Maintenance of pluripotency in mouse embryonic stem cells cultivated in stirred microcarrier cultures

The development of efficient and reproducible culture systems for embryonic stem (ES) cells is an essential pre‐requisite for regenerative medicine. Culture scale‐up ensuring maintenance of cell pluripotency is a central issue, because large amounts of pluripotent cells must be generated to warrant that differentiated cells deriving thereof are transplanted in great amounts and survive the procedure. This study aimed to develop a robust scalable cell expansion system, using a murine embryonic stem cell line that is feeder‐dependent and adapted to serum‐free medium, thus representing a more realistic model for human ES cells. We showed that high concentrations of murine ES cells can be obtained in stirred microcarrier‐based spinner cultures, with a 10‐fold concentration of cells per volume of medium and a 5‐fold greater cell concentration per surface area, as compared to static cultures. No differences in terms of pluripotency and differentiation capability were observed between cells grown in traditional static systems and cells that were replated onto the traditional system after being expanded on microcarriers in the stirred system. This was verified by morphological analyses, quantification of cells expressing important pluripotency markers (Oct‐4, SSEA‐1, and SOX2), karyotype profile, and the ability to form embryoid bodies with similar sizes, and maintaining their intrinsic ability to differentiate into all three germ layers. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

胚胎干细胞向造血干/祖细胞定向诱导分化的研究进展

胚胎干细胞(embryonic stem cell,ES细胞)是指由胚胎内细胞团(inner cell mass,ICM)细胞经体外抑制培养而筛选得到的细胞,具有无限增殖潜能,在体外可以向造血细胞分化,有可能为造血干细胞移植和血细胞输注开辟新的来源．此外,ES细胞向造血干/祖细胞的定向诱导分化也为阐明哺乳动物造血发育的细胞和分子机制提供了良好的体外模型．对ES细胞向造血干/祖细胞定向分化的研究进展进行了综述．     

Cryopreservation by slow cooling with DMSO diminished production of Oct-4 pluripotency marker in human embryonic stem cells

We tested a "standard" cryopreservation protocol (slow cooling with 10% DMSO) on the human embryonic stem cell (hESC) line H9 containing an Oct-4 (POU5F1) promoter-driven, enhanced green fluorescent protein (EGFP) reporter to monitor maintenance of pluripotency. Cells were cooled to -80 degrees C in cryovials and then transferred to a -80 degrees C freezer. Cells were held at -80 degrees C for 3 days ("short-term storage") or 3 months ("long-term storage"). Vials were thawed in a +36 degrees C water bath and cells were cultured for 3, 7, or 14 days. Propidium iodide (PI) was used to assess cell viability by flow cytometry. Control cells were passaged on the same day that the frozen cells were thawed. The majority of cells in control hESC cultures were Oct-4 positive and almost 99% of EGFP+ cells were alive as determined by exclusion of PI. In contrast, the frozen cells, even after 3 days of culture, contained only 50% live cells, and only 10% were EGFP-positive. After 7 days in culture, the proportion of dead cells decreased and there was an increase in the Oct-4-positive population but microscopic examination revealed large patches of EGFP-negative cells within clusters of colonies even after 14 days of culturing. After 3 months of storage at -80 degrees C the deleterious effect of freezing was even more pronounced: the samples regained a quantifiable number of EGFP-positive cells only after 7 days of culturing following thawing. It is concluded that new protocols and media are required for freezing hESC and safe storage at -80 degrees C as well as studies of the mechanisms of stress-related events associated with cell cryopreservation.     

Link between embryonic stem cell pluripotency and homologous allelic pairing of Oct4 loci

The Oct4 gene is a master regulator of the pluripotent properties of embryonic stem cells (ESCs). Recently, Oct4 loci were shown to frequently localize in close proximity to one another during the early stage of cellular differentiation, implicating this event as an important prerequisite step for ESCs to exert their full differentiation potential. Although the differentiation capacity of embryonal carcinoma cells (ECCs), such as F9 and P19 ECC lines, is severely restricted compared with ESCs, ECCs bear a highly similar expression profile to that of ESCs including expression of Oct4 and other pluripotency marker genes. Therefore, we examined whether allelic pairing of Oct4 loci also occurs during differentiation of F9 and P19 ECCs. Our data clearly demonstrate that this event is only observed within ESCs, but not ECCs, subjected to induction of differentiation, indicating transient allelic pairing of Oct4 loci as a specific feature of pluripotent ESCs. Moreover, our data revealed that this pairing did not occur broadly across chromosome 17, which carries the Oct4 gene, but occurred locally between Oct4 loci, suggesting that Oct4 loci somehow exert a driving force for their allelic pairing.     

