In vitro differentiation of mouse ES cells into hepatocytes with coagulation factors VIII and IX expression profiles

Coagulation factors II, V, VII, VIII, IX and X are produced by hepatocytes. So factors VIII and IX deficiencies, which result in hemophilia A and B, have the potential to respond to cellular re- place-ment therapy. Embryonic stem (ES) cells provide a unique source for therapeutic applications. Here, E14 mouse ES cells have been induced into hepatocytes in vitro. Morphology revealed that ES-derived hepatic-like cells were round or polyhedral shaped with distinct boundary of individual cells, and some arranged in trabeculae. These cells expressed endodermal- or liver-specific mRNA ——transthyretin (TTR), α1-anti-trypsin (AAT), α-fetoprotein (AFP), albumin (ALB), glucose-6- phoshpatase (G6P) and tyrosine aminotransferase (TAT). Approximately (85.1±0.5)% of the ES-de- rived cells was stained positive green with ICG uptake. These cells were also stained magenta as a result of PAS reaction. In this paper, expression of coagulation factors VIII and IX mRNA in the ES-derived cells is documented. Therefore, ES cells might be developed as substitute donor cells for the therapy of coagulation factor deficiencies.     

In vitro differentiation of mouse ES cells into hepatocytes with coagulation factors VIII and IX expression profiles

Coagulation factors II, V, VII, VIII, IX and X are produced by hepatocytes. So factors VIII and IX deficiencies, which result in hemophilia A and B, have the potential to respond to cellular replacement therapy. Embryonic stem (ES) cells provide a unique source for therapeutic applications. Here, E14 mouse ES cells have been induced into hepatocytes in vitro. Morphology revealed that ES-derived hepatic-like cells were round or polyhedral shaped with distinct boundary of individual cells, and some arranged in trabeculae. These cells expressed endodermal-or liver-specific mRNA—transthyretin (TTR), α1-anti-trypsin (AAT), α-fetoprotein (AFP), albumin (ALB), glucose-6-phoshpatase (G6P) and tyrosine aminotransferase (TAT). Approximately (85.1±0.5)% of the ES-derived cells was stained positive green with ICG uptake. These cells were also stained magenta as a result of PAS reaction. In this paper, expression of coagulation factors VIII and IX mRNA in the ES-derived cells is documented. Therefore, ES cells might be developed as substitute donor cells for the therapy of coagulation factor deficiencies.     

Differential expression of the Wnt and Frizzled genes in Flk1+ cells derived from mouse ES cells

Embryonic stem (ES) cells have the potential to develop into various cell lineages including hemangioblasts (Flk1+), a common progenitor for hematopoietic and vascular endothelial cells. Previous studies indicate that Flk1+ cells, a marker for hemangioblast, can be derived from ES cell and that Flk1+ can be differentiated into hematopoietic or endothelial cells depending on culture conditions. We developed an improved in vitro system to generate Flk1+-enriched cultures from mouse ES cells and used this in vitro system to study the role of Wnt signalling in early endothelial progenitor cells. We determined the expression of the Wnt and Frizzled genes in Flk1+ cells derived from mouse ES cells. RT-PCR analyses identified significantly higher expression of non-canonical Wnt5a and Wnt11 genes in Flk1+ cells compared to Flk1- cells. In contrast, expression of canonical Wnt3a gene was reduced in Flk1+ cells. In addition, Frizzled2, Frizzled5 and Frizzled7 genes were also expressed at a higher level in Flk1+ cells. The differential expression of Wnt and Frizzled genes in Flk1+ cells provides a novel insight into the role of non-canonical Wnt signalling in vascular endothelial fate determination.     

源于129S1品系的小鼠胚胎干细胞系的建立及其生物学特性分析

从129S1小鼠早期胚胎的内细胞团分离、培养类胚胎样细胞,经反复传代,成功地建立了129S1小鼠胚胎干细胞系,命名为NM-2细胞系。形态学鉴定具有胚胎干细胞的典型形态特征,正常核型率为80％;呈碱性磷酸酶阳性、表达胚胎干细胞特异性转录因子OCT-4;体内分化后可形成源于三胚层的组织结构;经囊胚腔显微注射后所获得的子代个体中79％具有毛色嵌合表型;雄性嵌合个体中31％发生生殖腺嵌合;同时,通过育种观察到所有生殖腺嵌合体的子代小鼠表型正常。以上结果证实NM-2细胞系为一株具高生殖腺嵌合能力的小鼠胚胎干细胞系。     

