RESUMEN
Naïve pluripotency can be established in human pluripotent stem cells (hPSCs) by manipulation of transcription factors, signaling pathways, or a combination thereof. However, differences exist in the molecular and functional properties of naïve hPSCs generated by different protocols, which include varying similarities with pre-implantation human embryos, differentiation potential, and maintenance of genomic integrity. We show here that short treatment with two chemical agonists (2a) of nuclear receptors, liver receptor homologue-1 (LRH-1) and retinoic acid receptor gamma (RAR-γ), along with 2i/LIF (2a2iL) induces naïve-like pluripotency in human cells during reprogramming of fibroblasts, conversion of pre-established hPSCs, and generation of new cell lines from blastocysts. 2a2iL-hPSCs match several defined criteria of naïve-like pluripotency and contribute to human-mouse interspecies chimeras. Activation of TGF-ß signaling is instrumental for acquisition of naïve-like pluripotency by the 2a2iL induction procedure, and transient activation of TGF-ß signaling substitutes for 2a to generate naïve-like hPSCs. We reason that 2a2iL-hPSCs are an easily attainable system to evaluate properties of naïve-like hPSCs and for various applications.
Asunto(s)
Células Madre Pluripotentes , Animales , Blastocisto , Diferenciación Celular , Línea Celular , Humanos , Ratones , Receptores Citoplasmáticos y Nucleares/genética , Receptores de Ácido Retinoico , Receptor de Ácido Retinoico gammaRESUMEN
Pluripotent cells transiently develop during peri-implantation embryogenesis and have the capacity to convert into three embryonic lineages. Two typical states of pluripotency, naïve and primed, can be experimentally induced in vitro. The in vitro naïve state can be stabilized in response to environmental inductive cues via a unique transcriptional regulatory program. However, interference with various signaling pathways creates a spectrum of alternative pluripotent cells that display different functions and molecular expression patterns. Similarly, human naïve pluripotent cells can be placed into two main levels - intermediate and bona fide. Here, we discuss several culture conditions that have been used to establish naïve-associated gene regulatory networks in human pluripotent cells. We also describe different transcriptional patterns in various culture systems that are associated with these two levels of human naïve pluripotency.
Asunto(s)
Blastocisto/citología , Células Madre Embrionarias/citología , Regulación del Desarrollo de la Expresión Génica , Células Madre Pluripotentes/citología , Animales , Blastocisto/metabolismo , Diferenciación Celular , Células Madre Embrionarias/metabolismo , Humanos , Células Madre Pluripotentes/metabolismo , Transducción de SeñalRESUMEN
The ability of human embryonic stem cells (hESCs) to proliferate unlimitedly and give rise to all tissues makes these cells a promising source for cell replacement therapies. To realize the full potential of hESCs in cell therapy, it is necessary to interrogate regulatory pathways that influence hESC maintenance and commitment. Here, we reveal that pharmacological attenuation of p38 mitogen-activated protein kinase (p38-MAPK) in hESCs concomitantly augments some characteristics associated with pluripotency and the expressions of early lineage markers. Moreover, this blockage capacitates hESCs to differentiate towards an endoderm lineage at the expense of other lineages upon spontaneous hESC differentiation. Notably, hESCs pre-treated with p38-MAPK inhibitor exhibit significantly improved pancreatic progenitor directed differentiation. Together, our findings suggest a new approach to the robust endoderm differentiation of hESCs and potentially enables the facile derivation of various endoderm-derived lineages such as pancreatic cells.
