tissue factor (TF). Hence, iPSC-derived megakaryocytes may serve as an efficient cell source for a donor-independent generation of growth factor-rich lysates with a broad application potential in innovative cell culture systems and regenerative therapies.
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Project description:PurposeTo investigate the interests of the Japanese general public in the reproductive use of in vitro derived (IVD)-gametes.MethodsWe conducted an online survey and obtained answers from 3096 respondents.ResultsMore than half of the respondents agreed with the reproductive use of IVD-gametes by infertile heterosexual married couples but disagreed with the same use by unmarried or homosexual individuals. Nearly 70.0% disagreed with the use for designing a baby.DiscussionIn Japan at present, the use of IVD-gametes that deviate from societal values regarding reproduction and family prescribed by the conventional marriage system is unlikely to be accepted. It is also unlikely to be accepted for non-treatment purposes in reproduction.
| S-EPMC8609981 | biostudies-literature
Project description:IntroductionHuman induced pluripotent stem cells (hiPSCs) have been derived from various somatic cell types. Granulosa cells, a group of cells which surround oocytes and are obtained from the (normally discarded) retrieved egg follicles of women undergoing infertility treatment, are a possible cell source for induced pluripotent stem cell (iPSC) generation. Here, we explored the possibility of using human granulosa cells as a donor cell type for iPSC reprogramming, and compared granulosa cell-derived iPSCs (iGRAs) with those derived from other cell sources, to determine the potential ability of iGRA differentiation.MethodsGranulosa cells were collected from egg follicles retrieved from women undergoing infertility treatment. After short-term culture, the granulosa cells derived from different patients were mixed in culture, and infected with retroviruses encoding reprogramming factors. The resulting iPSC clones were selected and subjected to microsatellite DNA analysis to determine their parental origin. IGRAs were subjected to RT-PCR, immunofluorescence staining, and in vitro and in vivo differentiation assays to further establish their pluripotent characteristics.ResultsMicrosatellite DNA analysis was used to demonstrate that hiPSCs with different parental origins can be simultaneously reprogrammed by retroviral transfection of a mixed human granulosa cell population obtained from multiple individuals. The iGRAs resemble human embryonic stem cells (hESCs) in many respects, including morphological traits, growth requirements, gene and marker expression profiles, and in vitro and in vivo developmental propensities. We also demonstrate that the iGRAs express low levels of NLRP2, and differentiating iGRAs possess a biased differentiation potential toward the trophoblastic lineage. Although NLRP2 knockdown in hESCs promotes trophoblastic differentiation of differentiating hESCs, it does not result in exit from pluripotency. These results imply that NLRP2 may play a role in regulating the trophoblastic differentiation of human pluripotent stem cells.ConclusionsThese findings provide a means of generating iPSCs from multiple granulosa cell populations with different parental origins. The ability to generate iPSCs from granulosa cells not only enables modeling of infertility-associated disease, but also provides a means of identifying potential clinical interventions through iPSC-based drug screening.
| S-EPMC4430911 | biostudies-literature
Project description:AimTo ascertain to what extent the Japanese general public accept the creation and use of in vitro derived (IVD)-gametes derived from human-induced pluripotent stem cells.Materials & methodsWe conducted an online survey and obtained answers from 3096 respondents.Results78.6% of the respondents answered that they would accept the creation and use of IVD-gametes for research purposes, 51.7% answered that they would accept the creation and use of embryos with IVD-gametes for research purposes, and 25.9% answered that they would accept childbirth using embryos with IVD-gametes.DiscussionThe results that approximately half of the respondents answered that they would accept the creation of embryos with IVD-gametes, which has not been allowed in the current Japanese research guidelines, is astonishing.
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Project description:BackgroundCardiovascular progenitor cells (CPCs) have been identified within the developing mouse heart and differentiating pluripotent stem cells by intracellular transcription factors Nkx2.5 and Islet 1 (Isl1). Study of endogenous and induced pluripotent stem cell (iPSC)-derived CPCs has been limited due to the lack of specific cell surface markers to isolate them and conditions for their in vitro expansion that maintain their multipotency.Methodology/principal findingsWe sought to identify specific cell surface markers that label endogenous embryonic CPCs and validated these markers in iPSC-derived Isl1(+)/Nkx2.5(+) CPCs. We developed conditions that allow propagation and characterization of endogenous and iPSC-derived Isl1(+)/Nkx2.5(+) CPCs and protocols for their clonal expansion in vitro and transplantation in vivo. Transcriptome analysis of CPCs from differentiating mouse embryonic stem cells identified a panel of surface markers. Comparison of these markers as well as previously described surface markers revealed the combination of Flt1(+)/Flt4(+) best identified and facilitated enrichment for Isl1(+)/Nkx2.5(+) CPCs from embryonic hearts and differentiating iPSCs. Endogenous mouse and iPSC-derived Flt1(+)/Flt4(+) CPCs differentiated into all three cardiovascular lineages in vitro. Flt1(+)/Flt4(+) CPCs transplanted into left ventricles demonstrated robust engraftment and differentiation into mature cardiomyocytes (CMs).Conclusion/significanceThe cell surface marker combination of Flt1 and Flt4 specifically identify and enrich for an endogenous and iPSC-derived Isl1(+)/Nkx2.5(+) CPC with trilineage cardiovascular potential in vitro and robust ability for engraftment and differentiation into morphologically and electrophysiologically mature adult CMs in vivo post transplantation into adult hearts.
| S-EPMC3467279 | biostudies-literature
Project description:Impact statementOur work reinforces the role of extracellular matrix (ECM) density and matrix metalloprotease activity on the formation of microvasculature from induced pluripotent stem cell (iPSC)-derived vascular cells. The cell-matrix interactions discussed in this study underscore the importance of understanding the role of mechanoregulation and matrix degradation on vasculogenesis and can potentially drive the development of ECM-mimicking angiogenic biomaterials. Furthermore, our work has broader implications concerning the response of iPSC-derived cells to the mechanics of engineered microenvironments. An understanding of these interactions will be critical to creating physiologically relevant transplantable tissue replacements.
