Coculture Of Embryonic Stem Cells Research Articles

The Effects of Co-Culture of Embryonic Stem Cells with Neural Stem Cells on Differentiation.

Researching the technology for in vitro differentiation of embryonic stem cells (ESCs) into neural lineages is very important in developmental biology, regenerative medicine, and cell therapy. Thus, studies on in vitro differentiation of ESCs into neural lineages by co-culture are expected to improve our understanding of this process. A co-culture system has long been used to study interactions between cell populations, improve culture efficiency, and establish synthetic interactions between populations. In this study, we investigated the effect of a co-culture of ESCs with neural stem cells (NSCs) in two-dimensional (2D) or three-dimensional (3D) culture conditions. Furthermore, we examined the effect of an NSC-derived conditioned medium (CM) on ESC differentiation. OG2-ESCs lost the specific morphology of colonies and Oct4-GFP when co-cultured with NSC. Additionally, real-time PCR analysis showed that ESCs co-cultured with NSCs expressed higher levels of ectoderm markers Pax6 and Sox1 under both co-culture conditions. However, the differentiation efficiency of CM was lower than that of the non-conditioned medium. Collectively, our results show that co-culture with NSCs promotes the differentiation of ESCs into the ectoderm.

Differentiation of embryonic stem cells into a putative hair cell-progenitor cells via co-culture with HEI-OC1 cells

Several studies have shown how different cell lines can influence the differentiation of stem cells through co-culture systems. The House Ear Institute-Organ of Corti 1 (HEI-OC1) is considered an important cell line for in vitro auditory research. However, it is unknown if HEI-OC1 cells can promote the differentiation of embryonic stem cells (ESCs). In this study, we investigated whether co-culture of ESCs with HEI-OC1 cells promotes differentiation. To this end, we developed a co-culture system of mouse ESCs with HEI-OC1 cells. Dissociated or embryonic bodies (EBs) of ESCs were introduced to a conditioned and inactivated confluent layer of HEI-OC1 cells for 14 days. The dissociated ESCs coalesced into an EB-like form that was smaller than the co-cultured EBs. Contact co-culture generated cells expressing several otic progenitor markers as well as hair cell specific markers. ESCs and EBs were also cultured in non-contact setup but using conditioned medium from HEI-OC1 cells, indicating that soluble factors alone could have a similar effect. The ESCs did not form into aggregates but were still Myo7a-positive, while the EBs degenerated. However, in the fully differentiated EBs, evidence to prove mature differentiation of inner ear hair cell was still rudimentary. Nevertheless, these results suggest that cellular interactions between ESCs and HEI-OC1 cells may both stimulate ESC differentiation.

Liver sinusoidal endothelial cells promote lymphohematopoietic differentiation from murine embryonic stem cells: Role of soluble factors

Liver sinusoid endothelial cells (LSEC) constitute an in vitro and in vivo microenvironment for the proliferation and differentiation of hematopoietic stem cells (HSC). Previously, we have shown that LSEC support the survival and growth of murine embryonic stem cells (ESC). In this study, we investigated the capacity of LSEC to promote hematopoietic differentiation from the murine ESC cell line, CGR8. Undifferentiated ESC were cultured on LSEC monolayers in the absence of exogenous cytokines. After 10 and 20 days, cells were harvested and examined by their morphology, phenotype and capacity of hematopoietic colony formation. Microscopic observation of LSEC/ESC cocultures showed the presence of cobblestone areas formation, which indicates active hematopoiesis. Morphological analysis of cell from these foci showed the presence of hematopoietic cells at different stages of differentiation. Cells expressing B lymphoid markers (B220 and CD19) were detected by flow cytometry, and clonogenic assays showed the formation of CFU-pre B colonies. Similar results were observed when ESC were cultured with LSEC conditioned media. Myeloid precursors were also detected by the presence of CFU-GM colonies and cells expressing myeloid markers. These results indicate that LSEC provided an in vitro microenvironment mainly for B cell development, but also myeloid differentiation from ESC. Coculture of ESC with LSEC may constitute a very powerful tool to study the mechanisms involved in B cell generation from ESC.

