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1.
Neurotoxicol Teratol ; 83: 106943, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33221301

RESUMO

Prenatal ethanol exposure can result in loss of neural stem cells (NSCs) and decreased brain growth. Here, we assessed whether a noncoding RNA (ncRNA) related to the NSC self-renewal factor Oct4/Pou5f1, and transcribed from a processed pseudogene locus on mouse chromosome 9 (mOct4pg9), contributed to the loss of NSCs due to ethanol. Mouse fetal cortical-derived NSCs, cultured ex vivo to mimic the early neurogenic environment of the fetal telencephalon, expressed mOct4pg9 ncRNA at significantly higher levels than the parent Oct4/Pou5f1 mRNA. Ethanol exposure increased expression of mOct4pg9 ncRNA, but decreased expression of Oct4/Pou5f1. Gain- and loss-of-function analyses indicated that mOct4pg9 overexpression generally mimicked effects of ethanol exposure, resulting in increased proliferation and expression of transcripts associated with neural maturation. Moreover, mOct4pg9 associated with Ago2 and with miRNAs, including the anti-proliferative miR-328-3p, whose levels were reduced following mOct4pg9 overexpression. Finally, mOct4pg9 inhibited Oct4/Pou5f1-3'UTR-dependent protein translation. Consistent with these observations, data from single-cell transcriptome analysis showed that mOct4pg9-expressing progenitors lack Oct4/Pou5f1, but instead overexpress transcripts for increased mitosis, suggesting initiation of transit amplification. Collectively, these data suggest that the inhibitory effects of ethanol on brain development are explained, in part, by a novel ncRNA which promotes loss of NSC identity and maturation.


Assuntos
Etanol/toxicidade , Células-Tronco Fetais/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Fator 3 de Transcrição de Octâmero/genética , RNA não Traduzido/genética , Animais , Proteínas Argonautas/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/embriologia , Encéfalo/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Feminino , Transtornos do Espectro Alcoólico Fetal/genética , Transtornos do Espectro Alcoólico Fetal/metabolismo , Transtornos do Espectro Alcoólico Fetal/patologia , Células-Tronco Fetais/metabolismo , Células-Tronco Fetais/patologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Neurológicos , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Neurogênese/efeitos dos fármacos , Neurogênese/genética , Fator 3 de Transcrição de Octâmero/antagonistas & inibidores , Fator 3 de Transcrição de Octâmero/metabolismo , Gravidez , Efeitos Tardios da Exposição Pré-Natal/genética , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Efeitos Tardios da Exposição Pré-Natal/patologia , Pseudogenes , RNA não Traduzido/metabolismo , Análise de Célula Única
2.
Mol Ther ; 28(7): 1645-1657, 2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32353323

RESUMO

Retinal pigment epithelial (RPE) cell replacement therapy has provided promising outcomes in the treatment of retinal degenerative diseases (RDDs), but the resulting limited visual improvement has raised questions about graft survival and differentiation. Through combined treatment with vitamin C and valproic acid (together, VV), we activated human fetal RPE (fRPE) cells to become highly proliferative fetal RPE stem-like cells (fRPESCs). In this study, we report that SOX2 (SRY-box 2) activation contributed to mesenchymal-epithelial transition and elevated the retinal progenitor and mesenchymal stromal markers expressions of fRPESCs. These fRPESCs could differentiate into RPE cells, rod photoreceptors, and mesenchymal lineage progenies under defined conditions. Finally, fRPESCs were transplanted into the subretinal space of an RDD mouse model, and a photoreceptor rescue benefit was demonstrated. The RPE and rod photoreceptor differentiation of transplanted fRPESCs may account for the neural retinal recovery. This study establishes fRPESCs as a highly proliferative, multi-lineage differentiation potential (including RPE, rod photoreceptor, and mesenchymal lineage differentiation), mesenchymal-to-epithelial-transitioned retinal stem-like cell source for cell-based therapy of RDDs.


Assuntos
Ácido Ascórbico/farmacologia , Células-Tronco Fetais/transplante , Degeneração Retiniana/terapia , Epitélio Pigmentado da Retina/embriologia , Fatores de Transcrição SOXB1/metabolismo , Ácido Valproico/farmacologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal , Células-Tronco Fetais/citologia , Células-Tronco Fetais/efeitos dos fármacos , Células-Tronco Fetais/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Degeneração Retiniana/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Resultado do Tratamento , Regulação para Cima
3.
Stem Cell Rev Rep ; 16(3): 524-540, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32020407

RESUMO

Nongonadal tissues express luteinizing hormone-chorionic gonadotropin receptors (LHCG-R) which are essential for their growth during fetal development. Adult mesenchymal stem/stromal cells (MSCs) have been shown to express functional LHCG-R outside pregnancy conditions, making them susceptible to hCG stimulation. In the present study we tested the effect of hCG treatment on bone marrow (BM) derived adherent stem cells in vitro, isolated from a parous women, mother of male sons, in order to evaluate its effect on maternal MSCs and in the same time on fetal microchimeric stem cells (FMSCs), to better understand the outcomes of this safe and affordable treatment on cell proliferation and expression of pluripotency genes. Our study highlights the beneficial effects of hCG exposure on gene regulation in bone marrow adherent stem cells through the upregulation of pluripotency genes and selection of more primitive mesenchymal stem cells with a better differentiation potential. Validation of these effects on MSCs and FMSCs long after parturition in vivo represents a close perspective as it could set the premises of a new mobilization strategy for the stem cell transplantation procedures in the clinical setting.