Asunto(s)
Endodermo/citología , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Diferenciación Celular/efectos de los fármacos , Línea Celular , Endodermo/efectos de los fármacos , Células Madre Embrionarias Humanas/metabolismo , Humanos , Páncreas/citología , Páncreas/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Although males and females have a variety of sexually dimorphic features related to hormonal effects, the genetic basis of dimorphism relies on early embryo development. Two pluripotent states, naïve and primed, emerge during early mammalian development. Identification of signaling pathways that induce differences between these two states can help to modulate conversion of primed cells to naïve cells. Naïve cells have a shorter doubling time and longer survival than their primed counterparts when passaged as single cells. In this study, we sought to explore the role of Y chromosome genes on human pluripotent stem cells (hPSCs) by investigating differential expressions of the male-specific region of the Y chromosome (MSY) genes in primed and naïve cells. Interestingly, we found that several MSY genes, including SRY, showed higher expression levels in primed compared to naïve human embryonic stem cells (hESCs). We hypothesize that SRY prevents WNT/ß-catenin signaling by its interaction and inhibition of ß-catenin activation in the nucleus. Results of the loss-of-function approach conducted by depletion of SRY indicated increased expressions of pluripotency marker genes and alkaline phosphatase (ALP) activity in the primed cells. SRY reduction was associated with overexpression of WNT signaling target genes AXIN2, Brachury, TCF1, TBX2, and TBX3. We suggest that inhibition of SRY may result in activation of ß-catenin and up-regulation of the WNT signaling pathway, both of which are important to naïve conversion.
Asunto(s)
Cromosomas Humanos Y , Células Madre Pluripotentes/fisiología , Proteína de la Región Y Determinante del Sexo/genética , Biomarcadores , Células Cultivadas , Células Madre Embrionarias/citología , Células Madre Embrionarias/fisiología , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Células Madre Pluripotentes/citología , Transducción de Señal , Vía de Señalización Wnt/genética , beta Catenina/metabolismoRESUMEN
STUDY QUESTION: Can transforming growth factor ß (TGFß) inhibition promote ground state pluripotency of embryonic stem cells (ESCs) from single blastomeres (SBs) of cleavage embryos in different mouse stains? SUMMARY ANSWER: Small molecule suppression of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and TGFß signaling (designated as R2i) can enhance the generation of mouse ESCs from SBs of different cleavage stage embryos compared with the dual suppression of ERK1/2 and glycogen synthase kinase 3 (GSK3), designated as 2i, regardless of the strain of mouce. WHAT IS KNOWN ALREADY: It is known that chemical inhibition of TGFß promotes ground state pluripotency in the generation and sustenance of naïve ES cells from mouse blastocysts compared with the well-known 2i condition. However, the positive effect of this inhibition on mouse ESCs from early embryonic SBs remains obscure. STUDY DESIGN, SIZE, DURATION: We used 155 cleavage-stage mouse embryos to optimize the culture conditions for blastocyst development. Then, to assess the effects of R2i and 2i on ESC generation from SBs, we cultured isolated SBs in 2i and R2i for 10 days. SBs were replated under the same conditions to produce ESCs. In total, 46 embryos and 321 SBs from two- to eight-cell stages were recovered from NMRI and BALB/c mouse strains and used in this study. PARTICIPANTS/MATERIALS, SETTING, METHODS: Blastomeres from 2- to 8-cell stage mouse embryos were dispersed and individually seeded into a 96-well plates that included mitotically inactivated feeder cells. ESCs were generated in B27N2 deï¬ned medium supplemented with R2i or 2i. Randomly selected ESC lines, generated from SBs of each stage, were assessed for pluripotency and germ-line transmission. MAIN RESULTS AND THE ROLE OF CHANCE: We demonstrated that dual inhibition of ERK1/2 and TGFß (R2i) enhanced efficient blastocyst development and efficient establishment of ESCs from SB of 2- to 8-cell stage mouse embryos compared with the dual inhibition of ERK1/2 and GSK3 (2i) regardless of the embryonic stage and strain of mice. The proportions of SBs that produced ESC were 50-60 versus 20-30%. LIMITATIONS, REASONS FOR CAUTION: This study was done with mouse embryos, it is not known whether these findings are transferable to humans. WIDER IMPLICATIONS OF THE FINDINGS: These findings resulted in an increased efficiency of ESC generation from one biopsied blastomere for autogeneic or allogeneic matched pluripotent cells without the need to destroy viable embryos. The results also provided information about the developmental capacity of early embryonic blastomeres. STUDY FUNDING/COMPETING INTERESTS: This study was funded by grants provided from Royan Institute, the Iranian Council of Stem Cell Research and Technology and the Iran National Science Foundation. The authors have no conï¬ict of interest to declare.
