RESUMO
YAP plays a vital role in controlling growth and differentiation in various cell lineages. Although the expression of YAP in mice testicular and spermatogenic cells suggests its role in mammalian spermatogenesis, the role of YAP in the development of human male germ cells has not yet been determined. Using an in vitro model and a gene editing approach, we generated human spermatogonia stem cell-like cells (hSSLCs) from human embryonic stem cells (hESCs) and investigated the role of YAP in human spermatogenesis. The results showed that reducing YAP expression during the early stage of spermatogenic differentiation increased the number of PLZF+ hSSLCs and haploid spermatid-like cells. We also demonstrated that the up-regulation of YAP is essential for maintaining spermatogenic cell survival during the later stages of spermatogenic differentiation. The expression of YAP that deviates from this pattern results in a lower number of hSSLCs and an increased level of spermatogenic cell death. Taken together, our result demonstrates that the dynamic expression pattern of YAP is essential for human spermatogenesis. Modulating the level of YAP during human spermatogenesis could improve the production yield of male germ cells derived from hESCs, which could provide the optimization method for in vitro gametogenesis and gain insight into the application in the treatment of male infertility.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Diferenciação Celular , Células-Tronco Embrionárias Humanas , Espermatogênese , Fatores de Transcrição , Proteínas de Sinalização YAP , Masculino , Humanos , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Proteínas de Sinalização YAP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Espermatogônias/metabolismo , Espermatogônias/citologia , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica/genéticaRESUMO
Although current stem cell therapies exhibit promising potential, the extended process of employing autologous cells and the necessity for donor-host matching to avert the rejection of transplanted cells significantly limit the widespread applicability of these treatments. It would be highly advantageous to generate a pluripotent universal donor stem cell line that is immune-evasive and, therefore, not restricted by the individual's immune system, enabling unlimited application within cell replacement therapies. Before such immune-evasive stem cells can be moved forward to clinical trials, in vivo testing via transplantation experiments in immune-competent animals would be a favorable approach preceding preclinical testing. By using human stem cells in immune competent animals, results will be more translatable to a clinical setting, as no parts of the immune system have been altered, although in a xenogeneic setting. In this way, immune evasiveness, cell survival, and unwanted proliferative effects can be assessed before clinical trials in humans. The current study presents the generation and characterization of three human embryonic stem cell lines (hESCs) for xenogeneic transplantation in immune-competent mice. The major histocompatibility complexes I- and II-encoding genes, B2M and CIITA, have been deleted from the hESCs using CRISPR-Cas9-targeted gene replacement strategies and knockout. B2M was knocked out by the insertion of murine CD47. Human-secreted embryonic alkaline phosphatase (hSEAP) was inserted in a safe harbor site to track cells in vivo. The edited hESCs maintained their pluripotency, karyotypic normality, and stable expression of murine CD47 and hSEAP in vitro. In vivo transplantation of hESCs into immune-competent BALB/c mice was successfully monitored by measuring hSEAP in blood samples. Nevertheless, transplantation of immune-evasive hESCs resulted in complete rejection within 11 days, with clear immune infiltration of T-cells on day 8. Our results reveal that knockout of B2M and CIITA together with species-specific expression of CD47 are insufficient to prevent rejection in an immune-competent and xenogeneic context.
RESUMO
The differentiation of human pluripotent stem cells into ventral mesencephalic dopaminergic (DA) fate is relevant for the treatment of Parkinson's disease. Shortcuts to obtaining DA cells through direct reprogramming often include forced expression of the transcription factor LMX1A. Although reprogramming with LMX1A can generate tyrosine hydroxylase (TH)-positive cells, their regional identity remains elusive. Using an in vitro model of early human neural tube patterning, we report that forced LMX1A expression induced a ventral-to-dorsal fate shift along the entire neuroaxis with the emergence of roof plate fates despite the presence of ventralizing molecules. The LMX1A-expressing progenitors gave rise to grafts containing roof plate-derived choroid plexus cysts as well as ectopically induced TH-positive neurons of a forebrain identity. Early activation of LMX1A prior to floor plate specification was necessary for the dorsalizing effect. Our work suggests using caution in employing LMX1A for the induction of DA fate, as this factor may generate roof plate rather than midbrain fates.