Endothelial cells regulate cardiomyocyte development from embryonic stem cells

The molecules and environment that direct pluripotent stem cell differentiation into cardiomyocytes are largely unknown. Here, we determined a critical role of receptor tyrosine kinase, EphB4, in regulating cardiomyocyte generation from embryonic stem (ES) cells through endothelial cells. The number of spontaneous contracting cardiomyocytes, and the expression of cardiac-specific genes, including alpha-MHC and MLC-2V, was significantly decreased in EphB4-null ES cells. EphB4 was expressed in endothelial cells underneath contracting cardiomyocytes, but not in cardiomyocytes. Angiogenic inhibitors, including endostatin and angiostatin, inhibited endothelial cell differentiation and diminished cardiomyogenesis in ES cells. Generation of functional cardiomyocytes and the expression of cardiac-specific genes were significantly enhanced by co-culture of ES cells with human endothelial cells. Furthermore, the defects of cardiomyocyte differentiation in EphB4-deficient ES cells were rescued by human endothelial cells. For the first time, our study demonstrated that endothelial cells play an essential role in facilitating cardiomyocyte differentiation from pluripotent stem cells. EphB4 signaling is a critical component of the endothelial niche to regulate regeneration of cardiomyocytes.

Magnetic Separation of Cells in Coculture Systems Using Magnetite Cationic Liposomes

In tissue engineering, coculture systems have been employed for two major purposes: (1) construction of tissue and organ substitutes (e.g., coculture of parenchymal and nonparenchymal cells in liver tissue engineering) and (2) maintenance of cellular functions (e.g., coculture of embryonic stem cells with embryonic fibroblasts as the feeder cells). For the characterization and recovery of specific cell types, however, target cells have to be isolated from other cells. We report here a novel magnetic separation method to isolate target cells in coculture systems. In this method, target cells were cocultured with nontarget cells labeled with magnetite cationic liposomes (MCLs). Thus, when necessary, the MCL-labeled nontarget cells were magnetically removed from the coculture, resulting in negative isolation of the target cells. As the separation models, three deferent types of coculture systems were examined: rat hepatocytes with various MCL-labeled cells (mouse NIH3T3, STO, or human umbilical vein endothelial cells), human keratinocyte HaCaT cells with MCL-labeled NIH3T3 cells, and mouse embryonic stem cells with MCL-labeled STO cells. In these cocultures, target cells were separated with 94% purity and 98%recovery yield on average. This technique provides a promising approach to isolate and recover target cells for further analysis and application.

HoxA11 Is Expressed in the Developing Hemangioblast as Well as Early Hematopoietic Precursor Stem Cells.

Congenital amegakaryocytic thrombocytopenia with radio-ulnar synostosis is associated with mutations in the HOXA11 gene, suggesting that HoxA11 may play a role in megakaryocytic lineage commitment or differentiation. The HOX genes encode transcription factors that are involved in cellular differentiation in embryonic as well as adult tissues. Numerous studies have identified HOX genes as important regulators of various aspects of hematopoiesis including self-renewal, proliferation, differentiation and leukemogenesis. Our initial studies failed to identify the expression of HoxA11 in platelets, TPO-induced CD34+ umbilical cord stem cells or normal bone marrow. More recently our lab has detected a small amount of HoxA11 mRNA in cells isolated from unfractionated human cord blood, suggesting the expression of HoxA11 may occur in a small subset of early hematopoietic or stromal cells. To test this hypothesis we have employed a murine embryonic stem (ES) cell culture system. Co-culture of ES cells and the bone marrow stromal cell line, OP9, can give rise to primitive as well as definitive hematopoietic progenitors in the absence of leukemia inhibitory factor (LIF). By day 6, ES cells on OP9 can differentiate into mesodermal colonies, which contain a bi-potential progenitor known as the hemangioblast. The hemangioblast can further differentiate into either a hematopoietic or endothelial lineage. To determine when HoxA11 is expressed we have employed this model using green fluorescent protein (GFP) expressing ES cells grown on OP9 and differentiated into hematopoietic precursors in the absence of LIF. Nested RT-PCR revealed that HoxA11 mRNA is highly expressed in ES cells following 6 days (D6) on OP9. HoxA11 expression was restricted to D6 ES cells, as HoxA11 mRNA was not found in OP9 cells alone or ES cells differentiated on OP9 for 0, 3, or 9 days. RT-PCR revealed HoxA11 mRNA expression coincided with the expression of flk-1, a marker for the hemangioblast. Since HoxA11 expression is concurrent with hemangioblast differentiation, we sought to determine if the hemangioblast is the cell that expressed HoxA11. Using flow cytometery and fluorescence activated cell sorting (FACS) analysis we separated D6 ES cells into flk-1 positive (flk-1+) and negative (flk-1−) populations and investigated which population expressed HoxA11. Nested RT-PCR revealed that HoxA11 mRNA expression is found in both the flk-1+ and flk-1- fractions. We further analyzed these fractions by RT-PCR for SCL/Tal-1. SCL/Tal-1 is a transcription factor that plays a critical role in the commitment of mesoderm into hematopoietic progenitor cells. We find SCL/Tal-1 mRNA also expressed in both flk-1+ and flk-1- fractions, which parallels HoxA11 mRNA expression. These data suggest HoxA11 expression occurs in the flk-1+ hemangioblast but also possibly in a flk-1-/SCL+ hematopoietic precursor cell population. Current studies are underway to determine the cell fate and role of the HoxA11 expressing progenitor cell. Taken together, these data are the first findings of HoxA11 expression in early progenitor cells as well as the first evidence of controlled HoxA11 regulation during early hematopoietic development.