Assuntos
Células da Medula Óssea/citologia , Quimerismo/efeitos dos fármacos , Gonadotropina Coriônica/farmacologia , Células-Tronco Fetais/citologia , Células-Tronco Fetais/imunologia , Tolerância Imunológica/efeitos dos fármacos , Regeneração/efeitos dos fármacos , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Células da Medula Óssea/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Separação Celular , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Feminino , Células-Tronco Fetais/efeitos dos fármacos , Células-Tronco Fetais/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Osteogênese/efeitos dos fármacos , Osteogênese/genética
4.
Am J Physiol Cell Physiol ; 317(2): C348-C357, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31166709

RESUMO

Maternal endothelial dysfunction is a cental feature of preeclampsia (PE), a hypertensive disorder of pregnancy. Factors in the maternal circulation are thought to contribute to this endothelial dysfunction. Although understudied, factors in the fetal circulation may influence fetal endothelial cell interactions with endothelial progenitor cells as critical steps in placental angiogenesis. We hypothesize that cell-cell interactions that are important for pregnancy health are impaired by fetal serum from PE pregnancies and that 1,25(OH)2-vitamin D3 attenuates the negative effects of this serum on cell function. We tested the ability of fetal cord blood-derived endothelial progenitor cells [endothelial colony-forming cells (ECFCs)] to invade into established monolayers and capillary tubule-like structures of human fetal umbilical venous endothelial cells (HUVECs), while in the presence/absence of fetal cord serum from uncomplicated or PE pregnancies, and tested the ability of 1,25(OH)2-vitamin D3 to modulate the serum-mediated effects. PE cord serum reduced the invasion of fetal ECFCs into HUVEC monolayers or tubule networks. Vitamin D attenuated these effects of PE fetal serum on endothelial functional properties. Immunocytochemical studies revealed involvement of VE-cadherin contacts in interactions between ECFCs and mature fetal endothelial cells. PE cord serum reduces the ability of fetal endothelial progenitor cells to incorporate into fetal endothelial cell networks. Physiologic concentrations of vitamin D reverse these PE serum-mediated effects. These data appear consistent with lines of evidence that vitamin D has antipreeclampsia effects.


Assuntos
Calcitriol/farmacologia , Comunicação Celular/efeitos dos fármacos , Células Progenitoras Endoteliais/efeitos dos fármacos , Células-Tronco Fetais/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Pré-Eclâmpsia/tratamento farmacológico , Adulto , Estudos de Casos e Controles , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Meios de Cultivo Condicionados/metabolismo , Células Progenitoras Endoteliais/metabolismo , Feminino , Células-Tronco Fetais/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Pré-Eclâmpsia/metabolismo , Gravidez , Receptores de Calcitriol/agonistas , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Transdução de Sinais
5.
Alcohol Clin Exp Res ; 43(7): 1414-1426, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31009095

RESUMO

BACKGROUND: Neural stem cells (NSCs) generate most of the neurons of the adult brain in humans, during the mid-first through second-trimester period. This critical neurogenic window is particularly vulnerable to prenatal alcohol exposure, which can result in diminished brain growth. Previous studies showed that ethanol (EtOH) exposure does not kill NSCs, but, rather, results in their depletion by influencing cell cycle kinetics and promoting aberrant maturation, in part, by altering NSC expression of key neurogenic miRNAs. NSCs reside in a complex microenvironment rich in extracellular vesicles, shown to traffic miRNA cargo between cells. METHODS: We profiled the miRNA content of extracellular vesicles from control and EtOH-exposed ex vivo neurosphere cultures of fetal NSCs. We subsequently examined the effects of one EtOH-sensitive miRNA, miR-140-3p, on NSC growth, survival, and maturation. RESULTS: EtOH exposure significantly elevates levels of a subset of miRNAs in secreted extracellular vesicles. Overexpression of one of these elevated miRNAs, miR-140-3p, and its passenger strand relative, miR-140-5p, significantly increased the proportion of S-phase cells while decreasing the proportion of G0 /G1 cells compared to controls. In contrast, while miR-140-3p knockdown had minimal effects on the proportion of cells in each phase of the cell cycle, knockdown of miR-140-5p significantly decreased the proportion of cells in G2 /M phase. Furthermore, miR-140-3p overexpression, during mitogen-withdrawal-induced NSC differentiation, favors astroglial maturation at the expense of neural and oligodendrocyte differentiation. CONCLUSIONS: Collectively, the dysregulated miRNA content of extracellular vesicles following EtOH exposure may result in aberrant neural progenitor cell growth and maturation, explaining brain growth deficits associated with prenatal alcohol exposure.


Assuntos
Proliferação de Células/efeitos dos fármacos , Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Vesículas Extracelulares/metabolismo , Células-Tronco Fetais/efeitos dos fármacos , MicroRNAs/biossíntese , Células-Tronco Neurais/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Vesículas Extracelulares/efeitos dos fármacos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/efeitos dos fármacos , Mitose/efeitos dos fármacos , Gravidez
6.
Methods Mol Biol ; 1905: 3-8, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30536085

RESUMO

Mouse fetal liver includes abundant hepatic stem/progenitor cells (HSPCs). Easy expansion with passage of HSPCs is necessary to obtain steady data. However, it is often difficult to enrich only HSPCs, and HSPCs can die when usual trypsin is used for replating. Here, we introduce serum-free long-term culture with passage of HSPCs using fetal mouse liver without a cell sorter.


Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Fetais/citologia , Fígado/embriologia , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Células Cultivadas , Meios de Cultura Livres de Soro/farmacologia , Feminino , Células-Tronco Fetais/efeitos dos fármacos , Citometria de Fluxo , Fígado/citologia , Camundongos , Gravidez
7.
BMC Res Notes ; 11(1): 343, 2018 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-29843819

RESUMO

OBJECTIVE: Human amniotic epithelial cells (hAECs) are a novel source of stem cells and have immunomodulatory effects on both the innate and adoptive immune system. hAECs can differentiate into multiple cell lineages that make them a suitable cell source for regenerative medicine. These cells express multiple toll-like receptors (TLRs) and respond to various TLR ligands. This study aimed to evaluate the effect of lipopolysaccharide (LPS), a TLR4 ligand, on the level of immunomodulatory and immunostimulatory factors of hAECs. RESULTS: Our results indicated that LPS had the ability to up-regulate the expression of prostaglandin E2 synthase and transforming growth factor-beta1 in hAECs. However, there was no change in the level of interleukin-1beta, interleukin-6 and interleukin-10 in hAECs when were stimulated with LPS. In addition, we observed tumor necrosis factor-alpha was only expressed at very low level in some of hAECs samples which its expression was independent of the effects of LPS.


Assuntos
Âmnio/citologia , Citocinas/efeitos dos fármacos , Células Epiteliais , Células-Tronco Fetais , Fatores Imunológicos/farmacologia , Lipopolissacarídeos/farmacologia , Prostaglandina-E Sintases/efeitos dos fármacos , Adulto , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Feminino , Células-Tronco Fetais/efeitos dos fármacos , Células-Tronco Fetais/imunologia , Células-Tronco Fetais/metabolismo , Humanos , Gravidez
8.
J Cell Physiol ; 233(6): 4841-4851, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29150960

RESUMO

Epithelial Cell Adhesion Molecule (EpCAM), or CD326, is a trans-membrane glycoprotein expressed by multiple normal epithelia as well as carcinoma. Human hepatic stem cells and bile duct epithelium of the liver are EpCAM positive. In tumor cell lines, its intracellular domain can be released after cleavage of the extracellular domain. Within the cell nucleus, it induces cell proliferation, but cleavage depends on cell contact. Fragments of various lengths have been described in tumor cells. Despite its described important role in proliferation in tumor cells, there is not much known about the expression and role of EpCAM fragments in primary human liver cells. Here, we demonstrate that EpCAM protein fragments and function are considerable different between tumor cells, normal fetal and adult liver cells. Contrary to previously reported findings in tumor cells, gene knockdown or treatment with an inhibitor of the cleavage enzyme ADAM17 (TACE) rather increased cell numbers in primary human fetal liver-derived EpCAM-positive cells. EpCAM fragment sizes were not affected by treatment with inhibitor. Knockdown of EPCAM gene expression by siRNA in sorted cells did not significantly affect proliferation-associated genes or cell numbers. The intracellular domain could not be detected within cell nuclei of fetal and adult liver cells. In conclusion, signaling through the intracellular domain of EpCAM appears to be a mechanism that induces proliferation specifically in tumorigenic cells but not in normal primary EpCAM-positive liver cells.


Assuntos
Células-Tronco Adultas/metabolismo , Proliferação de Células , Neoplasias Colorretais/metabolismo , Molécula de Adesão da Célula Epitelial/metabolismo , Células-Tronco Fetais/metabolismo , Fígado/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fragmentos de Peptídeos/metabolismo , Transdução de Sinais , Proteína ADAM17/genética , Proteína ADAM17/metabolismo , Células-Tronco Adultas/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Molécula de Adesão da Célula Epitelial/genética , Células-Tronco Fetais/efeitos dos fármacos , Regulação da Expressão Gênica , Glicosilação , Células HT29 , Humanos , Ácidos Hidroxâmicos/farmacologia , Fígado/citologia , Fígado/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Cultura Primária de Células , Domínios Proteicos , Transdução de Sinais/efeitos dos fármacos
9.
Stem Cell Res ; 22: 54-60, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28600955

RESUMO

Poly (ADP-ribose) polymerase (PARP) family members are ubiquitously expressed and play a key role in cellular processes, including DNA repair and cell death/survival balance. Accordingly, PARP inhibition is an emerging pharmacological strategy for cancer and neurodegenerative diseases. Consistent evidences support the critical involvement of PARP family members in cell differentiation and phenotype maturation. In this study we used an oligodendrocyte precursor cells (OPCs) enriched system derived from fetal and adult brain to investigate the role of PARP in OPCs proliferation, survival, and differentiation. The PARP inhibitors PJ34, TIQ-A and Olaparib were used as pharmacological tools. The main results of the study are: (i) PARP mRNA expression and PARP activity are much higher in fetal than in adult-derived OPCs; (ii) the culture treatment with PARP inhibitors is cytotoxic for OPCs derived from fetal, but not from adult, brain; (iii) PARP inhibition reduces cell number, according to the inhibitory potency of the compounds; (iv) PARP inhibition effect on fetal OPCs is a slow process; (v) PARP inhibition impairs OPCs maturation into myelinating OL in fetal, but not in adult cultures, according to the inhibitory potency of the compounds. These results have implications for PARP-inhibition therapies for diseases and lesions of the central nervous system, in particular for neonatal hypoxic/ischemic encephalopathy.