Assuntos
Diferenciação Celular , Neurônios Dopaminérgicos , Células-Tronco Embrionárias Humanas , Proteínas com Homeodomínio LIM , Mesencéfalo , Fatores de Transcrição , Humanos , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/citologia , Proteínas com Homeodomínio LIM/metabolismo , Proteínas com Homeodomínio LIM/genética , Mesencéfalo/citologia , Mesencéfalo/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Padronização Corporal/genética , Tirosina 3-Mono-Oxigenase/metabolismo , Tirosina 3-Mono-Oxigenase/genética , Animais , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
(1) Background: We reviewed a stem cell-derived therapeutic strategy for advanced neovascular age-related macular degeneration (nAMD) using a human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE) monolayer delivered on a coated, synthetic basement membrane (BM)-the patch-and assessed the presence and distribution of hESC-RPE over 5 years following transplantation, as well as functional outcomes. (2) Methods: Two subjects with acute vision loss due to sub-macular haemorrhage in advanced nAMD received the hESC-RPE patch. Systematic immunosuppression was used peri-operatively followed by local depot immunosuppression. The subjects were monitored for five years with observation of RPE patch pigmentation, extension beyond the patch boundary into surrounding retina, thickness of hESC-RPE and synthetic BM and review for migration and proliferation of hESC-RPE. Visual function was also assessed. (3) Results: The two study participants showed clear RPE characteristics of the patch, preservation of some retinal ultrastructure with signs of remodelling, fibrosis and thinning on optical coherence tomography over the 5-year period. For both participants, there was evidence of pigment extension beyond the patch continuing until 12 months post-operatively, which stabilised and was preserved until 5 years post-operatively. Measurement of hESC-RPE and BM thickness over time for both cases were consistent with predefined histological measurements of these two layers. There was no evidence of distant RPE migration or proliferation in either case beyond the monolayer. Sustained visual acuity improvement was apparent for 2 years in both subjects, with one subject maintaining the improvement for 5 years. Both subjects demonstrated initial improvement in fixation and microperimetry compared to baseline, at year 1, although only one maintained this at 4 years post-intervention. (4) Conclusions: hESC-RPE patches show evidence of continued pigmentation, with extension, to cover bare host basement membrane for up to 5 years post-implantation. There is evidence that this represents functional RPE on the patch and at the patch border where host RPE is absent. The measurements for thickness of hESC-RPE and BM suggest persistence of both layers at 5 years. No safety concerns were raised for the hypothetical risk of RPE migration, proliferation or tumour formation. Visual function also showed sustained improvement for 2 years in one subject and 5 years in the other subject.
RESUMO
Per- and polyfluoroalkyl substances (PFAS) are extensively utilized in varieties of products and tend to accumulate in the human body including umbilical cord blood and embryos/fetuses. In this study, we conducted an assessment and comparison of the potential early developmental toxicity of perfluorooctanoic acid (PFOA), undecafluorohexanoic acid (PFHxA), heptafluorobutyric acid, perfluorooctanesulfonate (PFOS), perfluorohexanesulfonate, and perfluorobutyric acid at noncytotoxic concentrations relevant to human exposure using models based on human embryonic stem cells in both three-dimensional embryoid body (EB) and monolayer differentiation configurations. All six compounds influenced the determination of cell fate by disrupting the expression of associated markers in both models and, in some instances, even led to alterations in the formation of cystic EBs. The expression of cilia-related gene IFT122 was significantly inhibited. Additionally, PFOS and PFOA inhibited ciliogenesis, while PFOA specifically reduced the cilia length. Transcriptome analysis revealed that PFOS altered 1054 genes and disrupted crucial signaling pathways such as WNT and TGF-ß, which play integral roles in cilia transduction and are critical for early embryonic development. These results provide precise and comprehensive insights into the potential adverse health effects of these six PFAS compounds directly concerning early human embryonic development.
