RESUMO
Embryonic stem cells (ESCs) can differentiate into all somatic cell types, but the development of effective strategies to direct ESC fate is dependent upon defining environmental parameters capable of influencing cell phenotype. ESCs are commonly differentiated via cell aggregates referred to as embryoid bodies (EBs), but current culture methods, such as hanging drop and static suspension, yield relatively few or heterogeneous populations of EBs. Alternatively, rotary orbital suspension culture enhances EB formation efficiency, cell yield, and homogeneity without adversely affecting differentiation. Thus, the objective of this study was to systematically examine the effects of hydrodynamic conditions created by rotary orbital shaking on EB formation, structure, and differentiation. Mouse ESCs introduced to suspension culture at a range of rotary orbital speeds (20-60 rpm) exhibited variable EB formation sizes and yields due to differences in the kinetics of cell aggregation. Computational fluid dynamic analyses indicated that rotary orbital shaking generated relatively uniform and mild shear stresses (< or =2.5 dyn/cm(2)) within the regions EBs occupied in culture dishes, at each of the orbital speeds examined. The hydrodynamic conditions modulated EB structure, indicated by differences in the cellular organization and morphology of the spheroids. Compared to static culture, exposure to hydrodynamic conditions significantly altered the gene expression profile of EBs. Moreover, varying rotary orbital speeds differentially modulated the kinetic profile of gene expression and relative percentages of differentiated cell types. Overall, this study demonstrates that manipulation of hydrodynamic environments modulates ESC differentiation, thus providing a novel, scalable approach to integrate into the development of directed stem cell differentiation strategies.
Assuntos
Células-Tronco Embrionárias , Estresse Mecânico , Animais , Técnicas de Cultura de Células , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Camundongos , SuspensõesRESUMO
Embryonic stem (ES) cells hold great promise as a robust cell source for cell-based therapies and as a model of early embryonic development. Current experimental methods for differentiation of ES cells via embryoid body (EB) formation are either inherently incapable of larger-scale production or exhibit limited control over cell aggregation during EB formation and subsequent EB agglomeration. This report describes and characterizes a novel method for formation of EBs using rotary orbital motion that simultaneously addresses both concerns. EBs formed under rotary suspension conditions were compared with hanging-drop and static EBs for efficiency of EB formation, cell and EB yield, homogeneity of EB size and shape, and gene expression. A 20-fold enhancement in the number of cells incorporated into primitive EBs in rotary versus static conditions was detected after the first 12 hours, and a fourfold increase in total cell yield was achieved by rotary culture after 7 days. Morphometric analysis of EBs demonstrated formation and maintenance of a more uniform EB population under rotary conditions compared with hanging-drop and static conditions. Quantitative gene expression analysis indicated that rotary EBs differentiated normally, on the basis of expression of ectoderm, endoderm, and mesoderm markers. Increased levels of endoderm gene expression, along with cystic EB formation, indicated by histological examination, suggested that differentiation was accelerated in rotary EBs. Thus, the rotary suspension culture method can produce a highly uniform population of efficiently differentiating EBs in large quantities in a manner that can be easily implemented by basic research laboratories conducting ES cell differentiation studies.
Assuntos
Técnicas de Cultura de Células , Diferenciação Celular , Eficiência , Células-Tronco Embrionárias/citologia , Rotação , Esferoides Celulares/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Perfilação da Expressão Gênica , Camundongos , Esferoides Celulares/citologia , Fatores de TempoRESUMO
This study investigated the differential effects of ramped and steady applications of cyclic hydrostatic pressure (CHP) on chondrogenic differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs) in 3-dimensional culture in the absence of transforming growth factor-beta (TGF-beta). A custom hydrostatic pressure system was designed and manufactured. hMSCs were seeded in agarose and exposed to steady (7.5 MPa) or ramped (1 MPa to 7.5 MPa over a 14-day period) CHP for 4 h/d at f = 1 Hz for 14 days. Real-time reverse transcriptase polymerase chain reaction analysis was performed on days 0, 4, 9, and 14 to determine changes in messenger ribonucleic acid (mRNA) expression levels of Sox9, aggrecan, collagen I, and collagen II. Collagen II and aggrecan mRNA expression remained unchanged. Collagen I increased at day 4 in CHP specimens before decreasing to levels at or below same-day unloaded controls at days 9 and 14. On average, ramped and steady regimens of CHP increased Sox9, with the largest upregulation occurring at day 4 in response to steady pressure. These findings indicate that hydrostatic pressure may induce chondrogenesis in hMSC-seeded agarose constructs without TGF-beta, and that hMSCs are capable of withstanding high initial pressures that may initiate chondrogenesis faster than lower pressures.