Asunto(s)
Blastómeros/efectos de los fármacos , Embrión de Mamíferos/efectos de los fármacos , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Animales , Blastómeros/metabolismo , Diferenciación Celular , Técnicas de Cultivo de Embriones , Embrión de Mamíferos/citología , Desarrollo Embrionario/efectos de los fármacos , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Femenino , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB CRESUMEN
STUDY QUESTION: Could selected pluripotency-enhancing small molecules (SMs) lead to efficient derivation of human embryonic stem cells (hESCs) from cleavage embryos-derived single blastomeres (SBs)? SUMMARY ANSWER: Inhibition of glycogen synthase kinase ß (GSK3ß) and Rho-associated kinase (ROCK) signaling can enhance the derivation of hESCs from cleavage embryo-derived SBs. WHAT IS KNOWN ALREADY: Parameters involved in sustaining the pluripotency of biopsied blastomeres for generating hESCs without causing injury to a viable embryo have remained obscure. This research seeks to improve the culture conditions for increasing the efficiency of deriving hESCs from SBs from cleavage-stage embryos by using SMs. STUDY DESIGN, SIZE, DURATION: In order to identify SMs which may enhance hESC generation from SBs, 11 pluripotency-enhancing SMs were screened and CHIR99021 (CH), a GSK3ß inhibitor, was selected. To optimize culture condition in hESC generation from SMs, we used ROCK inhibitor Y27632 (Y) and basic fibroblast growth factor in combination with CH or its alternative, Kenpaullone, in different time courses over 12 days. We also assessed a critical time point for CH + Y treatment of cleavage embryos from 4- to 8-cell embryo. In total, 224 embryos and 1607 SBs were used in the study. PARTICIPANTS/MATERIALS, SETTING, METHODS: Blastomeres of fair and poor-quality from 6- to 8-cell stage human embryos were mechanically dispersed and individually seeded into a 96-well plate that was precoated with mitotically inactivated feeder cells. Derivation of hESC line from each SB was carried out in hESC defined medium supplemented with SMs. Randomly selected hESC lines were evaluated by immunostaining for pluripotency markers, karyotype analysis and differentiation potential into the three embryonic germ layer derivatives. MAIN RESULTS AND THE ROLE OF CHANCE: We found that 3 µM CH was the only SM that was capable of directing SBs from fair and poor-quality 6-8-cell embryos into hESC lines. The application of hESC-conditioned medium had no additive effect on hESC establishment from SBs. Also, we indicated that CH combined with Y improved hESC generation efficiency by up to 31%. By using of Kenpaullone as an alternative to CH, we confirmed the involvement of GSK3 inhibition in hESC derivation from SBs. Interestingly, by treatment of 4-cell embryos, these SMs could enhance the derivation efficiency of SB-derived hESC lines up to 73% and the maximum number of hESC lines from SBs of one embryo was achieved in this state. LIMITATIONS, REASONS FOR CAUTION: The low quality of the embryos used in this study most likely had an effect on hESC generation. Furthermore, although we attempted to minimize any differences in inter-embryo quality, we cannot exclude the possibility that small differences in starting quality between embryos may have contributed to the differences observed, other than the addition of SMs. WIDER IMPLICATIONS OF THE FINDINGS: This approach would allow the establishment of autogeneic or allogeneic matched cells from embryos fertilized in vitro without destroying them. STUDY FUNDING/COMPETING INTEREST(S): This study was financially supported by the National Elite Foundation and the Royan Institute for Stem Cell Biology and Technology. The authors have no conflict of interest to declare.