Assuntos
Fluorocarbonos , Células-Tronco Embrionárias Humanas , Humanos , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Fluorocarbonos/toxicidade , Diferenciação Celular/efeitos dos fármacosRESUMO
Corneal transplantation represents the primary therapeutic approach for managing corneal endothelial dysfunction, but corneal donors remain scarce. Anterior chamber cell injection emerges as a highly promising alternative strategy for corneal transplantation, with pluripotent stem cells (PSC) demonstrating considerable potential as an optimal cell source. Nevertheless, only a few studies have explored the differentiation of functional corneal endothelial-like cells originating from PSC. In this investigation, a chemical-defined protocol was successfully developed for the differentiation of functional corneal endothelial-like cells derived from human embryonic stem cells (hESC). The application of nicotinamide (NAM) exhibited a remarkable capability in suppressing the fibrotic phenotype, leading to the generation of more homogeneous and well-distinctive differentiated cells. Furthermore, NAM effectively suppressed the expression of genes implicated in endothelial cell migration and extracellular matrix synthesis. Notably, NAM also facilitated the upregulation of surface marker genes specific to functional corneal endothelial cells (CEC), including CD26 (-) CD44 (-â¼+-) CD105 (-) CD133 (-) CD166 (+) CD200 (-). Moreover, in vitro functional assays were performed, revealing intact barrier properties and Na+/K+-ATP pump functionality in the differentiated cells treated with NAM. Consequently, our findings provide robust evidence supporting the capacity of NAM to enhance the differentiation of functional CEC originating from hESC, offering potential seed cells for therapeutic interventions of corneal endothelial dysfunction.
Assuntos
Diferenciação Celular , Endotélio Corneano , Células-Tronco Embrionárias Humanas , Niacinamida , Humanos , Diferenciação Celular/efeitos dos fármacos , Niacinamida/farmacologia , Endotélio Corneano/metabolismo , Endotélio Corneano/citologia , Endotélio Corneano/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Células Cultivadas , Complexo Vitamínico B/farmacologia , Citometria de Fluxo , Movimento Celular/efeitos dos fármacos , Antígenos CD/metabolismo , Antígenos CD/genéticaRESUMO
BACKGROUND AIMS: Rheumatoid arthritis (RA) is characterized by an overactive immune system, with limited treatment options beyond immunosuppressive drugs or biological response modifiers. Human embryonic stem cell-derived mesenchymal stromal cells (hESC-MSCs) represent a novel alternative, possessing diverse immunomodulatory effects. In this study, we aimed to elucidate the therapeutic effects and underlying mechanisms of hESC-MSCs in treating RA. METHODS: MSC-like cells were differentiated from hESC (hESC-MSCs) and cultured in vitro. Cell proliferation was assessed using Cell Counting Kit-8 assay and Ki-67 staining. Flow cytometry was used to analyze cell surface markers, T-cell proliferation and immune cell infiltration. The collagen-induced arthritis (CIA) mouse model and bleomycin-induced model of lung fibrosis (BLE) were established and treated with hESC-MSCs intravenously for in vivo assessment. Pathological analyses, reverse transcription-quantitative polymerase chain reaction and Western blotting were conducted to evaluate the efficacy of hESC-MSCs treatment. RESULTS: Intravenous transplantation of hESC-MSCs effectively reduced inflammation in CIA mice in this study. Furthermore, hESC-MSC administration enhanced regulatory T cell infiltration and activation. Additional findings suggest that hESC-MSCs may reduce lung fibrosis in BLE mouse models, indicating their potential to mitigate complications associated with RA progression. In vitro experiments revealed a significant inhibition of T-cell activation and proliferation during co-culture with hESC-MSCs. In addition, hESC-MSCs demonstrated enhanced proliferative capacity compared with traditional primary MSCs. CONCLUSIONS: Transplantation of hESC-MSCs represents a promising therapeutic strategy for RA, potentially regulating T-cell proliferation and differentiation.
