Small RNAs, big potential: the role of MicroRNAs in stem cell function

MicroRNAs (miRNAs) are a newly discovered, yet powerful mechanism for regulating protein expression via mRNA translational inhibition. Loss of all miRNA function within mice leads to embryonic lethality with a loss of the stem cell population in the epiblast and failure to form a primitive streak. These data suggest that miRNAs play a major role in embryonic development. As critical regulation of protein expression is also important for controlling the balance between self-renewal and differentiation in stem cells, the study of miRNAs within this model system is rapidly expanding. New data suggest that stem cells have discrete miRNA expression profiles, which may account for, or contribute to, the intrinsic stem cell properties of self-renewal and pluripotency. Specifically, miRNAs have been implicated in downregulation of cell cycle checkpoint proteins during germ stem cell division. Other data demonstrate that changes in miRNA expression can promote or inhibit stem or progenitor cell differentiation within different cell lineages, including hematopoietic cells, cardiomyocytes, myoblasts, and neural cells. In this review we detail the established functional roles of miRNAs in the embryonic and adult stem cell model systems. Finally, we explore new techniques that exploit endogenous miRNA processing and function for applications in basic and clinical research.     

Sustained embryoid body formation and culture in a non-laborious three dimensional culture system for human embryonic stem cells

Pluripotent human embryonic stem cell (hESC) lines are a promising model system in developmental and tissue regeneration research. Differentiation of hESCs towards the three germ layers and finally tissue specific cell types is often performed through the formation of embryoid bodies (EBs) in suspension or hanging droplet culture systems. However, these systems are inefficient regarding embryoid body (EB) formation, structural support to the EB and long term differentiation capacity. The present study investigates if agarose, as a semi solid matrix, can facilitate EB formation and support differentiation of hESC lines. The results showed that agarose culture is able to enhance EB formation efficiency with 10% and increase EB growth by 300%. The agarose culture system was able to maintain expression of the three germ layers over 8 weeks of culture. All of the four hESC lines tested developed EBs in the agarose system although with a histological heterogeneity between cell lines as well as within cell lines. In conclusion, a 3-D agarose culture of spherical hESC colonies improves EB formation and growth in a cost effective, stable and non-laborious technique.     

Production of mouse embryoid bodies with hepatic differentiation potential by stirred tank bioreactor

Embryonic stem (ES) cells can differentiate into functional hepatic lineage cells, which can potentially be used in biomedicine. To obtain hepatic lineage cells from ES cells, embryoid bodies (EBs) must be formed. In this study, we developed an EB formation system using a spinner flask for mass production of EBs. ES cells were inoculated into the spinner flask, where they formed EBs within 4 d. The EBs were then transferred into an attached culture for hepatic differentiation. To verify the hepatic lineage cells, albumin secretion and hepatic-specific gene expression were examined. We found that EBs formed by either the spinner flask or hanging drops exhibited similar albumin secretion potential and hepatic-specific gene expression. We conclude that the spinner flask method can be used to produce mouse EBs that can be used to mass produce hepatic lineage cells for use in biomedicine.     

Production of Mouse Embryoid Bodies with Hepatic Differentiation Potential by Stirred Tank Bioreactor

Embryonic stem (ES) cells can differentiate into functional hepatic lineage cells, which can potentially be used in biomedicine. To obtain hepatic lineage cells from ES cells, embryoid bodies (EBs) must be formed. In this study, we developed an EB formation system using a spinner flask for mass production of EBs. ES cells were inoculated into the spinner flask, where they formed EBs within 4 d. The EBs were then transferred into an attached culture for hepatic differentiation. To verify the hepatic lineage cells, albumin secretion and hepatic-specific gene expression were examined. We found that EBs formed by either the spinner flask or hanging drops exhibited similar albumin secretion potential and hepatic-specific gene expression. We conclude that the spinner flask method can be used to produce mouse EBs that can be used to mass produce hepatic lineage cells for use in biomedicine.     