Asunto(s)
Blastómeros/química , Técnicas de Cultivo de Embriones , Células Madre Embrionarias/citología , Glucógeno Sintasa Quinasas/antagonistas & inhibidores , Quinasas Asociadas a rho/metabolismo , Amidas/farmacología , Benzazepinas/farmacología , Diferenciación Celular , Línea Celular , Embrión de Mamíferos/citología , Desarrollo Embrionario , Factor 2 de Crecimiento de Fibroblastos/farmacología , Humanos , Indoles/farmacología , Cariotipificación , Piridinas/farmacología , Pirimidinas/farmacología , Transducción de Señal/efectos de los fármacos , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
AIM: Parkinson's Disease (PD) is a common age-related neurodegenerative disorder with a rising prevalence. Human pluripotent stem cells have emerged as the most promising source of cells for midbrain dopaminergic (mDA) neuron replacement in PD. This study aimed to generate transplantable mDA progenitors for treatment of PD. MATERIALS AND METHODS: Here, we optimized and fine-tuned a differentiation protocol using a combination of small molecules and growth factors to induce mDA progenitors to comply with good manufacturing practice (GMP) guidelines based on our clinical-grade human embryonic stem cell (hESC) line. KEY FINDINGS: The resulting mDA progenitors demonstrated robust differentiation and functional properties in vitro. Moreover, cryopreserved mDA progenitors were transplanted into 6-hydroxydopamine-lesioned rats, leading to functional recovery. SIGNIFICANCE: We demonstrate that our optimized protocol using a clinical hESC line is suitable for generating clinical-grade mDA progenitors and provides the ground work for future translational applications.
Asunto(s)
Células Madre Embrionarias Humanas , Enfermedad de Parkinson , Células Madre Pluripotentes , Humanos , Ratas , Animales , Enfermedad de Parkinson/terapia , Enfermedad de Parkinson/metabolismo , Neuronas Dopaminérgicas/fisiología , Diferenciación Celular , Dopamina/metabolismo , Mesencéfalo/metabolismoRESUMEN
OBJECTIVE: Systemic sclerosis (SSc) is a connective tissue disease associated with vascular damage and multi organ fibrotic changes with unknown pathogenesis. Most SSc patients suffer from defective angiogenesis/vasculogenesis and cardiac conditions leading to high mortality rates. We aimed to investigate the cardiovascular phenotype of SSc by cardiogenic differentiation of SSc induced pluripotent stem cells (iPSC). MATERIALS AND METHODS: In this experimental study, we generated iPSC from two diffuse SSc patients, followed by successful differentiation into endothelial cells (ECs) and cardiomyocytes (CMs). RESULTS: SSc-derived EC (SSc-EC) expressed KDR, a nearly EC marker, similar to healthy control-EC (C1-EC). After sorting and culturing KDR+ cells, the resulting EC expressed CD31, a late endothelial marker, but vascular endothelial (VE)-cadherin expression markedly dropped resulting in a functional defect as reflected in tube formation failure of SSc-EC. Interestingly, upregulation of SNAI1 (snail family transcriptional repressor 1) was observed in SSc-EC which might underlie VE-cadherin downregulation. Furthermore, SSc-derived CM (SSc-CM) successfully expressed cardiacspecific markers including ion channels, resulting in normal physiological behavior and responsiveness to cardioactive drugs. CONCLUSION: This study provides an insight into impaired angiogenesis observed in SSc patients by evaluating in vitro cardiovascular differentiation of SSc iPSC.
RESUMEN
Human pluripotent stem cells (hPSCs) are commonly kept in a primed state but also able to acquire a more immature naive state under specific conditions in vitro. Acquisition of naive state changes several properties of hPSCs and might affect their contribution to embryonic development in vivo. However, the lack of an appropriate animal test system has made it difficult to assess potential differences for chimera formation between naive and primed hPSCs. Here, we report that the developing chicken embryo is a permissive host for hPSCs, allowing analysis of the pluripotency potential of hPSCs. Transplantation of naive-like and primed hPSCs at matched developmental stages resulted in robust chimerism. Importantly, the ability of naive-like but not of primed hPSCs to form chimera was substantially reduced when injected at non-matched developmental stages. We propose that contribution to chick embryogenesis is an informative and versatile test to identify different pluripotent states of hPSCs.