Assuntos
Artrite Reumatoide , Diferenciação Celular , Modelos Animais de Doenças , Células-Tronco Embrionárias Humanas , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Fibrose Pulmonar , Animais , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Artrite Reumatoide/terapia , Artrite Reumatoide/imunologia , Transplante de Células-Tronco Mesenquimais/métodos , Fibrose Pulmonar/terapia , Fibrose Pulmonar/patologia , Células-Tronco Embrionárias Humanas/citologia , Proliferação de Células , Inflamação/terapia , Inflamação/patologia , Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Artrite Experimental/terapia , Artrite Experimental/patologia , Artrite Experimental/imunologiaRESUMO
BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) hold great promise in the treatment of diabetic retinopathy (DR), as evidenced by increasing preclinical and clinical studies. However, the absence of standardized and industrialized clinical-grade donor cells hampers the continued development and large-scale clinical application of MSCs-based therapies for DR. Previously, we have identified a unique population of MSCs generated from a clinical-grade human embryonic stem cell (hESC) line under Good Manufacturing Practice conditions that could be a potential source to address the issues. Here, we investigated the therapeutic potential of the clinical-grade hESC line-derived MSCs (hESC-MSCs) on db/db mice with DR. METHODS: hESC-MSCs were initially characterized by morphological assessment, flow cytometry analysis and trilineage differentiation assays. These cells (5 × 106 cells) were then transplanted intravenously into 12-week-old db/db mice via tail vein, with phosphate-buffered saline transplantation and untreated groups used as controls. The retinal alterations in neural functions and microvascular perfusions, and inflammatory responses in peripheral blood and retina were evaluated at 4 and 6 weeks after transplantation using electroretinography, optical coherence tomography angiography and flow cytometry, respectively. Body weight and fasting blood glucose (FBG) levels were also measured to investigate their systemic implications. RESULTS: Compared with controls, intravenous transplantation of hESC-MSCs could significantly: (i) enhance impaired retinal electroretinography functions (including amplitudes of a-, b-wave and oscillatory potentials) at 4 weeks after transplantation; (ii) alleviate microvascular dysfunctions, especially in the inner retina with significance (including reducing non-perfusion area and increasing vascular area density) at 4 weeks after transplantation; (iii) decrease FBG levels at 4 weeks after transplantation and induce weight loss up to 6 weeks after transplantation and (iv) increase both peripheral blood and retinal interleukin-10 levels at 4 weeks after transplantation and modulate peripheral blood inflammatory cytokines and chemokines levels, such as monocyte chemotactic protein-1, up to 6 weeks after transplantation. CONCLUSIONS: The findings of our study indicated that intravenous transplantation of hESC-MSCs ameliorated retinal neural and microvascular dysfunctions, regulated body weight and FBG and modulated peripheral blood and retinal inflammation responses in a mouse model of DR. These results suggest that hESC-MSCs could be a potentially effective clinical-grade cell source for the treatment of DR.
Assuntos
Retinopatia Diabética , Células-Tronco Embrionárias Humanas , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Animais , Humanos , Retinopatia Diabética/terapia , Camundongos , Células-Tronco Embrionárias Humanas/citologia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Diferenciação Celular , Retina , Modelos Animais de Doenças , Diabetes Mellitus Experimental/terapiaRESUMO
Nervous system tumors, particularly brain tumors, represent the most common tumors in children and one of the most lethal tumors in adults. Despite decades of research, there are few effective therapies for these cancers. Although human nervous system tumor cells and genetically engineered mouse models have served as excellent platforms for drug discovery and preclinical testing, they have limitations with respect to accurately recapitulating important aspects of the pathobiology of spontaneously arising human tumors. For this reason, attention has turned to the deployment of human stem cell engineering involving human embryonic or induced pluripotent stem cells, in which genetic alterations associated with nervous system cancers can be introduced. These stem cells can be used to create self-assembling three-dimensional cerebral organoids that preserve key features of the developing human brain. Moreover, stem cell-engineered lines are amenable to xenotransplantation into mice as a platform to investigate the tumor cell of origin, discover cancer evolutionary trajectories and identify therapeutic vulnerabilities. In this article, we review the current state of human stem cell models of nervous system tumors, discuss their advantages and disadvantages, and provide consensus recommendations for future research.