Detection of tissue-specific effects by methotrexate on differentiating mouse embryonic stem cells

BACKGROUND: Pluripotent embryonic stem (ES) cells offer a unique possibility to monitor the differentiation of several cell types in vitro. This study attempts to identify marker genes during in vitro cell differentiation of murine ES cells and allow a prediction of chemical effects on cell differentiation of specific target tissues. The study focused on the expression pattern of key genes involved in cardiomyocyte and osteoblast differentiation: Oct-4, Brachyury, Nkx2.5, alpha myosin heavy chain, Cbfa1, and Osteocalcin. METHODS: Methotrexate was selected due to its well-characterized teratogenic effects. Several in vivo studies have demonstrated the specific interactions of methotrexate with bone formation whereas the cardiovascular system is not specifically affected after exposure to low concentration. The capability of murine ES cells to differentiate in vitro into cardiomyocytes as well as into osteoblasts have been used to demonstrate the target cell specificity in vitro, at non-cytotoxic concentration. RESULTS: Exposure of differentiating ES cells did not result in any gene profile modification of the selected cardiomyocyte specific genes, whereas the expression of osteoblast specific key genes, Cbfa1 and Osteocalcin, decreased. At the latter stages of skeletal differentiation we observed a 30% decrease in gene expression for Cbfa1 and a 60% decrease for Osteocalcin, with reference to the control. Early marker genes for undifferentiated cells and mesodermal cells were not modified after methotrexate treatment. CONCLUSIONS: These results show the possibility to integrate specific in vitro tests for teratogenicity in a test strategy for developmental toxicity.     

Chondrogenic differentiation of mouse embryonic stem cells promoted by mature chondrocytes

In order to direct embryonic stem (ES) cells to differentiate into chondrocytes, a chondrogenic envi-ronment provided by mature chondrocytes was investigated. Flk-1 positive cells sorted from pre-differentiated mouse ES cells were mixed with adult porcine articular chondrocytes, seeded on biodegradable scaffolds, and then implanted subcutaneously into nude mice. The cell-scaffold com-plexes formed cartilage tissues after 4 weeks, which was demonstrated by histology and anti-type II collagen antibody staining. Positive staining of mouse Major Histocompatibility Complex class I molecules confirmed that part of the chondrocytes were derived from mouse ES cells. The current study established a new approach for directing ES cell differentiation.     

Hepatic differentiation of murine embryonic stem cells.

Murine embryonic stem (ES) cells can replicate indefinitely in culture and can give rise to all tissues, including the germline, when reimplanted into a murine blastocyst. ES cells can also be differentiated in vitro into a wide range of cell types. We have utilized a liver-specific marker to demonstrate that murine ES cells can differentiate into hepatocytes in vitro. We have used ES cells carrying a gene trap vector insertion (I.114) into an ankyrin repeat-containing gene (Gtar) that we have previously shown provides an exclusive beta-galactosidase marker for the early differentiation of hepatocytes in vivo. beta-Galactosidase-positive cells were differentiated from I.114 ES cells in vitro. The identity of these cells was confirmed by the expression of the proteins alpha-fetoprotein, albumin, and transferrin and by the fact that they have an ultrastructural appearance consistent with that of embryonic hepatocytes. We propose that this model system of hepatic differentiation in vitro could be used to define factors that are involved in specification of the hepatocyte lineage. In addition, human ES cells have recently been derived and it has been proposed that they may provide a source of differentiated cell types for cell replacement therapies in the treatment of a variety of diseases.     

Self-renewal and differentiation of mouse embryonic stem cells as measured by Oct4 gene expression: Effects of lif,serum-free medium,retinoic acid,and dbcAMP

Summary In this study we examined the interplay between serum, leukemia inhibitory factor (LIF), retinoic acid, and dibutyrl cyclic adenosine monophosphate (dbcAMP) in affecting IOUD2 embryonic stem cell self-renewal and differentiation as assessed by Oct4 expression, and cell proliferation as measured by total cell protein. Removal of LIF, reduced levels of fetal calf serum (FCS), and addition of retinoic acid all induced embryonic stem cell differentiation as measured by reduced Oct4 expression. Lower levels of retinoic acid (0.1–10 nM) promoted the formation of epithelial-like cells, whereas higher levels (100–10,000 nM) favored differentiation into fibroblastic-like cells. The effects of dbcAMP varied with the presence or absence of FCS and LIF and the concentration of dbcAMP. In FCS-containing media, a low level of dbcAMP (100 μM) increased self-renewal in the absence of LIF, but it had no effect in its presence. In contrast, at higher concentrations (1000 μM dbcAMP), regardless of LIF, differentiation was promoted. A similar effect of dbcAMP was seen in the presence of retinoic acid. In media without FCS but with serum replacement supplements, there was no effect of dbcAMP. This study shows that the Oct4 expression system of IOUD2 cells provides a novel, simple method for quantifying cellular differentiation.     