Coculture of Embryonic Stem Cells with Pulmonary Mesenchyme: A Microenvironment That Promotes Differentiation of Pulmonary Epithelium

Coculture of stem/progenitor cells with mature cells or tissues can drive their differentiation toward required lineages. Thus, we hypothesized that coculture of murine embryonic stem (ES) cells with embryonic mesenchyme from distal lung promotes the differentiation of pneumocytes. Murine ES cells were differentiated to embryoid bodies (EBs) and cultured for 5 or 12 days with pulmonary mesenchyme from embryonic day 11.5 or 13.5 murine embryos, in direct contact or separated by a membrane. Controls included EBs cultured alone or with embryonic gut mesenchyme. Histology revealed epithelium-lined channels in directly cocultured EBs, whereas EBs grown alone showed little structural organization. The lining cells expressed cytokeratin and thyroid transcription factor 1, an early developmental marker in pulmonary epithelium. Differentiation of type II pneumocytes specifically was demonstrated by the presence of surfactant protein C (SP-C) in some of the epithelial cells. None of these markers was seen in EBs cultured alone or with embryonic gut mesenchyme. Indirect coculture of EBs with lung mesenchyme resulted in a 14-fold increase in SP-C gene expression. Thus, provision of an appropriate microenvironment, in the form of pulmonary mesenchyme, appears to promote the differentiation of ES cells toward lung epithelium. Our findings may have applications in regenerative medicine strategies and the engineering of lung tissue.

Photoreceptor cells from mouse ES cells by co-culture with chick embryonic retina

Degeneration of photoreceptors is a consistent and common endpoint in retinal diseases. Herein, we report the efficient induction of photoreceptor-like cells from mouse embryonic stem (ES) cells using chick embryonic retina tissue. Undifferentiated mouse ES cells were initially cultured in a preferential condition into a neural lineage, and ES cells were then co-cultured with chick embryonic day 6 (E6) retina tissues. After a 10-day co-culture, approximately 20% of the mouse ES derivatives became immuno-positive for rhodopsin. RT-PCR analysis demonstrated expression of the transcription factor crx and a distinct increase of rod photoreceptor-specific markers, IRBP and recoverin, after the start of the co-culture. These results indicate that co-culture of ES cells with chick embryonic retina tissue is a useful and efficient method for the induction of photoreceptor-like cells.

A New Mechanism of Communication between Stem Cells Involving Vertical Transfer of mRNA by Its Intracellular Delivery within Membrane-Derived Microvesicles.

Membrane-derived microvesicles (MV) are spherical membrane fragments that are released from eukaryoctic cells upon their activation. We reported that MV transfer cell membrane-derived receptors between cells and/or directly stimulate target cells by the ligands expressed on their surface (Blood 2001: 98;3143; AIDS 2003: 17;33). Recently we observed that embryonic stem cells (ES) shed MV into culture media and that co-culture of ES with hematopoietic stem cells enhances the latter's expansion. Similarly, co-culture of ES with somatic cells induces their dedifferentiation. Although the mechanisms responsible for these phenomena are not clear, we hypothesized that ES-derived MV (ESMV) could play an important role. To address this we isolated ESMV from murine (ES-D3) and two human (CCTL-12 and CCTL-14) embryonic cell lines and focused on molecules that may be responsible for epigenetic changes of cells co-cultured with ESMV. We found that ESMV as compared to the ES cells from which they originated are highly enriched in mRNA. This increase in mRNA content suggested a segregation mechanism that enriches ESMV in cytoplasmatic mRNA during their shedding from ES. Using real-time RT-PCR we found that ES-MV are highly enriched (x 103−107) in mRNA for early transcription factors that regulate self-renewal of stem cells (e.g., Oct-4, Gata-4, Rex-1 and Nanog). Intrigued by these observations we hypothesized that ESMV could penetrate the cells and deliver ES-derived mRNA and that this could be a novel mechanism for reprogramming target cells. Supporting our hypothesis we found (i) by confocal microscopy that ESMV do indeed penetrate the cells (e.g., BM-derived CD34+ cells or ES themselves), and (ii) by Western blot analysis that mRNA delivered to the target cells by ESMV is not trapped in the endosomal compartment but is delivered to the cytoplasm and actively transcribed into appropriate proteins (e.g., Oct-4). Based on these data we postulate that MV may transfer mRNA between the stem cells and play a role in vertical transfer of genetic information. Our recent similar data on MV derived from other cell types (normal and malignant) lend further support to this novel hypothesis and mechanism of cell to cell signaling/communication.