DNA damage response by single-strand breaks in terminally differentiated muscle cells and the control of muscle integrity

DNA single-strand breaks (SSB) formation coordinates the myogenic program, and defects in SSB repair in post-mitotic cells have been associated with human diseases. However, the DNA damage response by SSB in terminally differentiated cells has not been explored yet. Here we show that mouse post-mitotic muscle cells accumulate SSB after alkylation damage, but they are extraordinarily resistant to the killing effects of a variety of SSB-inducers. We demonstrate that, upon SSB induction, phosphorylation of H2AX occurs in myotubes and is largely ataxia telangiectasia mutated (ATM)-dependent. However, the DNA damage signaling cascade downstream of ATM is defective as shown by lack of p53 increase and phosphorylation at serine 18 (human serine 15). The stabilization of p53 by nutlin-3 was ineffective in activating the cell death pathway, indicating that the resistance to SSB inducers is due to defective p53 downstream signaling. The induction of specific types of damage is required to activate the cell death program in myotubes. Besides the topoisomerase inhibitor doxorubicin known for its cardiotoxicity, we show that the mitochondria-specific inhibitor menadione is able to activate p53 and to kill effectively myotubes. Cell killing is p53-dependent as demonstrated by full protection of myotubes lacking p53, but there is a restriction of p53-activated genes. This new information may have important therapeutic implications in the prevention of muscle cell toxicity.     

Generation of hybrid hepatocytes by cell fusion from monkey embryoid body cells in the injured mouse liver

Monkey embryonic stem (ES) cells have characteristics that are similar to human ES cells, and might be useful as a substitute model for preclinical research. When embryoid bodies (EBs) formed from monkey ES cells were cultured, expression of many hepatocyte-related genes including cytochrome P450 (Cyp) 3a and Cyp7a1 was observed. Hepatocytes were immunocytochemically observed using antibodies against albumin (ALB), cytokeratin-8/18, and α1-antitrypsin in the developing EBs. The in vitro differentiation potential of monkey ES cells into the hepatic lineage prompted us to examine the transplantability of monkey EB cells. As an initial approach to assess the repopulation potential, we transplanted EB cells into immunodeficient urokinase-type plasminogen activator transgenic mice that undergo liver failure. After transplantation, the hepatocyte colonies expressing monkey ALB were observed in the mouse liver. Fluorescence in-situ hybridization revealed that the repopulating hepatocytes arise from cell fusion between transplanted monkey EB cells and recipient mouse hepatocytes. In contrast, neither cell fusion nor repopulation of hepatocytes was observed in the recipient liver after undifferentiated ES cell transplantation. These results indicate that the differentiated cells in developing monkey EBs, but not contaminating ES cells, generate functional hepatocytes by cell fusion with recipient mouse hepatocytes, and repopulate injured mouse liver.     

The specific binding of peptide ligands to cardiomyocytes derived from mouse embryonic stem cells

Purification of pluripotent stem cell (PSC)‐derived cardiomyocytes is critical for the application of cardiomyocytes both in clinical and basic research. Finding a specific cell marker is a promising method for purifying induced cells. The present study employed phage display technology to search for particular cell markers that could bind specifically to PSC‐derived cardiomyocytes. After three rounds of biopanning, several peptides were obtained. The ELISA results show the no. 3 sequence peptide (QPFTTSLTPPAR), and other four sequences having a consensus motif [SS(Q)PPQ(S)], no. 9, 11, 14, and 10, have relatively high affinity and specificity to cardiomyocytes. Immunofluorescence confirmed that the selected peptides could bind specifically to the PSC‐derived cardiomyocytes. Competition tests with chemically synthesized peptides revealed the binding ability was caused by the peptide itself. Western blot analysis proved the phages were both bound to two 17 kDa cardiomyocyte membrane proteins and the no. 9 sequence showed a 55 kDa protein that was not observed in the no. 3 sequence. These results suggest that the selected peptides specifically target receptors on PSC‐derived cardiomyocyte membranes. The results will pave the way for further studies of cell surface markers and their applications, such as labeling, purification, and as vehicles for drug delivery. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Generation of hematopoietic cells from mouse pluripotent stem cells in a 3D culture system of self-assembling peptide hydrogel