Assuntos
Células-Tronco Fetais/citologia , Células-Tronco Fetais/enzimologia , Células Precursoras de Oligodendrócitos/citologia , Células Precursoras de Oligodendrócitos/enzimologia , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células-Tronco Fetais/efeitos dos fármacos , Camundongos , Células Precursoras de Oligodendrócitos/efeitos dos fármacos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Transdução de Sinais/efeitos dos fármacos
10.
Alcohol ; 60: 149-158, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28438527

RESUMO

Fetal alcohol spectrum disorders are a leading cause of intellectual disability worldwide. Previous studies have shown that developmental ethanol exposure results in loss of microRNAs (miRNAs), including miR-9, and loss of these miRNAs, in turn, mediates some of ethanol's teratogenic effects in the developing brain. We previously found that ethanol increased methylation at the miR-9-2 encoding gene locus in mouse fetal neural stem cells (NSC), advancing a mechanism for epigenetic silencing of this locus and consequently, miR-9 loss in NSCs. Therefore, we assessed the role of the BAF (BRG1/BRM-Associated Factor) complex, which disassembles nucleosomes to facilitate access to chromatin, as an epigenetic mediator of ethanol's effects on miR-9. Chromatin immunoprecipitation and DNAse I-hypersensitivity analyses showed that the BAF complex was associated with both transcriptionally accessible and heterochromatic regions of the miR-9-2 locus, and that disintegration of the BAF complex by combined knockdown of BAF170 and BAF155 resulted in a significant decrease in miR-9. We hypothesized that ethanol exposure would result in loss of BAF-complex function at the miR-9-2 locus. However, ethanol exposure significantly increased mRNA transcripts for maturation-associated BAF-complex members BAF170, SS18, ARID2, BAF60a, BRM/BAF190b, and BAF53b. Ethanol also significantly increased BAF-complex binding within an intron containing a CpG island and in the terminal exon encoding precursor (pre)-miR-9-2. These data suggest that the BAF complex may adaptively respond to ethanol exposure to protect against a complete loss of miR-9-2 in fetal NSCs. Chromatin remodeling factors may adapt to the presence of a teratogen, to maintain transcription of critical miRNA regulatory pathways.


Assuntos
Montagem e Desmontagem da Cromatina/efeitos dos fármacos , DNA Helicases/metabolismo , Etanol/toxicidade , Células-Tronco Fetais/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , MicroRNAs/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica/efeitos dos fármacos , Animais , Células Cultivadas , DNA Helicases/genética , Transtornos do Espectro Alcoólico Fetal/genética , Transtornos do Espectro Alcoólico Fetal/metabolismo , Transtornos do Espectro Alcoólico Fetal/patologia , Células-Tronco Fetais/metabolismo , Células-Tronco Fetais/patologia , Camundongos , MicroRNAs/genética , Complexos Multiproteicos , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Neurogênese/genética , Proteínas Nucleares/genética , Interferência de RNA , Fatores de Tempo , Fatores de Transcrição/metabolismo , Transfecção
11.
J Cell Physiol ; 232(8): 2186-2200, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27966782

RESUMO

This study was conducted to know the impact of cryopreservation on caprine fetal adnexa derived mesenchymal stem cells (MSCs) on the basic stem cell characteristics. Gravid caprine uteri (2-3 months) were collected from local abattoir to derive (amniotic fluid [cAF], amniotic sac [cAS], Wharton's jelly [cWJ], and cord blood [cCB]) MSCs and expanded in vitro. Cells were cryopreserved at 3rd passage (P3) using 10% DMSO. Post-thaw viability and cellular properties were assessed. Cells were expanded to determine growth kinetics, tri-lineage differentiation, localization, and molecular expression of MSCs and pluripotency markers; thereafter, these cells were transplanted in the full-thickness (2 × 2cm2 ) rat skin wound to determine their wound healing potential. The post-thaw (pt) growth kinetics study suggested that cWJ MSCs expanded more rapidly with faster population doubling time (PDT) than that of other fetal adnexa MSCs. The relative mRNA expression of surface antigens (CD73, CD90, and CD 105) and pluripotency markers (Oct4, KLF, and cMyc) was higher in cWJ MSCs in comparison to cAS, cAF, and cCB MSCs post-thaw. The percent wound contraction on 7th day was more than 50% for all the MSC-treated groups (pre and post-thaw), against 39.55% in the control group. On day 28th, 99% and more wound contraction was observed in cAF, cAF-pt, cAS-pt, cWJ, cWJ-pt, and cCB, MSCs with better scores for epithelization, neovascularization, and collagen characteristics at a non-significant level. It is concluded that these MSCs could be successfully cryopreserved without altering their stemness and wound healing properties. J. Cell. Physiol. 232: 2186-2200, 2017. © 2016 Wiley Periodicals, Inc.