Asunto(s)
Embrión de Pollo/metabolismo , Quimerismo/veterinaria , Células Madre Pluripotentes/trasplante , Animales , Diferenciación Celular , Linaje de la Célula , Embrión de Pollo/citología , Pollos , Desarrollo Embrionario , Edición Génica , Humanos , Proteínas con Homeodominio LIM/genética , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción/genética , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMEN
Differentiation of stem cells from a pluripotent to a committed state involves global changes in genome expression patterns, critically determined by chromatin structure and interactions of chromatin-binding proteins. The dynamics of chromatin structure are tightly regulated by multiple epigenetic mechanisms such as histone modifications and the incorporation of histone variants. In the current work, we induced neural differentiation of a human embryonal carcinoma stem cell line, NTERA2/NT2, by retinoic acid (RA) treatment, primarily according to two different methods of adherent cell culture (rosette formation) and suspension cell culture (EB formation) conditions, and histone modifications and variations were compared through these processes. Western blot analysis of histone extracts showed significant changes in the acetylation and methylation patterns of histone H3, and expression level of the histone variant H2A.Z, after RA treatment in both protocols. Using chromatin immunoprecipitation (ChIP) coupled with real-time PCR, it was shown that these epigenetic changes occurred on the regulatory regions of 4 marker genes (Oct4, Nanog, Nestin, and Pax6) in a culture condition dependent manner. This report demonstrates the dynamic interplay of histone modification and variation in regulating the gene expression profile, during stem cell differentiation and under different culture conditions.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Células Madre de Carcinoma Embrionario/metabolismo , Epigénesis Genética , Histonas/metabolismo , Tretinoina/farmacología , Western Blotting , Células Madre de Carcinoma Embrionario/citología , Células Madre de Carcinoma Embrionario/efectos de los fármacos , Humanos , Microscopía Fluorescente , Sistema Nervioso/citología , Reacción en Cadena de la PolimerasaRESUMEN
Bombay phenotype is one of the rare phenotypes in the ABO blood group system that fails to express ABH antigens on red blood cells. Nonsense or missense mutations in fucosyltransfrase1 (FUT1) and fucosyltransfrase2 (FUT2) genes are known to create this phenotype. This blood group is compatible with all other blood groups as a donor, as it does not express the H antigen on the red blood cells. In this study, we describe the establishment of human induced pluripotent stem cells (iPSCs) from the dermal fibroblasts of a Bombay blood-type individual by the ectopic expression of established transcription factors Klf4, Oct4, Sox2, and c-Myc. Sequence analyses of fibroblasts and iPSCs revealed a nonsense mutation 826C to T (276 Gln to Ter) in the FUT1 gene and a missense mutation 739G to A (247 Gly to Ser) in the FUT2 gene in the Bombay phenotype under study. The established iPSCs resemble human embryonic stem cells in morphology, passaging, surface and pluripotency markers, normal karyotype, gene expression, DNA methylation of critical pluripotency genes, and in-vitro differentiation. The directed differentiation of the iPSCs into hematopoietic lineage cells displayed increased expression of the hematopoietic lineage markers such as CD34, CD133, RUNX1, KDR, alpha-globulin, and gamma-globulin. Such specific stem cells provide an unprecedented opportunity to produce a universal blood group donor, in-vitro, thus enabling cellular replacement therapies, once the safety issue is resolved.
Asunto(s)
Sistema del Grupo Sanguíneo ABO , Línea Celular , Eritrocitos/citología , Hematopoyesis , Células Madre Pluripotentes/citología , Secuencia de Aminoácidos , Secuencia de Bases , Donantes de Sangre , Fibroblastos/citología , Fibroblastos/metabolismo , Fucosiltransferasas/genética , Expresión Génica , Humanos , Factor 4 Similar a Kruppel , Mutación Missense , Fenotipo , Células Madre Pluripotentes/metabolismo , Factores de Transcripción/genética , Galactósido 2-alfa-L-FucosiltransferasaRESUMEN
BACKGROUND: An essential prerequisite for the future widespread application of human induced pluripotent (hiPSCs) and embryonic stem cells (hESCs) is the development of efficient cryopreservation methods to facilitate their storage and transportation. METHODS: We developed a simple and effective freezing/thawing method of single dissociated hESCs and hiPSCs in a feeder-free culture in the presence of Rho-associated kinase (ROCK) inhibitor Y-27632. RESULTS: Exposure to ROCK inhibitor Y-27632 in freezing solution alone does not significantly enhance the post-thaw survival rate of single dissociated hESCs and hiPSCs. However, when ROCK inhibitor was added to both pre- and post-thaw culture media, there was an enhancement in the survival rate, which further increased when ROCK inhibitor was added to Matrigel as well. Under these treatments, hESCs and hiPSCs retained typical morphology, stable karyotype, expression of pluripotency markers and the potential to differentiate into derivatives of all three germ layers after long-term culture. CONCLUSIONS: This method is an effective cryopreservation procedure for single dissociated hESCs in feeder-free culture, which is also applicable for single dissociated hiPSCs using a ROCK inhibitor. The cloning efficiency of hiPSCs and hESCs improves when ROCK inhibitor is added both in Matrigel and in medium in comparison with conventional addition to medium. Therefore, we believe this method would be useful for current and future applications of the pluripotent stem cells.
