RESUMEN
Efforts to direct the specification of human pluripotent stem cells (hPSCs) to therapeutically important somatic cell types have focused on identifying proper combinations of soluble cues. Yet, whether exosomes, which mediate intercellular communication, play a role in the differentiation remains unexplored. We took a first step toward addressing this question by subjecting hPSCs to stage-wise specification toward cardiomyocytes (CMs) in scalable stirred-suspension cultures and collecting exosomes. Samples underwent liquid chromatography (LC)/mass spectrometry (MS) and subsequent proteomic analysis revealed over 300 unique proteins from four differentiation stages including proteins such as PPP2CA, AFM, MYH9, MYH10, TRA2B, CTNNA1, EHD1, ACTC1, LDHB, and GPC4, which are linked to cardiogenic commitment. There was a significant correlation of the protein composition of exosomes with the hPSC line and stage of commitment. Differentiating hPSCs treated with exosomes from hPSC-derived CMs displayed improved efficiency of CM formation compared to cells without exogenously added vesicles. Collectively, these results demonstrate that exosomes from hPSCs induced along the CM lineage contain proteins linked to the specification process with modulating effects and open avenues for enhancing the biomanufacturing of stem cell products for cardiac diseases.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Exosomas/metabolismo , Miocitos Cardíacos , Células Madre Pluripotentes , Proteoma/metabolismo , Línea Celular , Humanos , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismoRESUMEN
Functional heart cells and tissues sourced from human pluripotent stem cells (hPSCs) have great potential for substantially advancing treatments of cardiovascular maladies. Realization of this potential will require the development of cost-effective and tunable bioprocesses for manufacturing hPSC-based cell therapeutics. Here, we report the development of a xeno-free platform for guiding the cardiogenic commitment of hPSCs. The system is based on a fully defined, open-source formulation without complex supplements, which have varied and often undetermined effects on stem cell physiology. The formulation was used to systematically investigate factors inducing the efficient commitment to cardiac mesoderm of three hPSC lines. Contractile clusters of cells appeared within a week of differentiation in planar cultures and by day 13 over 80% of the cells expressed cardiac progeny markers such as TNNT2. In conjunction with expansion, this differentiation strategy was employed in stirred-suspension cultures of hPSCs. Scalable differentiation resulted in 0.4-2 million CMs/ml or â¼5-20 TNNT2-positive cells per seeded hPSC without further enrichment. Our findings will contribute to the engineering of bioprocesses advancing the manufacturing of stem cell-based therapeutics for heart diseases.
RESUMEN
Pluripotent stem cells can differentiate to any cell type and contribute to damaged tissue repair and organ function reconstitution. The scalable culture of pluripotent stem cells is essential to furthering the use of stem cell products in a wide gamut of applications such as screening of candidate drugs and cell replacement therapies. Human stem cell cultivation in stirred-suspension vessels enables the expansion of stem cells and the generation of differentiated progeny in quantities suitable for use in animal models and clinical studies. We describe methods of culturing human pluripotent stem cells in spinner flasks either as aggregates or on microcarriers. Techniques for assessing the quality of the culture and characterizing the cells based on the presentation of pertinent markers are also presented. Spinner flask culture with its relatively low capital and operating costs is appealing to laboratories interested in scaling up their production of stem/progenitor cells.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Células Madre Pluripotentes/citología , Agregación Celular , Técnicas de Cultivo de Célula/instrumentación , Diferenciación Celular , Línea Celular , Endodermo/citología , Endodermo/metabolismo , Citometría de Flujo/métodos , Humanos , Inmunohistoquímica/métodos , Cariotipificación/métodos , Mesodermo/citología , Mesodermo/metabolismo , Células Madre Pluripotentes/metabolismo , ARN/genética , ARN/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
Recent advances on human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have brought us closer to the realization of their clinical potential. Nonetheless, tissue engineering and regenerative medicine applications will require the generation of hPSC products well beyond the laboratory scale. This also mandates the production of hPSC therapeutics in fully-defined, xeno-free systems and in a reproducible manner. Toward this goal, we summarize current developments in defined media free of animal-derived components for hPSC culture. Bioinspired and synthetic extracellular matrices for the attachment, growth and differentiation of hPSCs are also reviewed. Given that most progress in xeno-free medium and substrate development has been demonstrated in two-dimensional rather than three dimensional culture systems, translation from the former to the latter poses unique difficulties. These challenges are discussed in the context of cultivation platforms of hPSCs as aggregates, on microcarriers or after encapsulation in biocompatible scaffolds.