Assuntos
Proliferação de Células/efeitos dos fármacos , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Criopreservação , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Células-Tronco Fetais/efeitos dos fármacos , Células-Tronco Fetais/transplante , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/efeitos dos fármacos , Ferida Cirúrgica/cirurgia , Cicatrização , Líquido Amniótico/citologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem da Célula , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Feminino , Sangue Fetal/citologia , Células-Tronco Fetais/metabolismo , Cabras , Xenoenxertos , Cinética , Masculino , Células-Tronco Mesenquimais/metabolismo , Fenótipo , Gravidez , Ratos Wistar , Ferida Cirúrgica/metabolismo , Ferida Cirúrgica/patologia , Geleia de Wharton/citologia
12.
J Neurosci ; 35(8): 3676-88, 2015 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-25716865

RESUMO

Therapeutic repair of myelin disorders may be limited by the relatively slow rate of human oligodendrocyte differentiation. To identify appropriate pharmacological targets with which to accelerate differentiation of human oligodendrocyte progenitors (hOPCs) directly, we used CD140a/O4-based FACS of human forebrain and microarray to hOPC-specific receptors. Among these, we identified CHRM3, a M3R muscarinic acetylcholine receptor, as being restricted to oligodendrocyte-biased CD140a(+)O4(+) cells. Muscarinic agonist treatment of hOPCs resulted in a specific and dose-dependent blockade of oligodendrocyte commitment. Conversely, when hOPCs were cocultured with human neurons, M3R antagonist treatment stimulated oligodendrocytic differentiation. Systemic treatment with solifenacin, an FDA-approved muscarinic receptor antagonist, increased oligodendrocyte differentiation of transplanted hOPCs in hypomyelinated shiverer/rag2 brain. Importantly, solifenacin treatment of engrafted animals reduced auditory brainstem response interpeak latency, indicative of increased conduction velocity and thereby enhanced functional repair. Therefore, solifenacin and other selective muscarinic antagonists represent new adjunct approaches to accelerate repair by engrafted human progenitors.


Assuntos
Células-Tronco Fetais/citologia , Antagonistas Muscarínicos/farmacologia , Bainha de Mielina/metabolismo , Oligodendroglia/citologia , Quinuclidinas/farmacologia , Regeneração , Tetra-Hidroisoquinolinas/farmacologia , Animais , Tronco Encefálico/citologia , Tronco Encefálico/fisiologia , Células Cultivadas , Proteínas de Ligação a DNA/genética , Potenciais Evocados Auditivos do Tronco Encefálico , Feminino , Células-Tronco Fetais/efeitos dos fármacos , Células-Tronco Fetais/metabolismo , Células-Tronco Fetais/transplante , Humanos , Masculino , Camundongos , Agonistas Muscarínicos/farmacologia , Bainha de Mielina/genética , Neurogênese , Antígenos O/genética , Antígenos O/metabolismo , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , Oligodendroglia/transplante , Prosencéfalo/citologia , Prosencéfalo/embriologia , Receptor Muscarínico M3 , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores Muscarínicos/genética , Receptores Muscarínicos/metabolismo , Succinato de Solifenacina
13.
J Hepatol ; 62(5): 1085-91, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25529619

RESUMO

BACKGROUND & AIMS: During pregnancy, acetaminophen is one of the very few medications recommended by physicians to treat fever or pain. Recent insights from epidemiological studies suggest an association between prenatal acetaminophen medication and an increased risk for development of asthma in children later in life. The underlying pathogenesis of such association is still unknown. METHODS: We aimed to develop a mouse model to provide insights into the effect of prenatal acetaminophen on maternal, fetal and adult offspring's health. The toxic effect of acetaminophen was studied in mice on 1) maternal liver; mirrored by biomarkers of liver injury, centrilobular necrosis, and infiltration of granulocytes; 2) fetal liver; reflected by the frequency of hematopoietic stem cells, and 3) postnatal health; evaluated by the severity of allergic airway inflammation among offspring. RESULTS: We observed an increased susceptibility towards acetaminophen-induced liver damage in pregnant mice compared to virgins. Moreover, hematopoietic stem cell frequency in fetal liver declined in response to acetaminophen. Furthermore, a greater severity of airway inflammation was observed in offspring of dams upon prenatal acetaminophen treatment, identified lung infiltration by leukocytes and eosinophil infiltration into the airways. CONCLUSION: Our newly developed mouse model on prenatal use of acetaminophen reflects findings from epidemiological studies in humans. The availability of this model will allow improvement in our understanding of how acetaminophen-related hepatotoxicity is operational in pregnant individuals and how an increased risk for allergic diseases in response to prenatal acetaminophen is mediated. Such insights, once available, may change the recommendations for prenatal acetaminophen use.


Assuntos
Acetaminofen , Asma , Doença Hepática Induzida por Substâncias e Drogas , Células-Tronco Fetais , Efeitos Tardios da Exposição Pré-Natal , Acetaminofen/administração & dosagem , Acetaminofen/efeitos adversos , Adulto , Filhos Adultos , Analgésicos não Narcóticos/administração & dosagem , Analgésicos não Narcóticos/efeitos adversos , Animais , Asma/etiologia , Asma/fisiopatologia , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Doença Hepática Induzida por Substâncias e Drogas/fisiopatologia , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Modelos Animais de Doenças , Feminino , Células-Tronco Fetais/efeitos dos fármacos , Células-Tronco Fetais/patologia , Humanos , Inflamação/etiologia , Inflamação/fisiopatologia , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Camundongos , Gravidez , Efeitos Tardios da Exposição Pré-Natal/etiologia , Efeitos Tardios da Exposição Pré-Natal/patologia , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Efeitos Tardios da Exposição Pré-Natal/prevenção & controle , Índice de Gravidade de Doença
14.
Proc Natl Acad Sci U S A ; 111(18): E1924-32, 2014 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-24753613