Spatial and temporal expression pattern of germ layer markers during human embryonic stem cell differentiation in embryoid bodies

Human embryonic stem cell (hESC) differentiation in embryoid bodies (EBs) provides a valuable tool to study the interplay of different germ layers and their influence on cell differentiation. The gene expression of the developing EBs has been shown in many studies, but the protein expression and the spatial composition of different germ layers in human EBs have not been systematically studied. The aim of the present work was to study the temporal and spatial organisation of germ layers based on the expression of mesoderm (Brachyury T), endoderm (AFP) and ectoderm (SOX1) markers during the early stages of differentiation in eight hESC lines. Tissue multi-array technology was applied to study the protein expression of a large number of EBs. According to our results, EB formation and the organisation of germ layers occurred in a similar manner in all the lines. During 12 days of differentiation, all the germ layer markers were present, but no obvious distinct trajectories were formed. However, older EBs were highly organised in structure. Pluripotency marker OCT3/4 expression persisted unexpectedly long in the differentiating EBs. Cavity formation was observed in the immunocytological sections, and caspase-3 expression was high, suggesting a role of apoptosis in hESC differentiation and/or EB formation. The expression of Brachyury T was notably low in all the lines, also those with the best cardiac differentiation capacity, while the expression of SOX1 was higher in some lines, suggesting that the neural differentiation propensity may be detectable already in the early stages of EB differentiation.     

Histone h1 depletion impairs embryonic stem cell differentiation

Pluripotent embryonic stem cells (ESCs) are known to possess a relatively open chromatin structure; yet, despite efforts to characterize the chromatin signatures of ESCs, the role of chromatin compaction in stem cell fate and function remains elusive. Linker histone H1 is important for higher-order chromatin folding and is essential for mammalian embryogenesis. To investigate the role of H1 and chromatin compaction in stem cell pluripotency and differentiation, we examine the differentiation of embryonic stem cells that are depleted of multiple H1 subtypes. H1c/H1d/H1e triple null ESCs are more resistant to spontaneous differentiation in adherent monolayer culture upon removal of leukemia inhibitory factor. Similarly, the majority of the triple-H1 null embryoid bodies (EBs) lack morphological structures representing the three germ layers and retain gene expression signatures characteristic of undifferentiated ESCs. Furthermore, upon neural differentiation of EBs, triple-H1 null cell cultures are deficient in neurite outgrowth and lack efficient activation of neural markers. Finally, we discover that triple-H1 null embryos and EBs fail to fully repress the expression of the pluripotency genes in comparison with wild-type controls and that H1 depletion impairs DNA methylation and changes of histone marks at promoter regions necessary for efficiently silencing pluripotency gene Oct4 during stem cell differentiation and embryogenesis. In summary, we demonstrate that H1 plays a critical role in pluripotent stem cell differentiation, and our results suggest that H1 and chromatin compaction may mediate pluripotent stem cell differentiation through epigenetic repression of the pluripotency genes.     

Spatial Pattern Dynamics of 3D Stem Cell Loss of Pluripotency via Rules-Based Computational Modeling

Pluripotent embryonic stem cells (ESCs) have the unique ability to differentiate into cells from all germ lineages, making them a potentially robust cell source for regenerative medicine therapies, but difficulties in predicting and controlling ESC differentiation currently limit the development of therapies and applications from such cells. A common approach to induce the differentiation of ESCs in vitro is via the formation of multicellular aggregates known as embryoid bodies (EBs), yet cell fate specification within EBs is generally considered an ill-defined and poorly controlled process. Thus, the objective of this study was to use rules-based cellular modeling to provide insight into which processes influence initial cell fate transitions in 3-dimensional microenvironments. Mouse embryonic stem cells (D3 cell line) were differentiated to examine the temporal and spatial patterns associated with loss of pluripotency as measured through Oct4 expression. Global properties of the multicellular aggregates were accurately recapitulated by a physics-based aggregation simulation when compared to experimentally measured physical parameters of EBs. Oct4 expression patterns were analyzed by confocal microscopy over time and compared to simulated trajectories of EB patterns. The simulations demonstrated that loss of Oct4 can be modeled as a binary process, and that associated patterns can be explained by a set of simple rules that combine baseline stochasticity with intercellular communication. Competing influences between Oct4+ and Oct4− neighbors result in the observed patterns of pluripotency loss within EBs, establishing the utility of rules-based modeling for hypothesis generation of underlying ESC differentiation processes. Importantly, the results indicate that the rules dominate the emergence of patterns independent of EB structure, size, or cell division. In combination with strategies to engineer cellular microenvironments, this type of modeling approach is a powerful tool to predict stem cell behavior under a number of culture conditions that emulate characteristics of 3D stem cell niches.     

