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1.
Stem Cell Res ; 56: 102548, 2021 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-34592601

RESUMO

In this study we isolated and enriched erythroid progenitor cells (EPCs) from a 10 ml peripheral blood sample from a 37-year old healthy Saudi donor. After expansion, these EPCs were reprogrammed using episomal plasmids to generate an induced pluripotent stem (iPS) cell line, KAIMRCi001-A. The pluripotency of this line was confirmed by measuring the expression of typical pluripotency markers and assessing differentiation potential in vitro.

3.
Curr Genomics ; 20(6): 438-452, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32194342

RESUMO

Numerous human disorders of the blood system would directly or indirectly benefit from therapeutic approaches that reconstitute the hematopoietic system. Hematopoietic stem cells (HSCs), either from matched donors or ex vivo manipulated autologous tissues, are the most used cellular source of cell therapy for a wide range of disorders. Due to the scarcity of matched donors and the difficulty of ex vivo expansion of HSCs, there is a growing interest in harnessing the potential of pluripotent stem cells (PSCs) as a de novo source of HSCs. PSCs make an ideal source of cells for regenerative medicine in general and for treating blood disorders in particular because they could expand indefinitely in culture and differentiate to any cell type in the body. However, advancement in deriving functional HSCs from PSCs has been slow. This is partly due to an incomplete understanding of the molecular mechanisms underlying normal hematopoiesis. In this review, we discuss the latest efforts to generate human PSC (hPSC)-derived HSCs capable of long-term engraftment. We review the regulation of the key transcription factors (TFs) in hematopoiesis and hematopoietic differentiation, the Homeobox (HOX) and GATA genes, and the interplay between them and microRNAs. We also propose that precise control of these master regulators during the course of hematopoietic differentiation is key to achieving functional hPSC-derived HSCs.

4.
PLoS One ; 13(5): e0196817, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29734353

RESUMO

Recent studies have suggested a role for the Cyclin Dependent Kinase-2 Associated Protein 1 (CDK2AP1) in stem cell differentiation and self-renewal. In studies with mouse embryonic stem cells (mESCs) derived from generated mice embryos with targeted deletion of the Cdk2ap1 gene, CDK2AP1 was shown to be required for epigenetic silencing of Oct4 during differentiation, with deletion resulting in persistent self-renewal and reduced differentiation potential. Differentiation capacity was restored in these cells following the introduction of a non-phosphorylatible form of the retinoblastoma protein (pRb) or exogenous Cdk2ap1. In this study, we investigated the role of CDK2AP1 in human embryonic stem cells (hESCs). Using a shRNA to reduce its expression in hESCs, we found that CDK2AP1 knockdown resulted in a significant reduction in the expression of the pluripotency genes, OCT4 and NANOG. We also found that CDK2AP1 knockdown increased the number of embryoid bodies (EBs) formed when differentiation was induced. In addition, the generated EBs had significantly higher expression of markers of all three germ layers, indicating that CDK2AP1 knockdown enhanced differentiation. CDK2AP1 knockdown also resulted in reduced proliferation and reduced the percentage of cells in the S phase and increased cells in the G2/M phase of the cell cycle. Further investigation revealed that a higher level of p53 protein was present in the CDK2AP1 knockdown hESCs. In hESCs in which p53 and CDK2AP1 were simultaneously downregulated, OCT4 and NANOG expression was not affected and percentage of cells in the S phase of the cell cycle was not reduced. Taken together, our results indicate that the knockdown of CDK2AP1 in hESCs results in increased p53 and enhances differentiation and favors it over a self-renewal fate.


Assuntos
Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias Humanas/fisiologia , Proteínas Supressoras de Tumor/genética , Animais , Ciclo Celular/genética , Proliferação de Células/genética , Corpos Embrioides/fisiologia , Técnicas de Silenciamento de Genes/métodos , Camadas Germinativas/fisiologia , Humanos , Camundongos , Proteína Homeobox Nanog/genética , Fator 3 de Transcrição de Octâmero/genética , RNA Interferente Pequeno/genética , Proteína do Retinoblastoma/genética , Proteína Supressora de Tumor p53/genética
5.
Sci Rep ; 7: 45577, 2017 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-28361959

RESUMO

Induced pluripotent stem (iPS) cells hold great potential for being a major source of cells for regenerative medicine. One major issue that hinders their advancement to clinic is the persistence of undifferentiated iPS cells in iPS-derived tissue. In this report, we show that the CDKs inhibitor, Dinaciclib, selectively eliminates iPS cells without affecting the viability of cardiac cells. We found that low nanomolar concentration of dinaciclib increased DNA damage and p53 protein levels in iPSCs. This was accompanied by negative regulation of the anti-apoptotic protein MCL-1. Gene knockdown experiments revealed that p53 downregulation only increased the threshold of dinaciclib induced apoptosis in iPS cells. Dinaciclib also inhibited the phosphorylation of Serine 2 of the C-terminal domain of RNA Polyemrase II through CDK9 inhibition. This resulted in the inhibition of transcription of MCL-1 and the pluripotency genes, NANOG and c-MYC. Even though dinaciclib caused a slight downregulation of MCL-1 in iPS-derived cardiac cells, the viability of the cells was not significantly affected, and beating iPS-derived cardiac cell sheet could still be fabricated. These findings suggest a difference in tolerance of MCL-1 downregulation between iPSCs and iPS-derived cardiac cells which could be exploited to eliminate remaining iPS cells in bioengineered cell sheet tissues.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Miócitos Cardíacos/efeitos dos fármacos , Compostos de Piridínio/farmacologia , Animais , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Quinase 9 Dependente de Ciclina/metabolismo , Regulação para Baixo/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Miócitos Cardíacos/metabolismo , Proteína Homeobox Nanog/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ratos , Ratos Nus , Transcrição Genética/efeitos dos fármacos
6.
Sci Rep ; 6: 21747, 2016 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-26888607