Preparation of animals with a high degree of chimerism by one-step coculture of embryonic stem cells and preimplantation embryos.

A single-step coculture procedure has been developed that can generate chimeric mice with high efficiency and reproducibility. The procedure involves culture of embryonic stem (ES) cells with 8- to 16-cell embryos in microwells to provide conditions for effective cell-cell-embryo contact. A suspension of ES cells is layered over the microwells, followed by transfer of an embryo without zona pellucida into each microwell. Following overnight culture, the blastocysts are transferred into pseudopregnant recipients. The method has several advantages due to its simplicity and reproducibility: (i) Over 90% of ES-cell contribution in new-borns can be obtained frequently and most of the chimeras display germ-line transmission. (ii) The procedure does not require specialized skills or expensive instruments. (iii) All the steps of embryo manipulation can be completed in a relatively short period of time; therefore, a large number of embryos can be manipulated simultaneously. The method was tested with three independent ES cell lines and two different strains of mice with similar results. The technique may be an alternative to microinjection of DNA into zygotes to prepare transgenic lines of small and large mammalian species.

Production of mice entirely derived from embryonic stem (ES) cell with many passages by coculture of ES cells with cytochalasin B induced tetraploid embryos.

Mice entirely derived from ES cells were obtained from aggregates of TT2 ES cells and cytochalasin B induced tetraploid embryos. Tetraploid embryos were cocultured with ES cells in a well on the Multiplate-Terasaki. After embryo transfer of the aggregates, the male newborns were recovered normally after Cesarean section and reached adulthood. The male mice exhibited complete pigmentation of the eye and coat, suggesting ES cell contributions alone. Alkaline phosphatase-1 analysis yielded no evidence of tetraploid cells in the kidney or liver. The TT2-derived males were fertile, produced normal offspring, and exclusively transmitted the TT2 genotype to their progeny. This result clearly shows that ES cells are able to support complete fetal development.

Production of chimeric mice by coculture of embryonic stem cells and zona-free embryos

Production of chimeric mice by coculture of embryonic stem cells and zona-free embryos

Elevation of Transforming Growth Factor .BETA.1 mRNA Level in ES-D3 Mouse Embryonic Stem Cells Cocultured with Balb/c3T3 A31 Fibroblasts.

Embryonic stem (ES) cells are a pluripotent cell line derived from the inner cell mass of the mouse blastocyst. The mRNA levels of several growth-related genes were examined in ES cells by Northern blot analysis under several different growth conditions. In a coculture of ES cells with the mouse fibroblast cell line, Balb/c3T3 A31, the expression level of transforming growth factor beta 1 (TGF-beta 1) mRNA was elevated for 1 to 3 days. In a coculture of ES cells with primary embryonic fibroblasts, only slight accumulation of TGF-beta 1 mRNA was observed for 6 to 24 h and soon attenuated. In situ hybridization analysis revealed that TGF-beta 1 transcripts accumulated first in the masses of ES cells and subsequently in Balb/c3T3 A31 cells adjacent to ES cells' masses. The ability of ES cells to induce TGF-beta 1 in Balb/c3T3 A31 cells was not mediated by soluble factors and was lost upon differentiation. ES cells on primary embryonic fibroblasts grew in undifferentiated form, but those on Balb/c3T3 A31 cells stopped growing and formed embryoid cystic bodies. We suggest that TGF-beta 1 mRNA induction in a coculture is triggered by an interaction between differentiating ES cells and Balb/c3T3 A31, and that this activity is limited to undifferentiated ES cells.