MicroRNA signatures of iPSCs and endoderm-derived tissues

MicroRNAs (miRNAs), small non-coding RNAs that fine-tune gene expression, play multiple roles in the cell, including cell fate specification. We have analyzed the differential expression of miRNAs during fibroblast reprogramming into induced pluripotent stem cells (iPSCs) and endoderm induction from iPSCs upon treatment with high concentrations of Activin-A. The reprogrammed iPSCs assumed an embryonic stem cell (ESC)-like miRNA signature, marked by the induction of pluripotency clusters miR-290–295 and miR-302/367 and conversely the downregulation of the let-7 family. On the other hand, endoderm induction in iPSCs resulted in the upregulation of 13 miRNAs. Given that the liver and the pancreas are common derivatives of the endoderm, analysis of the expression of these 13 upregulated miRNAs in hepatocytes and pancreatic islets revealed a tendency for these miRNAs to be expressed more in pancreatic islets than in hepatocytes. These observations provide insights into how differentiation may be guided more efficiently towards the endoderm and further into the liver or pancreas. Moreover, we also report novel miRNAs enriched for each of the cell types analyzed.     

miRNA let-7e targeting MMP9 is involved in adipose-derived stem cell differentiation toward epithelia

miRNA let-7e is involved in stem cell differentiation, and metalloproteinases are among its potential target genes. We hypothesized that the inhibitory action of let-7e on regulation of MMP9 expression could represent a crucial mechanism during differentiation of adipose-derived stem cells (ASCs). ASCs were differentiated with all-trans retinoic acid (ATRA) to promote differentiation, and the effect of let-7 silencing during differentiation was tested. Results indicate that ASCs cultured with ATRA differentiated into cells of the epithelial lineage. We found that ASCs cultured with ATRA or transfected with miRNA let-7e expressed epithelial markers such as cytokeratin-18 and early renal organogenesis markers such as Pax2, Wt1, Wnt4 and megalin. Conversely, the specific knockdown of miRNA let-7e in ASCs significantly decreased the expression of these genes, indicating its vital role during the differentiation process. Using luciferase reporter assays, we also showed that MMP9 is a direct target of miRNA let-7e. Thus, our results suggest that miRNA let-7e acts as a matrix metalloproteinase-9 (MMP9) inhibitor and differentiation inducer in ASCs.     

Confined 3D microenvironment regulates early differentiation in human pluripotent stem cells

The therapeutic potential of human pluripotent stem (hPS) cells is threatened, among various problems, by the difficulty to homogenously direct cell differentiation into specific lineages. The transition from hPSC into committed differentiated cells is accompanied by secretome activity, remodeling of extracellular matrix and self‐organization into germ layers. In this work, we aimed to investigate how different three‐dimensional microenvironments regulate the early differentiation of the three germ layers in human embryonic stem (hES) cells derived embryoid bodies. In particular, a permeable, biocompatible, hydrogel microwell array was specifically designed for recreating a confined niche in which EB secreted molecules accumulate in accordance with hydrogel diffusional cut‐off. Fluorescence recovery after photobleaching technique was performed to accurately evaluate hydrogel permeability, mesh size and diffusional cutoff for soluble molecules. Three different culture conditions of EB culture were analyzed: suspension, confinement in microwells of width/depth ratio 1:1 and 1:2. Results show that EBs cultured in microwells are viable and have comparable average size after 8 days culture. Whole genome microarrays show that significative differential gene expression was observed between suspension and confined EBs culture. In particular, EBs culture in microwells promotes the expression of genes involved in pattern specification processes, brain development, ectoderm and endoderm differentiation. On the contrary, suspension EBs express instead genes involved in mesoderm specification and heart development. These results suggest that local accumulation of EBs secreted molecules drives differentiation patterns, as confirmed by immunofluorescence of germ layer markers, in hydrogel confined EB culture from both hES cells and human induced pluripotent stem (hiPS) cells. Our findings highlight an additional potential role of biomaterial in controlling hPSC differentiation through secreted factor niche specification. Biotechnol. Bioeng. 2012; 109: 3119–3132. © 2012 Wiley Periodicals, Inc.     

