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1.
Mol Cell Biol ; 39(4)2019 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-30455250

RESUMEN

Spermatogonial stem cells (SSCs) are unipotent germ cells that are at the foundation of spermatogenesis and male fertility. However, the underlying molecular mechanisms governing SSC stemness and growth properties remain elusive. We have recently identified chromodomain helicase/ATPase DNA binding protein 1-like (Chd1l) as a novel regulator for SSC survival and self-renewal, but how these functions are controlled by Chd1l remains to be resolved. Here, we applied high-throughput small RNA sequencing to uncover the microRNA (miRNA) expression profiles controlled by Chd1l and showed that the expression levels of 124 miRNA transcripts were differentially regulated by Chd1l in SSCs. KEGG pathway analysis shows that the miRNAs that are differentially expressed upon Chd1l repression are significantly enriched in the pathways associated with stem cell pluripotency and proliferation. As a proof of concept, we demonstrate that one of the most highly upregulated miRNAs, miR-486, controls SSC stemness gene expression and growth properties. The matrix metalloproteinase 2 (MMP2) gene has been identified as a novel miR-486 target gene in the context of SSC stemness gene regulation and growth properties. Data from cotransfection experiments showed that Chd1l, miR-486, and MMP2 work in concert in regulating SSC stemness gene expression and growth properties. Finally, our data also revealed that MMP2 regulates SSC stemness gene expression and growth properties through activating ß-catenin signaling by cleaving N-cadherin and increasing ß-catenin nuclear translocation. Our data demonstrate that Chd1l-miR-486-MMP2 is a novel regulatory axis governing SSC stemness gene expression and growth properties, offering a novel therapeutic opportunity for treating male infertility.


Asunto(s)
Células Madre Germinales Adultas/metabolismo , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , MicroARNs/metabolismo , Células Madre Germinales Adultas/citología , Animales , Proliferación Celular/fisiología , Células Cultivadas , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Masculino , Metaloproteinasa 2 de la Matriz/genética , Ratones , MicroARNs/genética , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Espermatogénesis/fisiología , Espermatogonias/metabolismo , Testículo/citología , Testículo/metabolismo , Factores de Transcripción/metabolismo
2.
Methods Mol Biol ; 1341: 217-34, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26160454

RESUMEN

Embryonic stem cells (ESCs) differentiation via embryoid body (EB) formation is an established method that generates the three germ layers. However, EB differentiation poses several problems including formation of heterogeneous cell populations. Herein, we described a differentiation protocol on enhancing mesoderm derivation from murine ESCs (mESCs) using conditioned medium (CM) from HepG2 cells. We used this technique to direct hematopoiesis by generating "embryoid-like" colonies (ELCs) from murine (m) ESCs without following standard formation of EBs. Our CM-mESCs group yielded an almost fivefold increase in ELC formation (p ≤ 0.05) and higher expression of mesoderm genes;-Brachyury-T, Goosecoid, and Flk-1 compared with control mESCs group. Hematopoietic colony formation from CM-mESCs was also enhanced by twofold at days 7 and 14 with earlier colony commitment compared to control mESCs (p ≤ 0.05). This early clonogenic capacity was confirmed morphologically by the presence of nucleated erythrocytes and macrophages as early as day 7 in culture using standard 14-day colony-forming assay. Early expression of hematopoietic primitive (ζ-globin) and definitive (ß-globin) erythroid genes and proteins was also observed by day 7 in the CM-treated culture. These data indicate that hematopoietic cells more quickly differentiate from CM-treated, compared with those using standard EB approaches, and provide an efficient bioprocess platform for erythroid-specific differentiation of ESCs.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Eritropoyesis , Células Madre Embrionarias de Ratones/citología , Animales , Colorantes Azulados/análisis , Western Blotting/métodos , Medios de Cultivo Condicionados/metabolismo , Electroforesis en Gel de Poliacrilamida/métodos , Células Hep G2 , Humanos , Ratones , Células Madre Embrionarias de Ratones/metabolismo , ARN/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Coloración y Etiquetado/métodos
3.
Stem Cells Dev ; 23(22): 2720-9, 2014 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-24926614

RESUMEN

HepG2-conditioned medium (CM) facilitates early differentiation of murine embryonic stem cells (mESCs) into hematopoietic cells in two-dimensional cultures through formation of embryoid-like colonies (ELCs), bypassing embryoid body (EB) formation. We now demonstrate that three-dimensional (3D) cultures of alginate-encapsulated mESCs cultured in a rotating wall vessel bioreactor can be differentially driven toward definitive erythropoiesis and cardiomyogenesis in the absence of ELC formation. Three groups were evaluated: mESCs in maintenance medium with leukemia inhibitory factor (LIF, control) and mESCs cultured with HepG2 CM (CM1 and CM2). Control and CM1 groups were cultivated for 8 days in early differentiation medium with murine stem cell factor (mSCF) followed by 10 days in hematopoietic differentiation medium (HDM) containing human erythropoietin, m-interleukin (mIL)-3, and mSCF. CM2 cells were cultured for 18 days in HDM, bypassing early differentiation. In CM1, a fivefold expansion of hematopoietic colonies was observed at day 14, with enhancement of erythroid progenitors, hematopoietic genes (Gata-2 and SCL), erythroid genes (EKLF and ß-major globin), and proteins (Gata-1 and ß-globin), although ζ-globin was not expressed. In contrast, CM2 primarily produced beating colonies in standard hematopoietic colony assay and expressed early cardiomyogenic markers, anti-sarcomeric α-actinin and Gata-4. In conclusion, a scalable, automatable, integrated, 3D bioprocess for the differentiation of mESC toward definitive erythroblasts has been established. Interestingly, cardiomyogenesis was also directed in a specific protocol with HepG2 CM and hematopoietic cytokines making this platform a useful tool for the study of erythroid and cardiomyogenic development.