Asunto(s)
Criopreservación/métodos , Células Madre Embrionarias/citología , Células Madre Pluripotentes/citología , Técnicas de Cultivo de Célula , Diferenciación Celular , Supervivencia Celular , Células Cultivadas , Colágeno , Crioprotectores , Medios de Cultivo , Combinación de Medicamentos , Células Madre Embrionarias/metabolismo , Marcadores Genéticos , Humanos , Laminina , Células Madre Pluripotentes/metabolismo , ProteoglicanosRESUMEN
The generation of human induced pluripotent stem cells (hiPSCs) from an individual patient provides a unique tool for disease modeling, drug discovery, and cell replacement therapies. Patient-specific pluripotent stem cells can be expanded in vitro and are thus suitable for genetic manipulations. To date, several genetic liver disorders have been modeled using patient-specific hiPSCs. Here, we present the generation of corrected hepatocyte-like cells (HLCs) from hiPSCs of a familial hypercholesterolemia (FH) patient with a homozygous mutation in the low-density lipoprotein receptor (LDLR) gene. We generated hiPSCs from a patient with FH with the mutated gene encoding a truncated non-functional receptor. In order to deliver normal LDLR to the defective cells, we used a plasmid vector carrying the normal receptor ORF to genetically transform the hiPSCs. The transformed cells were expanded and directed toward HLCs. Undifferentiated defective hiPSCs and HLCs differentiated from the defective hiPSCs did not have the ability to uptake labeled low-density lipoprotein (LDL) particles. The differentiated transformed hiPSCs showed LDL-uptake ability and the correction of disease phenotype as well as expressions of hepatocyte-specific markers. The functionality of differentiated cells was also confirmed by indo-cyanine green (ICG) uptake assay, PAS staining, inducible cyp450 activity, and oil red staining. These data suggest that hiPSC technology can be used for generation of disease-corrected, patient-specific HLCs with potential value for disease modeling and drug discovery as well as cell therapy applications in future.
Asunto(s)
Trasplante de Células/métodos , Terapia Genética/métodos , Hepatocitos/citología , Hiperlipoproteinemia Tipo II/terapia , Células Madre Pluripotentes Inducidas/citología , Adolescente , Biomarcadores/análisis , Biomarcadores/metabolismo , Biotecnología , Femenino , Citometría de Flujo , Humanos , Inmunohistoquímica , Cariotipo , Mutación , Receptores de LDL/genética , TransfecciónRESUMEN
Mouse embryonic stem cells (ESCs) are pluripotent stem cell lines derived from pre-implantation embryos. The efficiency of mESC generation is affected by genetic variation in mice; that is, some mouse strains are refractory or non-permissive to ESC establishment. Developing an efficient method to derive mESCs from strains of various genetic backgrounds should be valuable for establishment of ESCs in various mammalian species. In the present study, we identified dual inhibition of TGF-ß and ERK1/2, by SB431542 and PD0325901, respectively led to the highly efficient and reproducible generation of mESC lines from NMRI, C57BL/6, BALB/c, DBA/2, and FVB/N strains, which previously considered refractory or non-permissive for ESC establishment. These mESCs expressed pluripotency markers and retained the capacity to differentiate into derivatives of all three germ layers. The evaluated lines exhibited high rates of chimerism when reintroduced into blastocysts. To our knowledge, this is the first report of efficient (100%) mESC lines generation from different genetic backgrounds. The application of these two inhibitors will not only solve the problems of mESC derivation but also clarifies new signaling pathways in pluripotent mESCs.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Células Madre Embrionarias/enzimología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Sistema de Señalización de MAP Quinasas , Factor de Crecimiento Transformador beta/metabolismo , Animales , Benzamidas/farmacología , Biomarcadores/metabolismo , Blastocisto/citología , Blastocisto/efectos de los fármacos , Blastocisto/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Medios de Cultivo/farmacología , Dioxoles/farmacología , Difenilamina/análogos & derivados , Difenilamina/farmacología , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Endogámicos , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/efectos de los fármacos , Células Madre Pluripotentes/metabolismo , Reproducibilidad de los Resultados , Bibliotecas de Moléculas Pequeñas/farmacología , Factores de TiempoRESUMEN