Assuntos
Neoplasias Encefálicas , Células-Tronco Pluripotentes Induzidas , Criança , Humanos , Animais , Camundongos , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/patologia , Neoplasias Encefálicas/patologia , Encéfalo/patologia , MutaçãoRESUMO
Per- and polyfluoroalkyl substances (PFAS) are a class of highly stable chemicals, widely used in everyday products, and widespread in the environment, even in pregnant women. While epidemiological studies have linked prenatal exposure to PFAS with atopic dermatitis in children, little is known about their toxic effects on skin development, especially during the embryonic stage. In this study, we utilized human embryonic stem cells to generate non-neural ectoderm (NNE) cells and exposed them to six PFAS (perfluorooctanoic acid (PFOA), undecafluorohexanoic acid (PFHxA), heptafluorobutyric acid (PFBA), perfluorooctane sulfonate (PFOS), perfluorohexane sulfonate (PFHxS) and perfluorobutyric acid (PFBS)) during the differentiation process to assess their toxicity to early skin development. Our results showed that PFOS altered the spindle-like morphology of NNE cells to a pebble-like morphology, and disrupted several NNE markers, including KRT16, SMYD1, and WISP1. The six PFAS had a high potential to cause hypohidrotic ectodermal dysplasia (HED) by disrupting the expression levels of HED-relevant genes. Transcriptomic analysis revealed that PFOS treatment produced the highest number (1156) of differentially expressed genes (DEGs) among the six PFAS, including the keratinocyte-related genes KRT6A, KRT17, KRT18, KRT24, KRT40, and KRT81. Additionally, we found that PFOS treatment disturbed several signaling pathways that are involved in regulating skin cell fate decisions and differentiation, including TGF-ß, NOTCH, Hedgehog, and Hippo signaling pathways. Interestingly, we discovered that PFOS inhibited, by partially interfering with the expression of cytoskeleton-related genes, the ciliogenesis of NNE cells, which is crucial for the intercellular transduction of the above-mentioned signaling pathways. Overall, our study suggests that PFAS can inhibit ciliogenesis and hamper the transduction of important signaling pathways, leading potential congenital skin diseases. It sheds light on the underlying mechanisms of early embryonic skin developmental toxicity and provides an explanation for the epidemiological data on PFAS. ENVIRONMENTAL IMPLICATION: We employed a model based on human embryonic stem cells to demonstrate that PFOS has the potential to elevate the risk of hypohidrotic ectodermal dysplasia. This is achieved by targeting cilia, inhibiting ciliogenesis, and subsequently disrupting crucial signaling pathways like TGF-ß, NOTCH, Hedgehog, and Hippo, during the early phases of embryonic skin development. Our study highlights the dangers and potential impacts of six PFAS pollutants on human skin development. Additionally, we emphasize the importance of closely considering PFHxA, PFBA, PFHxS, and PFBS, as they have shown the capacity to modify gene expression levels, albeit to a lesser degree.
Assuntos
Ácidos Alcanossulfônicos , Displasia Ectodérmica Anidrótica Tipo 1 , Poluentes Ambientais , Fluorocarbonos , Criança , Humanos , Feminino , Gravidez , Animais , Ouriços , Ácidos Alcanossulfônicos/toxicidade , Alcanossulfonatos , Poluentes Ambientais/toxicidade , Fluorocarbonos/toxicidade , Fator de Crescimento Transformador beta , MicrotúbulosRESUMO
Human embryonic stem cell (hESC)- and human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) therapies are promising alternatives for the treatment of retinal degenerative diseases caused by RPE degeneration. The generation of autologous RPE cells from human adult donors, which has the advantage of avoiding immune rejection and teratoma formation, is an alternative cell resource to gain mechanistic insight into and test potential therapies for RPE degenerative diseases. Here, we found that limbal stem cells (LSCs) from hESCs and adult primary human limbus have the potential to produce RPE cells and corneal stromal stem cells (CSSCs). We showed that hESC-LSC-derived RPE cells (LSC-RPE) expressed RPE markers, had a phagocytic function, and synthesized tropical factors. Furthermore, during differentiation from LSCs to RPE cells, cells became pigmented, accompanied by a decrease in the level of LSC marker KRT15 and an increase in the level of RPE marker MITF. The Wnt signaling pathway plays a role in LSC-RPE fate transition, promotes MITF expression in the nucleus, and encourages RPE fate transition. In addition, we also showed that primary LSCs (pLSCs) from adult human limbus similar to hESC-LSC could generate RPE cells, which was supported by the co-expression of LSC and RPE cell markers (KRT15/OTX2, KRT15/MITF), suggesting the transition from pLSC to RPE cells, and typical polygonal morphology, melanization, RPE cell marker genes expression (TYR, RPE65), tight junction formation by ZO-1 expression, and the most crucial phagocytotic function. On the other hand, both hESC-LSCs and pLSCs also differentiated into CSSCs (LSC-CSSCs) that expressed stem cell markers (PAX6, NESTIN), presented MSC features, including surface marker expression and trilineage differentiation capability, like those in human CSSCs. Furthermore, the capability of pLSC-CSSC to differentiate into cells expressing keratocyte marker genes (ALDH3A1, PTGDS, PDK4) indicated the potential to induce keratocytes. These results suggest that the adult pLSC is an alternative cell resource, and its application provides a novel potential therapeutic avenue for preventing RPE dysfunction-related retinal degenerative diseases and corneal scarring.
Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco do Limbo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Diferenciação Celular , Células Epiteliais/metabolismo , Pigmentos da Retina/metabolismoRESUMO
Human embryonic stem cell (hESC)-derived midbrain dopaminergic (mDA) cell transplantation is a promising therapeutic strategy for Parkinson's disease (PD). Here, we present the derivation of high-purity mDA progenitors from clinical-grade hESCs on a large scale under rigorous good manufacturing practice (GMP) conditions. We also assessed the toxicity, biodistribution, and tumorigenicity of these cells in immunodeficient rats in good laboratory practice (GLP)-compliant facilities. Various doses of mDA progenitors were transplanted into hemi-parkinsonian rats, and a significant dose-dependent behavioral improvement was observed with a minimal effective dose range of 5,000-10,000 mDA progenitor cells. These results provided insights into determining a low cell dosage (3.15 million cells) for human clinical trials. Based on these results, approval for a phase 1/2a clinical trial for PD cell therapy was obtained from the Ministry of Food and Drug Safety in Korea, and a clinical trial for treating patients with PD has commenced.
Assuntos
Células-Tronco Embrionárias Humanas , Doença de Parkinson , Humanos , Ratos , Animais , Doença de Parkinson/terapia , Distribuição Tecidual , Neurônios Dopaminérgicos , Transplante de Células-Tronco/métodos , Mesencéfalo , Dopamina , Diferenciação CelularRESUMO
Human embryonic stem cells (hESCs) cultured in media containing bone morphogenic protein 4 (BMP4; B) differentiate into trophoblast-like cells. Supplementing media with inhibitors of activin/nodal signaling (A83-01) and of fibroblast growth factor 2 (PD173074) suppresses mesoderm and endoderm formation and improves specification of trophoblast-like lineages, but with variable effectiveness. We compared differentiation in four BMP4-containing media: mTeSR1-BMP4 only, mTeSR1-BAP, basal medium with BAP (basal-BAP), and a newly defined medium, E7-BAP. These media variably drive early differentiation towards trophoblast-like lineages with upregulation of early trophoblast markers CDX2 and KRT7 and downregulation of pluripotency markers (OCT4 and NANOG). As expected, based on differences between media in FGF2 and its inhibitors, downregulation of mesendoderm marker EOMES was variable between media. By day 7, only hESCs grown in E7-BAP or basal-BAP expressed HLA-G protein, indicating the presence of cells with extravillous trophoblast characteristics. Expression of HLA-G and other differentiation markers (hCG, KRT7, and GCM1) was highest in basal-BAP, suggesting a faster differentiation in this medium, but those cultures were more inhomogeneous and still expressed some endodermal and pluripotency markers. In E7-BAP, HLA-G expression increased later and was lower. There was also a low but maintained expression of some C19MC miRNAs, with more CpG hypomethylation of the ELF5 promoter, suggesting that E7-BAP cultures differentiate slower along the trophoblast lineage. We conclude that while all protocols drive differentiation into trophoblast lineages with varying efficiency, they have advantages and disadvantages that must be considered when selecting a protocol for specific experiments.