Asunto(s)
Alginatos/metabolismo , Diferenciación Celular/fisiología , Citocinas/metabolismo , Células Madre Embrionarias/fisiología , Eritropoyesis/fisiología , Células Madre Hematopoyéticas/metabolismo , Desarrollo de Músculos/fisiología , Animales , Técnicas de Cultivo de Célula/métodos , Línea Celular , Línea Celular Tumoral , Medios de Cultivo Condicionados/metabolismo , Células Madre Embrionarias/metabolismo , Factores de Unión al ADN Específico de las Células Eritroides/metabolismo , Ácido Glucurónico/metabolismo , Células Madre Hematopoyéticas/fisiología , Células Hep G2 , Ácidos Hexurónicos/metabolismo , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/metabolismo , Interleucina-3/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/fisiología , Factor de Células Madre/metabolismo , Globinas beta/metabolismo , Globinas zeta/metabolismo
4.
Stem Cells Dev ; 21(17): 3152-61, 2012 Nov 20.
Artículo en Inglés | MEDLINE | ID: mdl-22587789

RESUMEN

Embryonic stem cell (ESC) differentiation via embryoid body (EB) formation is an established method that generates the 3 germ layers. However, EB differentiation poses several problems including formation of heterogeneous cell populations. Previously, we have enhanced mesoderm derivation from murine ESCs (mESCs) using conditioned medium (CM) from HepG2 cells. We used this technique to direct hematopoiesis by generating "embryoid-like" colonies (ELCs) from mESCs without standard formation of EBs. Two predifferentiation conditions were tested: (1) mESCs cultured 3 days in standard predifferentiation medium (control) and (2) mESCs cultured 3 days in HepG2 CM (CM-mESCs). Both groups were then exposed to primary differentiation for 8 days (ELC-formation period) and 14 days of hematopoietic differentiation. Enhanced mesoderm formation was observed in the CM-mESC group with an almost 5-fold increase in ELC formation (P ≤ 0.05) and higher expression of mesoderm genes-Brachyury-T, Goosecoid, and Flk-1-compared with those of control mESCs. Hematopoietic colony formation by CM-mESCs was also enhanced by 2-fold at days 7 and 14 with earlier colony commitment compared with those of control mESCs (P ≤ 0.05). This early clonogenic capacity was confirmed morphologically by the presence of nucleated erythrocytes and macrophages as early as day 7 in CM-mESC culture using standard 14-day colony-forming assay. Early expression of hematopoietic primitive (ζ-globin) and definitive (ß-globin) erythroid genes and proteins was also observed by day 7 in CM-mESC cultures. These data indicate that hematopoietic cells more quickly differentiate from CM-mESCs, compared with those using standard EB approaches, and provide an efficient bioprocess platform for erythroid-specific differentiation of ESCs.


Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/citología , Eritrocitos/citología , Células Precursoras Eritroides/citología , Animales , Técnicas de Cultivo de Célula/métodos , Linaje de la Célula , Ensayo de Unidades Formadoras de Colonias/métodos , Medios de Cultivo Condicionados/metabolismo , Células Madre Embrionarias/metabolismo , Eritrocitos/metabolismo , Células Precursoras Eritroides/metabolismo , Eritropoyesis , Proteínas Fetales/genética , Proteínas Fetales/metabolismo , Células Hep G2 , Humanos , Macrófagos/citología , Macrófagos/metabolismo , Mesodermo/citología , Mesodermo/metabolismo , Ratones , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Factores de Tiempo , Globinas beta/genética , Globinas beta/metabolismo , Globinas zeta/genética , Globinas zeta/metabolismo
5.
J R Soc Interface ; 6(32): 209-32, 2009 Mar 06.
Artículo en Inglés | MEDLINE | ID: mdl-19033137

RESUMEN

In recent years, the potential of stem cell research for tissue engineering-based therapies and regenerative medicine clinical applications has become well established. In 2006, Chung pioneered the first entire organ transplant using adult stem cells and a scaffold for clinical evaluation. With this a new milestone was achieved, with seven patients with myelomeningocele receiving stem cell-derived bladder transplants resulting in substantial improvements in their quality of life. While a bladder is a relatively simple organ, the breakthrough highlights the incredible benefits that can be gained from the cross-disciplinary nature of tissue engineering and regenerative medicine (TERM) that encompasses stem cell research and stem cell bioprocessing. Unquestionably, the development of bioprocess technologies for the transfer of the current laboratory-based practice of stem cell tissue culture to the clinic as therapeutics necessitates the application of engineering principles and practices to achieve control, reproducibility, automation, validation and safety of the process and the product. The successful translation will require contributions from fundamental research (from developmental biology to the 'omics' technologies and advances in immunology) and from existing industrial practice (biologics), especially on automation, quality assurance and regulation. The timely development, integration and execution of various components will be critical-failures of the past (such as in the commercialization of skin equivalents) on marketing, pricing, production and advertising should not be repeated. This review aims to address the principles required for successful stem cell bioprocessing so that they can be applied deftly to clinical applications.


Asunto(s)
Medicina Regenerativa/métodos , Células Madre/citología , Ingeniería de Tejidos/métodos , Animales , Humanos , Andamios del Tejido
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