Conserved and divergent paths that regulate self-renewal in mouse and human embryonic stem cells

The past few years have seen remarkable progress in our understanding of embryonic stem cell (ES cell) biology. The necessity of examining human ES cells in culture, coupled with the wealth of genomic data and the multiplicity of cell lines available, has enabled researchers to identify critical conserved pathways regulating self-renewal and identify markers that tightly correlate with the ES cell state. Comparison across species has suggested additional pathways likely to be important in long-term self-renewal of ES cells including heterochronic genes, microRNAs, genes involved in telomeric regulation, and polycomb repressors. In this review, we have discussed information on molecules known to be important in ES cell self-renewal or blastocyst development and highlighted known differences between mouse and human ES cells. We suggest that several additional pathways required for self-renewal remain to be discovered and these likely include genes involved in antisense regulation, microRNAs, as well as additional global repressive pathways and novel genes. We suggest that cross species comparisons using large-scale genomic analysis tools are likely to reveal conserved and divergent paths required for ES cell self-renewal and will allow us to derive ES lines from species and strains where this has been difficult.     

Combined activin A/LiCl/Noggin treatment improves production of mouse embryonic stem cell-derived definitive endoderm cells

Induction of definitive endoderm (DE) cells is a prerequisite for the whole process of embryonic stem (ES) cells differentiating into hepatic or pancreatic progenitor cells. We have established an efficient method to induce mouse ES cell-derived DE cells in suspension embryonic body (EB) culture. Similar to previous studies, mouse ES cell-derived DE cells, which were defined as Cxcr4(+) c-Kit(+) , Cxcr4(+) E-cadherin(+) cells or Cxcr4(+) PDGFRa(-) cells, could be induced in the serum-free EBs at Day 4 of induction. The activations of Wnt, Nodal, and FGF signaling pathways in differentiating EBs promoted DE cell differentiation, while activation of BMP4 signaling inhibited the process. In the present study, we found that chemical activation of canonical Wnt signaling pathway by LiCl could synergize with Activin A-mediated Nodal signaling pathway to promote induction of DE cells, and inhibition of Bmp4 signaling by Noggin along with Activin A/LiCl further improved the efficiency of DE cell differentiation. The derived DE cells were proved for their capacities to become hepatic progenitor cells or pancreatic progenitor cells. In conclusion, we significantly improved the efficiency of generating mouse ES cell-derived DE cells by combined Activin A/LiCl/Noggin treatment. Our work will be greatly helpful to generate ES cell-derived hepatic cells and ES cell-derived pancreatic cells for future regenerative medicine.     

Chondrocytes derived from mouse embryonic stem cells

Our knowledge of cellular differentiation processes during chondro- and osteogenesis, in particular the complex interaction of differentiation factors, is still limited. We used the model system of embryonic stem (ES) cell differentiation in vitro via cellular aggregates, so called embryoid bodies (EBs), to analyze chondrogenic and osteogenic differentiation. ES cells differentiated into chondrocytes and osteocytes throughout a series of developmental stages resembling cellular differentiation events during skeletal development in vivo. A lineage from pluripotent ES cells via mesenchymal, prechondrogenic cells, chondrocytes and hypertrophicchondrocytes up to osteogenic cells was characterized. Furthermore, we found evidence for another osteogenic lineage, bypassing the chondrogenic stage. Together our results suggest that this in vitro system will be helpful to answer so far unacknowledged questions regarding chondrogenic and osteogenic differentiation. For example, we isolated an as yet unknown cDNA fragment from ES cell-derived chondrocytes, which showed a developmentally regulated expression pattern during EB differentiation. Considering ES cell differentiation as an alternative approach for cellular therapy, we used two different methods to obtain pure chondrocyte cultures from the heterogenous EBs. First, members of the transforming growth factor (TGF)-β family were applied and found to modulate chondrogenic differentiation but were not effective enough to produce sufficient amounts of chondrocytes. Second, chondrocytes were isolated from EBs by micro-manipulation. These cells initially showed dedifferentiation into fiboblastoid cells in culture, but later redifferentiated into mature chondrocytes. However, a small amount of chondrocytes isolated from EBs transdifferentiated into other mesenchymal cell types, indicating that chondrocytes derived from ES cells posses a distinct differentiation plasticity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Platelets generated from human embryonic stem cells are functional in vitro and in the microcirculation of living mice

Platelets play an essential role in hemostasis and atherothrombosis. Owing to their short storage time, there is constant demand for this life-saving blood component. In this study, we report that it is feasible to generate functional megakaryocytes and platelets from human embryonic stem cells (hESCs) on a large scale. Differential-interference contrast and electron microscopy analyses showed that ultrastructural and morphological features of hESC-derived platelets were indistinguishable from those of normal blood platelets. In functional assays, hESC-derived platelets responded to thrombin stimulation, formed microaggregates, and facilitated clot formation/retraction in vitro. Live cell microscopy demonstrated that hESC-platelets formed lamellipodia and filopodia in response to thrombin activation, and tethered to each other as observed in normal blood. Using real-time intravital imaging with high-speed video microscopy, we have also shown that hESC-derived platelets contribute to developing thrombi at sites of laser-induced vascular injury in mice, providing the first evidence for in vivo functionality of hESC-derived platelets. These results represent an important step toward generating an unlimited supply of platelets for transfusion. Since platelets contain no genetic material, they are ideal candidates for early clinical translation involving human pluripotent stem cells.