Assuntos
Células-Tronco Embrionárias Humanas , Humanos , Ativinas/farmacologia , Ativinas/metabolismo , Proteína Morfogenética Óssea 4/farmacologia , Diferenciação Celular , Células-Tronco Embrionárias/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Antígenos HLA-G , Células-Tronco Embrionárias Humanas/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Trofoblastos/metabolismoRESUMO
Glycosphingolipids (GSLs) display diverse functions during embryonic development. Here, we examined the GSL profiles of extracellular vesicles (EVs) secreted from human embryonic stem cells (hESCs) and investigated their functions in priming macrophages to enhance immune tolerance of embryo implantation. When peripheral blood mononuclear cells were incubated with ESC-secreted EVs, globo-series GSLs (GHCer, SSEA3Cer, and SSEA4Cer) were transferred via EVs into monocytes/macrophages. Incubation of monocytes during their differentiation into macrophages with either EVs or synthetic globo-series GSLs induced macrophages to exhibit phenotypic features that imitate immune receptivity, i.e., macrophage polarization, augmented phagocytic activity, suppression of T cell proliferation, and the increased trophoblast invasion. It was also demonstrated that decidual macrophages in first-trimester tissues expressed globo-series GSLs. These findings highlight the role of globo-series GSLs via transfer from EVs in priming macrophages to display decidual macrophage phenotypes, which may facilitate healthy pregnancy.
Assuntos
Glicoesfingolipídeos , Leucócitos Mononucleares , Gravidez , Feminino , Humanos , Macrófagos , Diferenciação Celular , Tolerância ImunológicaRESUMO
BACKGROUND: Research on human pluripotent stem cells (hPSCs) has shown tremendous progress in cell-based regenerative medicine. Corneal endothelial dysfunction is associated with the loss and degeneration of corneal endothelial cells (CECs), rendering cell replacement a promising therapeutic strategy. However, comprehensive preclinical assessments of hPSC-derived CECs for this cell therapy remain a challenge. RESULTS: Here we defined an adapted differentiation protocol to generate induced corneal endothelial cells (iCECs) consistently and efficiently from clinical-grade human embryonic stem cells (hESCs) with xeno-free medium and manufactured cryopreserved iCECs. Cells express high levels of typical CECs markers and exhibit transendothelial potential properties in vitro typical of iCECs. After rigorous quality control measures, cells meeting all release criteria were available for in vivo studies. We found that there was no overgrowth or tumorigenicity of grafts in immunodeficient mice. After grafting into rabbit models, the surviving iCECs ameliorated edema and recovered corneal opacity. CONCLUSIONS: Our work provides an efficient approach for generating iCECs and demonstrates the safety and efficacy of iCECs in disease modeling. Therefore, clinical-grade iCECs are a reliable source for future clinical treatment of corneal endothelial dysfunction.
RESUMO
Autosomal recessive Stargardt disease (STGD1) is an inherited retinal degenerative disease associated with a mutated ATP-binding cassette, subfamily A, member 4 (ABCA4) gene. STGD1 is the most common form of juvenile macular degeneration with onset in late childhood to early or middle adulthood and causes progressive, irreversible visual impairment and blindness. No effective treatment is currently available. In the present article, we review the most recent updates in clinical trials targeting the management of STGD1, including gene therapy, small molecule therapy, and stem cell therapy. In gene therapy, dual adeno-associated virus and non-viral vectors have been successful in delivering the human ABCA4 gene in preclinical studies. For pharmaceutical therapies ALK-001, deuterated vitamin A shows promise with preliminary data for phase 2 trial, demonstrating a decreased atrophy growth rate after two years. Stem cell therapy using human pluripotent stem cell-derived retinal pigment epithelium cells demonstrated long-term safety three years after implantation and visual acuity improvements in the first two years after initiation of therapy. Many other treatment options have ongoing investigations and clinical trials. While multiple potential interventions have shown promise in attenuating disease progression, further exploration is necessary to demonstrate treatment safety and efficacy.