Induced pluripotent stem cells (iPSCs) provide an invaluable resource for drug or toxicology screening, medical research and patient-specific cell therapy. However, the potential applications of iPSCs are largely dependent on the degree of similarity between iPSCs and embryonic stem cells (ESCs). In the present study, we analyzed the proteome of human ESCs and hiPSCs with different genetic background. We carried out an orthogonal contrast analysis of the proteome pattern of two human ESC lines (Royan H5 and Royan H6) and two hiPSC lines from a normal individual, three hiPSC lines from a normal individual with Bombay blood group phenotype, and two hiPSC lines from a patient with tyrosinemia. Forty-nine protein spots showed statistically significant differences between two human ESC lines and seven human iPSCs. Mass spectrometry analysis resulted in the identification of 48 proteins belonging to different biological processes, including cytoskeleton organization, energy and metabolic processes, protein synthesis and processing, signal transduction, cell growth and proliferation, cellular trafficking, transcription, calcium binding and immune response. Our results showed that hESCs and hiPSCs had subtle differences at the proteome level thus warranting more detailed and systematic examinations of these cells.
Asunto(s)
Células Madre Embrionarias/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Proteoma/análisis , Sistema del Grupo Sanguíneo ABO/genética , Línea Celular , Células Cultivadas , Análisis por Conglomerados , Regulación hacia Abajo , Electroforesis en Gel Bidimensional , Células Madre Embrionarias/química , Humanos , Células Madre Pluripotentes Inducidas/química , Espectrometría de Masas , Fenotipo , Proteoma/química , Proteoma/clasificación , Proteómica , Tirosinemias/genéticaRESUMEN
We describe a new, efficient protocol that involves the serial addition of noggin, basic fibroblast growth factor (bFGF), retinoic acid, and sonic hedgehog (Shh) for the differentiation of human induced pluripotent stem cells (hiPSC) to retinal pigmented epithelium (RPE) in a serum- and feeder-free adherent condition. hiPSC-RPE cells exhibited RPE morphology and specific molecular markers. Additionally, several hiPSC lines were generated from retinal-specific patients with Leber's congenital amaurosis, Usher syndrome, two patients with retinitis pigmentosa, and a patient with Leber's hereditary optic neuropathy. The RPE cells generated from these disease-specific hiPSCs expressed specific markers by the same RPE lineage-directed differentiation protocol. These findings indicate a new short-term, simple, and efficient protocol for differentiation of hiPSCs to RPE cells. Such specific retinal disease-specific hiPSCs offer an unprecedented opportunity to recapitulate normal and pathologic formation of human retinal cells in vitro, thereby enabling pharmaceutical screening, and potentially autologous cell replacement therapies for retinal diseases.
Asunto(s)
Diferenciación Celular , Células Madre Pluripotentes Inducidas/fisiología , Enfermedades de la Retina/patología , Epitelio Pigmentado de la Retina/patología , Adolescente , Adulto , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Técnicas de Cultivo de Célula , Forma de la Célula , Células Cultivadas , Niño , Femenino , Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Cariotipo , Masculino , Persona de Mediana Edad , Fenotipo , Reacción en Cadena en Tiempo Real de la Polimerasa , Epitelio Pigmentado de la Retina/metabolismoRESUMEN
Here we describe a simple and efficient human embryonic stem (ES) and induced pluripotent stem (iPS) cells cryopreservation protocol. This protocol involves the use of Rho-associated kinase (ROCK) inhibitor, Y-27632, for the feeder-free dissociated cells. The addition of ROCK inhibitor to both pre- and post-thaw culture media enhanced the cloning efficiency. The presence of Y-27632 in Matrigel further increased the cloning efficiency. As compared with other available protocols for human ES and iPS cells cryopreservation, our protocol differs in the technical simplicity, high cloning efficiency and post-thawing passaging. We believe that this protocol could be a generally applicable and robust platform for feeder-free cryopreservation and the expansion of present and future applications of human ES and iPS cells. The treatment with ROCK inhibitor, cell harvesting and the freezing-thawing process usually takes about 2 h excluding overnight incubation at -80 degrees C.