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Project description:Mesenchymal stem cells (MSCs) have received significant attention in recent years due to their large potential for cell therapy. Indeed, they secrete a wide variety of immunomodulatory factors of interest for the treatment of immune-related disorders and inflammatory diseases. MSCs can be extracted from multiple tissues of the human body. However, several factors may restrict their use for clinical applications: the requirement of invasive procedures for their isolation, their limited numbers, and their heterogeneity according to the tissue of origin or donor. In addition, MSCs often present early signs of replicative senescence limiting their expansion in vitro, and their therapeutic capacity in vivo. Due to the clinical potential of MSCs, a considerable number of methods to differentiate induced pluripotent stem cells (iPSCs) into MSCs have emerged. iPSCs represent a new reliable, unlimited source to generate MSCs (MSCs derived from iPSC, iMSCs) from homogeneous and well-characterized cell lines, which would relieve many of the above mentioned technical and biological limitations. Additionally, the use of iPSCs prevents some of the ethical concerns surrounding the use of human embryonic stem cells. In this review, we analyze the main current protocols used to differentiate human iPSCs into MSCs, which we classify into five different categories: MSC Switch, Embryoid Body Formation, Specific Differentiation, Pathway Inhibitor, and Platelet Lysate. We also evaluate common and method-specific culture components and provide a list of positive and negative markers for MSC characterization. Further guidance on material requirements to produce iMSCs with these methods and on the phenotypic features of the iMSCs obtained is added. The information may help researchers identify protocol options to design and/or refine standardized procedures for large-scale production of iMSCs fitting clinical demands.
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Project description:To assess the utility of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as an in vitro proarrhythmia model, we evaluated the concentration dependence and sources of variability of electrophysiologic responses to 28 drugs linked to low, intermediate, and high torsades de pointes (TdP) risk categories using two commercial cell lines and standardized protocols in a blinded multisite study using multielectrode array or voltage-sensing optical approaches. Logistical and ordinal linear regression models were constructed using drug responses as predictors and TdP risk categories as outcomes. Three of seven predictors (drug-induced arrhythmia-like events and prolongation of repolarization at either maximum tested or maximal clinical exposures) categorized drugs with reasonable accuracy (area under the curve values of receiver operator curves ?0.8). hiPSC-CM line, test site, and platform had minimal influence on drug categorization. These results demonstrate the utility of hiPSC-CMs to detect drug-induced proarrhythmic effects as part of the evolving Comprehensive In Vitro Proarrhythmia Assay paradigm.
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Project description:Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) hold promise for therapeutic applications. To serve these functions, the hiPSC-CM must recapitulate the electrophysiologic properties of native adult cardiomyocytes. This study examines the electrophysiologic characteristics of hiPSC-CM between 11 and 121 days of maturity. Embryoid bodies (EBs) were generated from hiPS cell line reprogrammed with Oct4, Nanog, Lin28 and Sox2. Sharp microelectrodes were used to record action potentials (AP) from spontaneously beating clusters (BC) micro-dissected from the EBs (n?=?103; 37°C) and to examine the response to 5 µM E-4031 (n?=?21) or BaCl(2) (n?=?22). Patch-clamp techniques were used to record I(Kr) and I(K1) from cells enzymatically dissociated from BC (n?=?49; 36°C). Spontaneous cycle length (CL) and AP characteristics varied widely among the 103 preparations. E-4031 (5 µM; n?=?21) increased Bazett-corrected AP duration from 291.8±81.2 to 426.4±120.2 msec (p<0.001) and generated early afterdepolarizations in 8/21 preparations. In 13/21 BC, E-4031 rapidly depolarized the clusters leading to inexcitability. BaCl(2), at concentrations that selectively block I(K1) (50-100 µM), failed to depolarize the majority of clusters (13/22). Patch-clamp experiments revealed very low or negligible I(K1) in 53% (20/38) of the cells studied, but presence of I(Kr) in all (11/11). Consistent with the electrophysiological data, RT-PCR and immunohistochemistry studies showed relatively poor mRNA and protein expression of I(K1) in the majority of cells, but robust expression of I(Kr.) In contrast to recently reported studies, our data point to major deficiencies of hiPSC-CM, with remarkable diversity of electrophysiologic phenotypes as well as pharmacologic responsiveness among beating clusters and cells up to 121 days post-differentiation (dpd). The vast majority have a maximum diastolic potential that depends critically on I(Kr) due to the absence of I(K1). Thus, efforts should be directed at producing more specialized and mature hiPSC-CM for future therapeutic applications.
| S-EPMC3396384 | biostudies-literature
Project description:Summary Multiple strategies have been developed to efficiently differentiate human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Here, we describe a protocol for measuring three key functional parameters of hiPSC-CMs, including contractile function, calcium (Ca2+) handling, and action potential. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2021). Graphical abstract Highlights • Automated measurement of hiPSC-CM contractile function• Single-cell calcium imaging of hiPSC-CMs loaded with Fura-2 AM• Imaging-based action potential measurement of hiPSC-CMs Multiple strategies have been developed to efficiently differentiate human-induced pluripotent stem-derived cardiomyocytes (hiPSC-CMs). Here, we describe a protocol for measuring three key functional parameters of hiPSC-CMs, including contractile function, calcium (Ca2+) handling, and action potential.
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