RESUMO
BACKGROUND: New disease-modifying ways to treat Parkinson's disease (PD) may soon become a reality with intracerebral transplantation of cell products produced from human embryonic stem cells (hESCs). The aim of this study was to assess what factors influence preferences of patients with PD regarding stem-cell based therapies to treat PD in the future. METHODS: Patients with PD were invited to complete a web-based discrete choice experiment to assess the importance of the following attributes: (i) type of treatment, (ii) aim of treatment, (iii) available knowledge of the different types of treatments, (iv) effect on symptoms, and (v) risk for severe side effects. Latent class conditional logistic regression models were used to determine preference estimates and heterogeneity in respondents' preferences. RESULTS: A substantial difference in respondents' preferences was observed in three latent preference patterns (classes). "Effect on symptoms" was the most important attribute in class 1, closely followed by "type of treatment," with medications as preferred to other treatment alternatives. Effect on symptoms was also the most important attribute in class 2, with treatment with hESCs preferred over other treatment alternatives. Likewise for class 3, that mainly focused on "type of treatment" in the decision-making. Respondents' class membership was influenced by their experience in treatment, side effects, and advanced treatment therapy as well as religious beliefs. CONCLUSIONS: Most of the respondents would accept a treatment with products emanating from hESCs, regardless of views on the moral status of embryos. Preferences of patients with PD may provide guidance in clinical decision-making regarding treatments deriving from stem cells.
Assuntos
Comportamento de Escolha , Doença de Parkinson , Humanos , Doença de Parkinson/terapia , Preferência do Paciente , Modelos Logísticos , Células-Tronco EmbrionáriasRESUMO
The differentiation of pluripotent stem cells has been used to study disease mechanisms and development. We previously described a method for differentiating human pluripotent stem cells (hPSCs) into salivary gland epithelial progenitors (SGEPs). Here, cystic fibrosis transmembrane conductance regulator (CFTR) knockout hPSCs were differentiated into SGEPs derived from CFTR knockout hESCs (CF-SGEPs) using the same protocol to investigate whether the hPSC-derived SGEPs can model the characteristics of CF. CF-a disease that affects salivary gland (SG) function-is caused by mutations of the CFTR gene. Firstly, we successfully generated CFTR knockout hPSCs with reduced CFTR protein expression using the CRISPR-Cas9 system. After 16 days of differentiation, the protein expression of CFTR decreased in SGEPs derived from CFTR knockout hESCs (CF-SGEPs). RNA-Seq revealed that multiple genes modulating SG development and function were down-regulated, and positive regulators of inflammation were up-regulated in CF-SGEPs, correlating with the salivary phenotype of CF patients. These results demonstrated that CFTR suppression disrupted the differentiation of hPSC-derived SGEPs, which modeled the SG development of CF patients. In summary, this study not only proved that the hPSC-derived SGEPs could serve as manipulable and readily accessible cell models for the study of SG developmental diseases but also opened up new avenues for the study of the CF mechanism.
RESUMO
BACKGROUND: Huntington's disease (HD) is a motor and cognitive neurodegenerative disorder due to prominent loss of striatal medium spiny neurons (MSNs). Cell replacement using human embryonic stem cells (hESCs) derivatives may offer new therapeutic opportunities to replace degenerated neurons and repair damaged circuits. METHODS: With the aim to develop effective cell replacement for HD, we assessed the long-term therapeutic value of hESC-derived striatal progenitors by grafting the cells into the striatum of a preclinical model of HD [i.e., adult immunodeficient rats in which the striatum was lesioned by monolateral injection of quinolinic acid (QA)]. We examined the survival, maturation, self-organization and integration of the graft as well as its impact on lesion-dependent motor alterations up to 6 months post-graft. Moreover, we tested whether exposing a cohort of QA-lesioned animals to environmental enrichment (EE) could improve graft integration and function. RESULTS: Human striatal progenitors survived up to 6 months after transplantation and showed morphological and neurochemical features typical of human MSNs. Donor-derived interneurons were also detected. Grafts wired in both local and long-range striatal circuits, formed domains suggestive of distinct ganglionic eminence territories and displayed emerging striosome features. Moreover, over time grafts improved complex motor performances affected by QA. EE selectively increased cell differentiation into MSN phenotype and promoted host-to-graft connectivity. However, when combined to the graft, the EE paradigm used in this study was insufficient to produce an additive effect on task execution. CONCLUSIONS: The data support the long-term therapeutic potential of ESC-derived human striatal progenitor grafts for the replacement of degenerated striatal neurons in HD and suggest that EE can effectively accelerate the maturation and promote the integration of human striatal cells.