In vitro generation of hematopoietic stem cells from pluripotent stem cells (PSCs) can be regarded as novel therapeutic approaches for replacing bone marrow transplantation without immune rejection or graft versus host disease. To date, many different approaches have been evaluated in terms of directing PSCs toward different hematopoietic cell types, yet, low efficiency and no function restrict the further hematopoietic differentiation study, our research aims to develop a three dimension (3D) hematopoietic differentiation approach that serves as recapitulation of embryonic development in vitro to a degree of complexity not achievable in a two dimension culture system. We first found that mouse PSCs could be efficiently induced to hematopoietic differentiation with an expression of hematopoietic makers, such as c-kit, CD41, and CD45 within self-assembling peptide hydrogel. Colony-forming cells assay results suggested mouse PSCs (mPSCs) could be differentiated into multipotential progenitor cells and 3D induction system derived hematopoietic colonies owned potential of differentiating into lymphocyte cells. In addition, in vivo animal transplantation experiment showed that mPSCs (CD45.2) could be embedded into nonobese diabetic/severe combined immunodeficiency mice (CD45.1) with about 3% engraftment efficiency after 3 weeks transplantation. This study demonstrated that we developed the 3D induction approach that could efficiently promote the hematopoietic differentiation of mPSCs in vitro and obtained the multipotential progenitors that possessed the short-term engraftment potential.     

Pax7 reveals a greater frequency and concentration of satellite cells at the ends of growing skeletal muscle fibers.

The main sites of longitudinal growth in skeletal muscle are the ends of the fibers. This study tests the hypothesis that satellite cells (SCs) are at a greater frequency (#SC nuclei/all nuclei within basal laminae) and concentration (closer together) within growing fiber ends of posthatch chicken pectoralis. SCs were localized by their Pax7 expression, and fiber ends were identified by their retention of neonatal myosin heavy chains and small cross-sectional profiles. Whereas SC frequency decreased from about 20% at 9 days posthatch to <5% at 115 days, fiber ends retained a frequency of approximately 16%. Calculated mean area of sarcolemma per SC revealed higher concentrations of SCs at fiber ends. There was also a strong inverse correlation between SC frequency and fiber profile cross-sectional size throughout development. This study suggests that SCs at fiber ends play a key role in the longitudinal growth of muscle fibers, and that fiber profile size may impact SC distribution.     

Derivation and induction of the differentiation of animal ES cells as well as human pluripotent stem cells derived from fetal membrane

We succeeded in the derivation and maintenance of pluripotent embryonic stem (ES) cells from equine and bovine blastocysts. These cells expressed markers that are characteristics of mouse ES cells, namely, alkaline phosphatase, stage-specific embryonic antigen 1, STAT 3 and Oct 4. We confirmed the pluripotential ability of these cells, which were able to undergo somatic differentiation in vitro to neural progenitors and to endothelial or hematopoietic lineages. We were able to use bovine ES cells as a source of nuclei for nuclear transfer and we generated cloned cattle with a higher frequency of pregnancies to term than has been achieved with somatic cells. On the other hand, we established human fetal membrane derived stem cell lines by the colonial cloning techniques using MEMalpha culture medium containing 10 ng/ml of EGF, 10 ng/ml of LIF and 10% fetal bovine serum (FBS). These cells appeared to maintain normal karyotype in vitro and expressed markers characteristics of stem cells. Furthermore, these cells contributed to the formation of chimeric murine embryoid bodies and gave rise to all three germ layers in vitro. Results from animal ES cells and human fetal membrane derived stem cells clearly demonstrate that these cells might be used for providing different types of cells for regenerative medicine as well as used for targeted genetic manipulation of the genome.     

Generation of a novel germline stem cell line expressing a germline‐specific reporter in the mouse

Germline stem (GS) cells are stem cell lines derived from postnatal male germline cells. Remarkably, GS cells can form functional spermatozoa when transplanted into infertile host mouse testes, indicating that GS cells have spermatogonial stem cell (SSC) activity. As GS cells are the only type with SSC activity, they are most suitable for in vitro studies on male germ cell differentiation. However, GS cells can deviate from the germ cell state to become other types of cells, depending on culture conditions. Therefore, it is desirable to have a monitor system to ensure that GS cells are kept at the germ cell state in culture. Here, we established GS cell lines from neonatal testes of transgenic mice that express the fluorescent protein, Venus, whose gene expression is driven by the promoter of Mvh (mouse Vasa homolog), a gene highly specific to mammalian germ cells. This novel cell line has genuine GS cell properties equivalent to existing GS lines, including the ability to generate viable offspring. This Mvh–Venus GS cell line, to our knowledge, is the first one expressing a germ cell‐specific reporter. This valuable resource should provide new opportunities for studies on male germ cell differentiation. genesis 51:498–505. © 2013 Wiley Periodicals, Inc.     

