A therapy-grade protocol for differentiation of pluripotent stem cells into mesenchymal stem cells using platelet lysate as supplement.

INTRODUCTION: Mesenchymal stem cells (MSCs) are a promising source of cells for regenerative therapies. Although they can be isolated easily from several tissues, cell expansion is limited since their properties are lost with successive passages. Hence, pluripotent derived MSCs (PD-MSCs) arise as a suitable alternative for MSC production. Nevertheless, at present, PD-MSC derivation protocols are either expensive or not suitable for clinical purposes. METHODS: In this work we present a therapy-grade, inexpensive and simple protocol to derive MSCs from pluripotent stem cells (PSCs) based on the use of platelet lysate (PL) as medium supplement. RESULTS: We showed that the PD-MSCPL expressed multiple MSC markers, including CD90, CD73, CD105, CD166, and CD271, among others. These cells also show multilineage differentiation ability and immunomodulatory effects on pre-stimulated lymphocytes. Thorough characterization of these cells showed that a PD-MSCPL resembles an umbilical cord (UC) MSC and differs from a PSC in surface marker and extracellular matrix proteins and integrin expression. Moreover, the OCT-4 promoter is re-methylated with mesenchymal differentiation comparable with the methylation levels of UC-MSCs and fibroblasts. Lastly, the use of PL-supplemented medium generates significantly more MSCs than the use of fetal bovine serum. CONCLUSIONS: This protocol can be used to generate a large amount of PD-MSCs with low cost and is compatible with clinical therapies.

Diferenciación en cardiomiocitos a partir de células madre embrionarias humanas / Differentiation of Human Embryonic Stem Cells into Cardiomyocytes

Introducción Las células madre son motivo de intensa investigación debido a la posibilidad de su utilización en el tratamiento de numerosas enfermedades, en particular las cardiovasculares. La diferenciación de células madre embrionarias humanas en cardiomiocitos se ha realizado exitosamente in vitro. Se han establecido métodos de cultivo y diferenciación, señales involucradas en la cardiogénesis y los cardiomiocitos generados se han utilizado en modelos de regeneración miocárdica. Sin embargo, aún quedan muchos interrogantes que se están investigando activamente. Objetivo Desarrollar una metodología que permita el cultivo de células embrionarias y su diferenciación en cardiomiocitos. Material y métodos Se utilizaron cuatro líneas de células madre embrionarias humanas. Se cultivaron y diferenciaron a través de los métodos publicados previamente en la bibliografía. El estado indiferenciado y la diferenciación en cardiomiocitos se verificaron por medio de inmunomarcación fluorescente y RT-PCR. Resultados La metodología utilizada permitió cultivar las células y mantenerlas en estado indiferenciado. Aunque con eficacia dispar, se logró la diferenciación en cardiomiocitos de las cuatro líneas celulares utilizadas. La confirmación se realizó por medio de la expresión de factores de transcripción miocárdicos y proteínas estructurales cardíacas. Conclusiones El cultivo y la diferenciación de células madre embrionarias humanas fue posible en nuestro sistema. Estos resultados preliminares nos impulsan a continuar y a desarrollar nuestros métodos con células pluripotentes inducidas.

Background The role of stem cells in the treatment of several conditions, especially heart diseases, is under permanent investigation. Human embryonic stem cells have been successfully differentiated in vitro into cardiomyocytes. Methods of cell culture and cardiomyocyte differentiation are well established; signals regulating cardiogenesis have been identified and the cardiomyocytes generated have been used in models of myocardial regeneration. However, several questions still remain and are currently under active investigation. Objective To develop a culture system that is suitable for the induction of embryonic stem cells to cardiomyocyte differentiation. Material and Methods Four human embryonic stem cell lines were used. The cells were cultured and differentiation was induced using methods previously described. The presence of cells in an undifferentiated state and cardiomyocyte differentiation was detected by immunohistochemical studies (fluorescent staining) and RT-PCR. Results The methodology used allowed stem cells growth in the culture, and maintained them in an undifferentiated state. Cardiomyocyte differentiation was achieved in the four cell lines used, yet with uneven efficacy. This was confirmed by the expression of myocardial transcription factors and heart structural proteins. Conclusions Our system allowed human embryonic stem cell growth and differentiation in the culture. These preliminary results encourage us to continue developing our methods with induced pluripotent stem cells.