RESUMO

Fetal growth plays a role in programming of adult cardiometabolic disorders, which in men, are associated with lowered testosterone levels. Fetal growth and fetal androgen exposure can also predetermine testosterone levels in men, although how is unknown, because the adult Leydig cells (ALCs) that produce testosterone do not differentiate until puberty. To explain this conundrum, we hypothesized that stem cells for ALCs must be present in the fetal testis and might be susceptible to programming by fetal androgen exposure during masculinization. To address this hypothesis, we used ALC ablation/regeneration to identify that, in rats, ALCs derive from stem/progenitor cells that express chicken ovalbumin upstream promoter transcription factor II. These stem cells are abundant in the fetal testis of humans and rodents, and lineage tracing in mice shows that they develop into ALCs. The stem cells also express androgen receptors (ARs). Reduction in fetal androgen action through AR KO in mice or dibutyl phthalate (DBP) -induced reduction in intratesticular testosterone in rats reduced ALC stem cell number by ∼40% at birth to adulthood and induced compensated ALC failure (low/normal testosterone and elevated luteinizing hormone). In DBP-exposed males, this failure was probably explained by reduced testicular steroidogenic acute regulatory protein expression, which is associated with increased histone methylation (H3K27me3) in the proximal promoter. Accordingly, ALCs and ALC stem cells immunoexpressed increased H3K27me3, a change that was also evident in ALC stem cells in fetal testes. These studies highlight how a key component of male reproductive development can fundamentally reprogram adult hormone production (through an epigenetic change), which might affect lifetime disease risk.


Assuntos
Células-Tronco Adultas/fisiologia , Androgênios/fisiologia , Desenvolvimento Fetal/fisiologia , Células Intersticiais do Testículo/fisiologia , Células-Tronco Adultas/efeitos dos fármacos , Animais , Callithrix , Linhagem da Célula/fisiologia , Dibutilftalato/toxicidade , Feminino , Desenvolvimento Fetal/efeitos dos fármacos , Células-Tronco Fetais/efeitos dos fármacos , Células-Tronco Fetais/fisiologia , Humanos , Técnicas In Vitro , Células Intersticiais do Testículo/efeitos dos fármacos , Hormônio Luteinizante/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Modelos Animais , Gravidez , Ratos , Ratos Transgênicos , Ratos Wistar , Receptores Androgênicos/deficiência , Receptores Androgênicos/genética , Receptores Androgênicos/fisiologia , Regeneração , Testículo/embriologia , Testículo/fisiologia , Testosterona/deficiência , Testosterona/fisiologia
15.
Clin Immunol ; 152(1-2): 68-76, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24607604

RESUMO

RATIONALE: Cord blood eosinophil/basophil progenitor cells (Eo/B) of high risk infants have been shown to predict respiratory illnesses in infancy. Here we investigated this association in a population-based cohort. Furthermore, we analysed whether newborns Th1/Th2 balance and prenatal environmental exposure impact Eo/B recruitment. METHODS: In a sub-cohort of the LINA study cord blood mononuclear cells were used for methylcellulose assays to assess Eo/B differentiation. Questionnaires were recorded during pregnancy and annually thereafter. Volatile organic compounds were measured during pregnancy and cord blood cytokines after ex vivo stimulation. RESULTS: Cord blood IL-4 and IL-13 positively correlated with Eo/B. Tobacco smoke related benzene was also positively associated with Eo/B. Enhanced Eo/B numbers increased the risk for wheezing within the first 24 months. CONCLUSIONS: The association between cord blood Eo/B and respiratory illnesses is not restricted to high-risk children. Prenatal environmental exposure and a Th2 milieu at birth contribute to Eo/B recruitment.


Assuntos
Basófilos/imunologia , Eosinófilos/imunologia , Células-Tronco Fetais/imunologia , Infecções Respiratórias/imunologia , Compostos Orgânicos Voláteis/efeitos adversos , Basófilos/citologia , Basófilos/efeitos dos fármacos , Diferenciação Celular , Estudos de Coortes , Exposição Ambiental , Eosinófilos/citologia , Eosinófilos/efeitos dos fármacos , Feminino , Sangue Fetal/citologia , Células-Tronco Fetais/citologia , Células-Tronco Fetais/efeitos dos fármacos , Humanos , Lactente , Recém-Nascido , Masculino , Gravidez , Efeitos Tardios da Exposição Pré-Natal/genética , Efeitos Tardios da Exposição Pré-Natal/imunologia , Infecções Respiratórias/genética , Inquéritos e Questionários , Equilíbrio Th1-Th2
16.
Arch Toxicol ; 88(8): 1537-48, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24599297