Asunto(s)
Criopreservación/métodos , Células Madre Embrionarias , Células Madre Pluripotentes Inducidas , Amidas , Técnicas de Cultivo de Célula , Proliferación Celular , Supervivencia Celular , Colágeno , Medios de Cultivo , Combinación de Medicamentos , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/enzimología , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/enzimología , Laminina , Inhibidores de Proteínas Quinasas , Proteoglicanos , Piridinas , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
Optimization and development of better defined culture methods for human embryonic and induced pluripotent stem cells (hESCs and hiPSCs) will provide an invaluable contribution to the field of regenerative medicine. However, one problem is the vulnerability of hESCs and hiPSCs to apoptosis that causes a low plating efficiency upon passaging. Herein, we have developed a novel hESCs and hiPSCs culture technique that uses ROCK inhibitor (ROCKi) Y-27632 (10 microM) in Matrigel-coated dishes in both serum- and feeder-free culture conditions. This increases plating efficiency during enzymatic and mechanical passaging as compared to its presence solely in culture medium. Under these conditions, hESCs (three lines) and hiPSCs (two lines) retain their typical morphology, a stable karyotype, express pluripotency markers and have the potential to differentiate into derivatives of all three germ layers after long-term culture. Real-time RT-PCR analysis of stemness-related integrins (alphaV, alpha6, and beta1) has demonstrated that their expression increases in the presence of ROCKi. Similar plating efficiencies have been obtained in both hESCs and hiPSCs with a lower concentration of Y-27632 (800 nM) and another ROCKi (HA-1077/Fasudil), thus ruling out the non-specific effects of Y-27632. These results show that addition of ROCKi in the extracellular matrix can increase the plating efficiency of hESCs and hiPSCs during passaging of clusters. This is due not only to an anti-apoptotic effect, but also to an increase in the ECM-cells interaction. Therefore, we believe this method will be useful for both current and future applications of these pluripotent stem cells.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Colágeno/metabolismo , Células Madre Embrionarias/efectos de los fármacos , Matriz Extracelular/metabolismo , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Laminina/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteoglicanos/metabolismo , Quinasas Asociadas a rho/antagonistas & inhibidores , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Amidas/farmacología , Apoptosis/efectos de los fármacos , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Forma de la Célula/efectos de los fármacos , Portadores de Fármacos , Combinación de Medicamentos , Células Madre Embrionarias/enzimología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Células Madre Pluripotentes Inducidas/enzimología , Integrina alfa6/genética , Integrina alfaV/genética , Integrina beta1/genética , Cariotipificación , Piridinas/farmacología , ARN Mensajero/metabolismo , Quinasas Asociadas a rho/metabolismoRESUMEN
In this study, we focused on the derivation of human embryonic stem cell (hESC) from preimplantation genetic screening (PGS)-analyzed and preimplantation genetic diagnosis (PGD)-analyzed embryos. Out of 62 fresh PGD/PGS-analyzed embryos, 22 embryos reached the blastocyst stage. From 12 outgrowth blastocysts, we derived four hESC lines onto a feeder layer. Surprisingly, karyotype analysis showed that hESC lines derived from aneuploid embryos had diploid female karyotype. One hESC line was found to carry a balanced Robertsonian translocation. All the cell lines showed hESC markers and had the pluripotent ability to differentiate into derivatives of the three embryonic germ layers. The established lines had clonal propagation with 22-31% efficiency in the presence of ROCK inhibitor. These results further indicate that hESC lines can be derived from PGD/PGS-analyzed embryos that are destined to be discarded and can serve as an alternative source for normal euploid lines.