RESUMO

The development of a suitable strategy for eliminating remaining undifferentiated cells is indispensable for the use of human-induced pluripotent stem (iPS) cell-derived cells in regenerative medicine. Here, we show for the first time that TRPV-1 activation through transient culture at 42 °C in combination with agonists is a simple and useful strategy to eliminate iPS cells from bioengineered cardiac cell sheet tissues. When human iPS cells were cultured at 42 °C, almost all cells disappeared by 48 hours through apoptosis. However, iPS cell-derived cardiomyocytes and fibroblasts maintained transcriptional and protein expression levels, and cardiac cell sheets were fabricated after reducing the temperature. TRPV-1 expression in iPS cells was upregulated at 42 °C, and iPS cell death at 42 °C was TRPV-1-dependent. Furthermore, TRPV-1 activation through thermal or agonist treatment eliminated iPS cells in cardiac tissues for a final concentration of 0.4% iPS cell contamination. These findings suggest that the difference in tolerance to TRPV-1 activation between iPS cells and iPS cell-derived cardiac cells could be exploited to eliminate remaining iPS cells in bioengineered cell sheet tissues, which will further reduce the risk of tumour formation.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Engenharia Tecidual/métodos , Diferenciação Celular , Células Cultivadas , Técnicas de Cocultura , Fibroblastos/citologia , Fibroblastos/metabolismo , Temperatura Alta , Humanos , Miócitos Cardíacos/metabolismo , Canais de Cátion TRPV/agonistas
7.
PLoS One ; 10(3): e0120782, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25785833

RESUMO

Cyclin Dependent Kinase-2 Associated Protein-1 (CDK2AP1) is known to be a tumor suppressor that plays a role in cell cycle regulation by sequestering monomeric CDK2, and targeting it for proteolysis. A reduction of CDK2AP1 expression is considered to be a negative prognostic indicator in patients with oral squamous cell carcinoma and also associated with increased invasion in human gastric cancer tissue. CDK2AP1 overexpression was shown to inhibit growth, reduce invasion and increase apoptosis in prostate cancer cell lines. In this study, we investigated the effect of CDK2AP1 downregulation in primary human dermal fibroblasts. Using a short-hairpin RNA to reduce its expression, we found that knockdown of CDK2AP1 in primary human fibroblasts resulted in reduced proliferation and in the induction of senescence associated beta-galactosidase activity. CDK2AP1 knockdown also resulted in a significant reduction in the percentage of cells in the S phase and an accumulation of cells in the G1 phase of the cell cycle. Immunocytochemical analysis also revealed that the CDK2AP1 knockdown significantly increased the percentage of cells that exhibited γ-H2AX foci, which could indicate presence of DNA damage. CDK2AP1 knockdown also resulted in increased mRNA levels of p53, p21, BAX and PUMA and p53 protein levels. In primary human fibroblasts in which p53 and CDK2AP1 were simultaneously downregulated, there was: (a) no increase in senescence associated beta-galactosidase activity, (b) decrease in the number of cells in the G1-phase and increase in number of cells in the S-phase of the cell cycle, and (c) decrease in the mRNA levels of p21, BAX and PUMA when compared with CDK2AP1 knockdown only fibroblasts. Taken together, this suggests that the observed phenotype is p53 dependent. We also observed a prominent increase in the levels of ARF protein in the CDK2AP1 knockdown cells, which suggests a possible role of ARF in p53 stabilization following CDK2AP1 knockdown. Altogether, our results show that knockdown of CDK2AP1 in primary human fibroblasts reduced proliferation and induced premature senescence, with the observed phenotype being p53 dependent.


Assuntos
Senescência Celular/genética , Fibroblastos/citologia , Técnicas de Silenciamento de Genes , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Proteínas Reguladoras de Apoptose/genética , Ciclo Celular/genética , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/genética , Dano ao DNA , Humanos , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno/genética , Proteína Supressora de Tumor p14ARF/metabolismo , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Proteína X Associada a bcl-2/genética
8.
Crit Rev Biomed Eng ; 37(4-5): 321-53, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-20528730

RESUMO

Multiple neurodegenerative disorders typically result from irrevocable damage and improper functioning of specialized neuronal cells or populations of neuronal cells. These disorders have the potential to contribute to an already overburdened health care system unless the progression of neurodegeneration can be altered. Progress in understanding neurodegenerative cell biology has been hampered by a lack of predictive and, some would claim, relevant cellular models. Additionally, the research needed to develop new drugs and determine methods for repair or replacement of damaged neurons is severely hampered by the lack of an adequate in vitro human neuron cell-based model. In this context, pluripotent stem cells and neural progenitors and their properties including unlimited proliferation, plasticity to generate other cell types, and a readily available source of cells--pose an excellent alternative to ex vivo primary cultures or established immortalized cell lines in contributing to our understanding of neurodegenerative cell biology and our ability to analyze the therapeutic or cytotoxic effects of chemicals, drugs, and xenobiotics. Many questions that define the underlying "genesis" of the neuronal death in these disorders also remain unanswered, with evidence suggesting a key role for mitochondrial dysfunction. The assessment of stem cells, neural progenitors, and engineered adult cells can provide useful insights into neuronal development and neurodegenerative processes. Finally, the potential for a combination of cell- and gene-based therapeutics for neurodegenerative disorders is also discussed.


Assuntos
Modelos Animais de Doenças , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/cirurgia , Transplante de Células-Tronco/métodos , Animais , Humanos
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