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.     

Naive and primed murine pluripotent stem cells have distinct miRNA expression profiles

Over the last years, the microRNA (miRNA) pathway has emerged as a key component of the regulatory network of pluripotency. Although clearly distinct states of pluripotency have been described in vivo and ex vivo, differences in miRNA expression profiles associated with the developmental modulation of pluripotency have not been extensively studied so far. Here, we performed deep sequencing to profile miRNA expression in naive (embryonic stem cell [ESC]) and primed (epiblast stem cell [EpiSC]) pluripotent stem cells derived from mouse embryos of identical genetic background. We developed a graphical representation method allowing the rapid identification of miRNAs with an atypical profile including mirtrons, a small nucleolar RNA (snoRNA)-derived miRNA, and miRNAs whose biogenesis may differ between ESC and EpiSC. Comparison of mature miRNA profiles revealed that ESCs and EpiSCs exhibit very different miRNA signatures with one third of miRNAs being differentially expressed between the two cell types. Notably, differential expression of several clusters, including miR290-295, miR17-92, miR302/367, and a large repetitive cluster on chromosome 2, was observed. Our analysis also showed that differentiation priming of EpiSC compared to ESC is evidenced by changes in miRNA expression. These dynamic changes in miRNAs signature are likely to reflect both redundant and specific roles of miRNAs in the fine-tuning of pluripotency during development.     

Generation of a multi-layer muscle fiber sheet from mouse ES cells by the spermine action at specific timing and concentration

Here, we show that spermine can induce the generation of a multi-layer muscle fiber sheet (MMFS) in mouse embryonic stem (ES) cells. ES cells were cultured by the hanging drop method and embryoid bodies (EBs) that formed after 2 days of culture were transferred to a 24-well dish (1 EB/well) containing differentiation medium. EBs cultured in the absence of spermine showed no evidence of differentiation of contractile muscle fibers. In contrast, the addition of spermine (0.5-1.0 mM) for 24 hr on day 12 of culture was found to result in the formation of contractile muscle fibers around the EBs by day 17, with further differentiation into MMFS by day 32. We found that spermine could only induce muscle cell differentiation in EBs during a limited period of culture. Moreover, high concentrations of spermine inhibited muscle fiber generation. Histochemical analysis showed that the MMFS induced by spermine had a heterogeneous architecture. Heart muscle cells appeared to be predominant in some regions, as evidenced by the expression of the markers atrial natriuretic peptide (ANP) and connexin 40 (Cx40), while skeletal muscle appeared to predominate in other regions, as indicated by the expression of MyoD. DNA array analysis showed specific enhancement of expression of muscle cell genes, supporting our conclusion that spermine induces differentiation of muscle cells in vitro.     

应用旋转生物反应器批量制备拟胚体的研究

以小鼠胚胎干细胞(ES-D3)为模型,应用新型细胞培养系统——STLV型旋转生物反应器(rotarycellculturesystem,RCCS)建立一种批量制备拟胚体(embryoidbodies,EBs)的新方法,研究不同细胞接种密度及培养时间对RCCS内EBs产生效率的影响。为了进一步研究该制备方法是否对EBs的分化潜能产生影响,对照传统方法制备的EBs,利用形态学及RT-PCR方法测定经旋转生物反应器制备的EBs在自发性或诱导条件下(1%DMSO)向心肌细胞的分化能力。结果表明:ES-D3在RCCS内能够高效形成EBs,与传统的直接悬浮法比较,其EBs的形成效率可达到后者的2倍。1×104个/ml为最佳细胞接种密度,培养时间也是在RCCS制备EBs过程中的重要因素之一,培养第4~5天为最佳收获EBs的时间。与悬滴法制备的EBs比较,该方法制备的EBs分化为心肌细胞的潜能未改变。由此,应用旋转生物反应器可以高效制备EBs,该方法制备的EBs可以用于发育生物学等基础及应用领域的相关研究。