Isolation and enrichment of skeletal muscle progenitor cells from mouse bone marrow

There is great interest in the therapeutic potential of non-hematopoietic stem cells obtained from bone marrow called mesenchymal stem cells (MSCs). Rare myogenic progenitor cells in MSC cultures have been shown to convert into skeletal muscle cells in vitro and also in vivo after transplantation of bone marrow into mice. To be clinically useful, however, isolation and expansion of myogenic progenitor cells is important to improve the efficacy of cell transplantation in generating normal skeletal muscle cells. We introduced into MSCs obtained from mouse bone marrow, a plasmid vector in which an antibiotic (Zeocin) resistance gene is driven by MyoD and Myf5 enhancer elements, which are selectively active in skeletal muscle progenitor cells. Myogenic precursor cells were then isolated by antibiotic selection, expanded in culture, and shown to differentiate appropriately into multinucleate myotubes in vitro. Our results show that using a genetic selection strategy, an enriched population of myogenic progenitor cells, which will be useful for cell transplantation therapies, can be isolated from MSCs.     

Inhibition of gut pacemaker cell formation from mouse ES cells by the c-kit inhibitor

Using an embryoid body (EB) culture system, we developed a functional organ-like cluster, a "gut", from mouse embryonic stem (ES) cells (ES gut). Each ES gut exhibited various types of spontaneous movements. In these spontaneously contracting ES guts, dense distributions of interstitial cells of Cajal (ICC) (c-kit, a transmembrane receptor that has tyrosine kinase activity, positive cells; gut pacemaker cells) and smooth muscle cells were discernibly identified. By adding Glivec 10(-5)M, a tyrosine kinase receptor c-kit inhibitor, only during EB formation, we for the first time succeeded in suppressing in vitro formation of ICC in the ES gut. The ES gut without ICC did not exhibit any movements. However, it appeared that Glivec 10(-6)-10(-7)M rather increased number of ES guts with spontaneous movements associated with increase of intracellular Ca(2+) concentration ([Ca(2+)](i)). These results suggest ICC is critical for in vitro formation of ES guts with spontaneous movements.     

Purification and cell-surface marker characterization of quiescent satellite cells from murine skeletal muscle by a novel monoclonal antibody

A novel monoclonal antibody, SM/C-2.6, specific for mouse muscle satellite cells was established. SM/C-2.6 detects mononucleated cells beneath the basal lamina of skeletal muscle, and the cells co-express M-cadherin. Single fiber analyses revealed that M-cadherin+ mononucleated cells attaching to muscle fibers are stained with SM/C-2.6. SM/C-2.6+ cells, which were freshly purified by FACS from mouse skeletal muscle, became MyoD+ in vitro in proliferating medium, and the cells differentiated into desmin+ and nuclear-MyoD+ myofibers in vitro when placed under differentiation conditions. When the sorted cells were injected into mdx mouse muscles, donor cells differentiated into muscle fibers. Flow cytometric analyses of SM/C-2.6+ cells showed that the quiescent satellite cells were c-kit-, Sca-1-, CD34+, and CD45-. More, SM/C-2.6+ cells were barely included in the side population but in the main population of cells in Hoechst dye efflux assay. These results suggest that SM/C-2.6 identifies and enriches quiescent satellite cells from adult mouse muscle, and that the antibody will be useful as a powerful tool for the characterization of cellular and molecular mechanisms of satellite cell activation and proliferation.     

Bridging the gap from frog research to human therapy: A tale of neural differentiation in Xenopus animal caps and human pluripotent cells

Over the last decade, much progress has been made toward an understanding of the mechanism of regulation of neural differentiation. In this article, following a brief overview of neural induction research, I would like to discuss the potential contribution of basic embryological research to the progress of human therapeutic development in the present and future, focusing on the medical application of in vitro differentiation of neural tissues. This kind of linkage between basic and medical research will probably be strengthened even more by the recent emergence of human induced pluripotent stem cells. Human pluripotent stem cells are powerful tools for bridging the gap from our accumulated knowledge of embryology to regenerative medicine, as well as to a wide spectrum of medical and pharmaceutical research and development. In this commentary, I describe these issues with a particular emphasis on the contributions made by Japanese scientists.