Assuntos
Condrócitos/citologia , Condrócitos/metabolismo , Condrogênese/fisiologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , RNA Mensageiro/metabolismo , Engenharia Tecidual/métodos , Adolescente , Adulto , Materiais Biocompatíveis/química , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Técnicas de Cultura de Células/métodos , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Humanos , Masculino , Mecanotransdução Celular/efeitos dos fármacos , Mecanotransdução Celular/fisiologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Pressão , Sefarose/química , Fator de Crescimento Transformador beta/administração & dosagemRESUMO
Pluripotent stem cell differentiation recapitulates aspects of embryonic development, including the regulation of morphogenesis and cell specification via precise spatiotemporal signaling. The assembly and reorganization of cadherins within multicellular aggregates may similarly influence ß-catenin signaling dynamics and the associated cardiomyogenic differentiation of pluripotent embryonic stem cells (ESCs). In this study, dynamic changes in ß-catenin expression and transcriptional activity were analyzed in response to altered cell adhesion kinetics during embryoid body (EB) formation and differentiation. Modulation of intercellular adhesion kinetics by rotary orbital mixing conditions led to temporal modulation of T-cell factor/lymphoid enhancer-binding factor activity, as well as changes in the spatial localization and phosphorylation state of ß-catenin expression. Slower rotary speeds, which promoted accelerated ESC aggregation, resulted in the early accumulation of nuclear dephosphorylated ß-catenin, which was followed by a decrease in ß-catenin transcriptional activity and an increase in the gene expression of Wnt inhibitors such as Dkk-1. In addition, EBs that exhibited increased ß-catenin transcriptional activity at early stages of differentiation subsequently demonstrated increased expression of genes related to cardiomyogenic phenotypes, and inhibition of the Wnt pathway during the initial 4 days of differentiation significantly decreased cardiomyogenic gene expression. Together, the results of this study indicate that the expression and transcriptional activity of ß-catenin are temporally regulated by multicellular aggregation kinetics of pluripotent ESCs and influence mesoderm and cardiomyocyte differentiation.
Assuntos
Células-Tronco Embrionárias/metabolismo , Mesoderma/metabolismo , Miócitos Cardíacos/metabolismo , Células-Tronco Pluripotentes/metabolismo , beta Catenina/metabolismo , Animais , Caderinas/metabolismo , Adesão Celular/fisiologia , Diferenciação Celular , Linhagem Celular , Corpos Embrioides/metabolismo , Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Camundongos , Desenvolvimento Muscular/fisiologia , Fosforilação , Transdução de Sinais , Transcrição Gênica , Ativação Transcricional , Proteínas Wnt/antagonistas & inibidores , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , beta Catenina/biossíntese , beta Catenina/genéticaRESUMO
Stem cells possess the unique capacity to differentiate into many clinically relevant somatic cell types, making them a promising cell source for tissue engineering applications and regenerative medicine therapies. However, in order for the therapeutic promise of stem cells to be fully realized, scalable approaches to efficiently direct differentiation must be developed. Traditionally, suspension culture systems are employed for the scale-up manufacturing of biologics via bioprocessing systems that heavily rely upon various types of bioreactors. However, in contrast to conventional bench-scale static cultures, large-scale suspension cultures impart complex hydrodynamic forces on cells and aggregates due to fluid mixing conditions. Stem cells are exquisitely sensitive to environmental perturbations, thus motivating the need for a more systematic understanding of the effects of hydrodynamic environments on stem cell expansion and differentiation. This article discusses the interdependent relationships between stem cell aggregation, metabolism, and phenotype in the context of hydrodynamic culture environments. Ultimately, an improved understanding of the multifactorial response of stem cells to mixed culture conditions will enable the design of bioreactors and bioprocessing systems for scalable directed differentiation approaches.
Assuntos
Técnicas de Cultura de Células/métodos , Hidrodinâmica , Células-Tronco/citologia , Células Cultivadas , Humanos , Células-Tronco/metabolismoRESUMO
Embryonic stem cells (ESCs) can differentiate into all somatic cell types, including cardiomyocytes, which may be used for regenerative cardiac cell therapies. ESCs are commonly differentiated via cell aggregates known as embryoid bodies (EBs), but current cardiomyogenic differentiation methods, such as formation via hanging drops, yield relatively small numbers of EBs and differentiated cells. On the other hand, batch culture methods, like static suspension, yield increased numbers of EBs and cells, but typically exhibit less overall cardiomyogenic differentiation. The objective of this study was to determine if rotary orbital suspension culture, which produces EBs resembling hanging drops, was capable of enhancing cardiomyogenic differentiation compared to static suspension culture. Similar to hanging drops, rotary suspension culture significantly increased the proportion of spontaneously contracting EBs compared to static suspension culture. The gene expression of mesoderm (Brachyury-T) and cardiac transcription factors (Gata4, Nkx2.5, and Mef2c), as well as sarcomeric muscle proteins (alpha-MHC and MLC-2v) was increased within EBs cultured in rotary suspension conditions. Rotary orbital culture also yielded a greater percentage of EBs that were immunoreactive for alpha-sarcomeric actin protein compared to static suspension, and augmented the average percentage of alpha-sarcomeric actin-positive cells detected via flow cytometry. These results demonstrate that rotary orbital suspension culture enhances endogenous cardiomyogenesis of EBs and therefore could benefit the development of regenerative cardiac therapies.