RESUMO

Polybrominated diphenyl ethers (PBDEs) are bioaccumulating flame retardants found in rising concentrations in human tissue. Epidemiological and animal studies have raised concern for their potential to induce developmental neurotoxicity (DNT). Considering the essential role of calcium homeostasis in neurodevelopment, PBDE-induced disturbance of intracellular calcium concentration ([Ca(2+)]i) may underlie PBDE-induced DNT. To test this hypothesis, we investigated acute effects of BDE-47 and 6-OH-BDE-47 on [Ca(2+)]i in human neural progenitor cells (hNPCs) and unraveled involved signaling pathways. Short-time differentiated hNPCs were exposed to BDE-47, 6-OH-BDE-47, and multiple inhibitors/stimulators of presumably involved signaling pathways to determine possible effects on [Ca(2+)]i by single-cell microscopy with the fluorescent dye Fura-2. Initial characterization of calcium signaling pathways confirmed the early developmental stage of hNPCs. In these cells, BDE-47 (2 µM) and 6-OH-BDE-47 (0.2 µM) induce [Ca(2+)]i transients. This increase in [Ca(2+)]i is due to extracellular Ca(2+) influx and intracellular release of Ca(2+), mainly from the endoplasmic reticulum (ER). While extracellular Ca(2+) seems to enter the cytoplasm upon 6-OH-BDE-47 by interfering with the cell membrane and independent of Ca(2+) ion channels, ER-derived Ca(2+) is released following activation of protein lipase C and inositol 1,4,5-trisphosphate receptor, but independently of ryanodine receptors. These findings illustrate that immature developing hNPCs respond to low concentrations of 6-OH-BDE-47 by an increase in [Ca(2+)]i and provide new mechanistic explanations for such BDE-induced calcium disruption. Thus, these data support the possibility of a critical window of PBDE exposure, i.e., early human brain development, which has to be acknowledged in risk assessment.


Assuntos
Cálcio/metabolismo , Células-Tronco Fetais/efeitos dos fármacos , Éteres Difenil Halogenados/toxicidade , Homeostase/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Bifenil Polibromatos/toxicidade , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Células Cultivadas , Células-Tronco Fetais/metabolismo , Idade Gestacional , Homeostase/fisiologia , Humanos , Potenciais da Membrana/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neurogênese/efeitos dos fármacos , Neurogênese/fisiologia , Técnicas de Patch-Clamp , Cultura Primária de Células
17.
Cryobiology ; 68(2): 244-50, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24530510

RESUMO

A previous study demonstrated that disaccharides, antioxidants, and caspase inhibitors can be used in freezing solutions to reduce the concentration of Me2SO from the current standard of 10% (v/v) to 5% (v/v) or 2.5% and to eliminate fetal bovine serum (FBS) for the cryopreservation of human amniotic fluid-derived stem cells (AFSCs). Hence, this study investigated whether an irreversible inhibitor of caspase enzymes, benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone (zVAD-fmk), could be used in post-thaw culture media to increase the survival rate of AFSCs. Our results showed that AFSCs cryopreserved in freezing solution containing trehalose, catalase, and 5% (v/v) Me2SO and then supplemented with zVAD-fmk in the post-thaw culture media showed similar post-thawing viability, proliferation, and apoptosis than cells cryopreserved in the control solution (10% (v/v) Me2SO and 20% FBS). The caspase-3 activity in all the cryopreservation solutions tested was similar to that of the control. Caspase-3, caspase-8, caspase-9, and PARP expression was not found in the cryopreserved cells. In addition, no difference was found in the survival rate and apoptosis between short-term (3weeks) and long-term (1year) storage of AFSCs cryopreserved in the solutions used in this study. The results of the present study demonstrate that recovery of cryopreserved cells was enhanced by using a caspase inhibitor in the post-thaw culture media.


Assuntos
Clorometilcetonas de Aminoácidos/farmacologia , Apoptose/efeitos dos fármacos , Crioprotetores/farmacologia , Células-Tronco Fetais/efeitos dos fármacos , Líquido Amniótico/citologia , Western Blotting , Inibidores de Caspase/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Criopreservação/métodos , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
18.
J Neuroimmune Pharmacol ; 9(3): 340-53, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24469921

RESUMO

Neurological complications in opioid abusing Human Immunodeficiency Virus-1 (HIV-1) patients suggest enhanced neurodegeneration as compared to non-drug abusing HIV-1 infected population. Neural precursor cells (NPCs), the multipotent cells of the mammalian brain, are susceptible to HIV-1 infection and as opiates also perturb their growth kinetics, detailed mechanistic studies for their co-morbid exposure are highly warranted. Using a well characterized in vitro model of human fetal brain-derived neural precursor cells, we investigated alterations in NPC properties at both acute and chronic durations. Chronic morphine and Tat treatment attenuated proliferation in NPCs, with cells stalled at G1-phase of the cell cycle. Furthermore HIV-Tat and morphine exposure increased activation of extracellular signal-regulated kinase-1/2 (ERK1/2), enhanced levels of p53 and p21, and decreased cyclin D1 and Akt levels in NPCs. Regulated by ERK1/2 and p53, p21 was found to be indispensible for Tat and morphine mediated cell cycle arrest. Our study elaborates on the cellular and molecular machinery in NPCs and provides significant mechanistic details into HIV-drug abuse co-morbidity that may have far reaching clinical consequences both in pediatric as well as adult neuroAIDS.


Assuntos
Pontos de Checagem do Ciclo Celular/fisiologia , Infecções por HIV , Sistema de Sinalização das MAP Quinases/fisiologia , Células-Tronco Neurais/fisiologia , Transtornos Relacionados ao Uso de Substâncias , Proteína Supressora de Tumor p53/biossíntese , Proteínas rho de Ligação ao GTP/biossíntese , Proteínas Reguladoras de Apoptose/toxicidade , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Comorbidade , Células-Tronco Fetais/efeitos dos fármacos , Células-Tronco Fetais/fisiologia , Infecções por HIV/epidemiologia , Infecções por HIV/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Morfina/toxicidade , Células-Tronco Neurais/efeitos dos fármacos , Proteínas Recombinantes de Fusão/toxicidade , Transtornos Relacionados ao Uso de Substâncias/epidemiologia , Transtornos Relacionados ao Uso de Substâncias/metabolismo , Survivina , Produtos do Gene tat do Vírus da Imunodeficiência Humana/toxicidade
19.
FASEB J ; 27(12): 4853-65, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23995291

RESUMO

Functional smooth muscle engineering requires isolation and expansion of smooth muscle cells (SMCs), and this process is particularly challenging for visceral smooth muscle tissue where progenitor cells have not been clearly identified. Herein we showed for the first time that efficient SMCs can be obtained from human amniotic fluid stem cells (hAFSCs). Clonal lines were generated from c-kit(+) hAFSCs. Differentiation toward SM lineage (SMhAFSCs) was obtained using a medium conditioned by PDGF-BB and TGF-ß1. Molecular assays revealed higher level of α smooth muscle actin (α-SMA), desmin, calponin, and smoothelin in SMhAFSCs when compared to hAFSCs. Ultrastructural analysis demonstrated that SMhAFSCs also presented in the cytoplasm increased intermediate filaments, dense bodies, and glycogen deposits like SMCs. SMhAFSC metabolism evaluated via mass spectrometry showed higher glucose oxidation and an enhanced response to mitogenic stimuli in comparison to hAFSCs. Patch clamp of transduced hAFSCs with lentiviral vectors encoding ZsGreen under the control of the α-SMA promoter was performed demonstrating that SMhAFSCs retained a smooth muscle cell-like electrophysiological fingerprint. Eventually SMhAFSCs contractility was evident both at single cell level and on a collagen gel. In conclusion, we showed here that hAFSCs under selective culture conditions are able to give rise to functional SMCs.


Assuntos
Líquido Amniótico/citologia , Diferenciação Celular , Linhagem da Célula , Células-Tronco Fetais/citologia , Células-Tronco Multipotentes/citologia , Miócitos de Músculo Liso/citologia , Actinas/genética , Actinas/metabolismo , Potenciais de Ação , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Meios de Cultivo Condicionados/farmacologia , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Desmina/genética , Desmina/metabolismo , Células-Tronco Fetais/efeitos dos fármacos , Células-Tronco Fetais/metabolismo , Células-Tronco Fetais/fisiologia , Glucose/metabolismo , Glicogênio/metabolismo , Humanos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Células-Tronco Multipotentes/efeitos dos fármacos , Células-Tronco Multipotentes/metabolismo , Células-Tronco Multipotentes/fisiologia , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/fisiologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Calponinas
20.
Alcohol Clin Exp Res ; 37(10): 1657-67, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23800254

RESUMO

BACKGROUND: Fetal alcohol exposure produces multiorgan defects, making it difficult to identify underlying etiological mechanisms. However, recent evidence for ethanol (EtOH) sensitivity of the miRNA miR-9 suggests one mechanism, whereby EtOH broadly influences development. We hypothesized that loss of miR-9 function recapitulates aspects of EtOH teratology. METHODS: Zebrafish embryos were exposed to EtOH during gastrulation, or injected with anti-miR-9 or nonsense control morpholinos during the 2-cell stage of development and collected between 24 and 72 hours postfertilization (hpf). We also assessed the expression of developmentally important, and known miR-9 targets, FGFR-1, FOXP2, and the nontargeted transcript, MECP2. Methylation at CpG islands of mammalian miR-9 genes was assessed in fetal murine neural stem cells (mNSCs) by methylation-specific PCR, and miRNA processing assessed by qRT-PCR for pre-miR-9 transcripts. RESULTS: EtOH treatment and miR-9 knockdown resulted in similar cranial defects including microcephaly. Additionally, EtOH transiently suppressed miR-9, as well as FGFR-1 and FOXP2, and alterations in miR-9 expression were correlated with severity of EtOH-induced teratology. In mNSCs, EtOH increased CpG dinucleotide methylation at the miR-9-2 locus and accumulation of pre-miR-9-3. CONCLUSIONS: EtOH exerts regulatory control at multiple levels of miR-9 biogenesis. Moreover, early embryonic loss of miR-9 function recapitulated the severe range of teratology associated with developmental EtOH exposure. EtOH also disrupts the relationship between miR-9 and target gene expression, suggesting a nuanced relationship between EtOH and miRNA regulatory networks in the developing embryo. The implications of these data for the expression and function of mature miR-9 warrant further investigation.


Assuntos
Epigênese Genética/fisiologia , Etanol/toxicidade , Células-Tronco Fetais/fisiologia , MicroRNAs/fisiologia , Células-Tronco Neurais/fisiologia , Teratogênese/fisiologia , Animais , Epigênese Genética/efeitos dos fármacos , Células-Tronco Fetais/efeitos dos fármacos , Técnicas de Silenciamento de Genes/métodos , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/efeitos dos fármacos , Distribuição Aleatória , Teratogênese/efeitos dos fármacos , Peixe-Zebra
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