Shiro Baba

Generation of Pluripotent Stem Cells from Neonatal Mouse Testis

Although germline cells can form multipotential embryonic stem (ES)/embryonic germ (EG) cells, these cells can be derived only from embryonic tissues, and such multipotent cells have not been available from neonatal gonads. Here we report the successful establishment of ES-like cells from neonatal mouse testis. These ES-like cells were phenotypically similar to ES/EG cells except in their genomic imprinting pattern. They differentiated into various types of somatic cells in vitro under conditions used to induce the differentiation of ES cells and produced teratomas after inoculation into mice. Furthermore, these ES-like cells formed germline chimeras when injected into blastocysts. Thus, the capacity to form multipotent cells persists in neonatal testis. The ability to derive multipotential stem cells from the neonatal testis has important implications for germ cell biology and opens the possibility of using these cells for biotechnology and medicine.

The effects of cardioactive drugs on cardiomyocytes derived from human induced pluripotent stem cells

Developing effective drug therapies for arrhythmic diseases is hampered by the fact that the same drug can work well in some individuals but not in others. Human induced pluripotent stem (iPS) cells have been vetted as useful tools for drug screening. However, cardioactive drugs have not been shown to have the same effects on iPS cell-derived human cardiomyocytes as on embryonic stem (ES) cell-derived cardiomyocytes or human cardiomyocytes in a clinical setting. Here we show that current cardioactive drugs affect the beating frequency and contractility of iPS cell-derived cardiomyocytes in much the same way as they do ES cell-derived cardiomyocytes, and the results were compatible with empirical results in the clinic. Thus, human iPS cells could become an attractive tool to investigate the effects of cardioactive drugs at the individual level and to screen for individually tailored drugs against cardiac arrhythmic diseases.

Induced pluripotent stem cells from patients with human fibrodysplasia ossificans progressiva show increased mineralization and cartilage formation

BackgroundAbnormal activation of endochondral bone formation in soft tissues causes significant medical diseases associated with disability and pain. Hyperactive mutations in the bone morphogenetic protein (BMP) type 1 receptor ACVR1 lead to fibrodysplasia ossificans progressiva (FOP), a rare genetic disorder characterized by progressive ossification in soft tissues. However, the specific cellular mechanisms are unclear. In addition, the difficulty obtaining tissue samples from FOP patients and the limitations in mouse models of FOP hamper our ability to dissect the pathogenesis of FOP.MethodsTo address these challenges and develop a “disease model in a dish”, we created human induced pluripotent stem cells (iPS cells) derived from normal and FOP dermal fibroblasts by two separate methods, retroviral integration or integration-free episomal vectors. We tested if the ability to contribute to different steps of endochondral bone formation was different in FOP vs. control iPS cells.ResultsRemarkably, FOP iPS cells showed increased mineralization and enhanced chondrogenesis in vitro. The mineralization phenotypes could be suppressed with a small-molecule inhibitor of BMP signaling, DMH1. Our results indicate that the FOP ACVR1 R206H mutation favors chondrogenesis and increases mineral deposition in vitro.ConclusionsOur findings establish a FOP disease cell model for in vitro experimentation and provide a proof-of-concept for using human iPS cell models to understand human skeletal disorders.

Identification of cardiac stem cells with FLK1, CD31, and VE-cadherin expression during embryonic stem cell differentiation

We evaluated the expression of the FLK1, one of the lateral mesoderm early markers where cardiogenesis occurs, to characterize and isolate cardiac stem/progenitor cells from ES cells. Dissociated cells from embryoid bodies (EBs) on day 3, 4, or 5 were collected into two subpopulations with or without FLK1 expression and coculture on OP9 stromal cells was continued to examine whether contracting colonies came out or not. FLK1+ cells from EBs at days 3 and 4 formed spontaneous contracting colonies more efficiently than FLK1− cells on the same days, but not at day 5. Most contracting cardiac colonies derived from FLK1 cells mainly on day 4 were detected on endothelial cells along with hematopoietic cells. Further characterization of cells with these capabilities into three lineages revealed the FLK1+ CD31−VE‐cadherin‐ phenotype. Our findings indicate that FLK1+ cells, especially FLK1+ CD31−VE‐cadherin− cells, could act as cardiohemangioblasts to form cardiac cells as well as endothelial cells and hematopoietic cells.—Iida, M., Heike, T., Yoshimoto, M., Baba, S., Doi, H., Nakahat, T. Identification of cardiac stem cells with FLK1, CD31, and VE‐cadherin expression during embryonic stem cell differentiation. FASEB J. 19, 371–378 (2005)

Cell line-dependent differentiation of induced pluripotent stem cells into cardiomyocytes in mice

AIMS Mouse and human fibroblasts can be directly reprogrammed to pluripotency by the ectopic expression of four transcription factors (Oct3/4, Sox2, Klf4, and c-Myc) to yield induced pluripotent stem (iPS) cells. iPS cells can be generated even without the expression of c-Myc. The present study examined patterns of differentiation of mouse iPS cells into cardiomyocytes in three different cell lines reprogrammed by three or four factors. METHODS AND RESULTS During the induction of differentiation on feeder-free gelatinized dishes, genes involved in cardiogenesis were expressed as in embryonic stem cells and myogenic contraction occurred in two iPS cell lines. However, in one iPS cell line (20D17) generated by four factors, the expression of cardiac-specific genes and the beating activity were extremely low. Treating iPS cells with trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, increased Nkx2.5 expression in all iPS cell lines. While the basal Nkx2.5 expression was very low in 20D17, the TSA-induced increase was the greatest. TSA also induced the expression of contractile proteins in 20D17. Furthermore, we demonstrated the increased mRNA level of Oct3/4 and nuclear protein level of HDAC4 in 20D17 compared with the other two iPS cell lines. DNA microarray analysis identified genes whose expression is up- or down-regulated in 20D17. CONCLUSIONS Mouse iPS cells differentiate into cardiomyocytes in a cell line-dependent manner. TSA induces myocardial differentiation in mouse iPS cells and might be useful to overcome cell line variation in the differentiation efficiency.

Calcium Transients Closely Reflect Prolonged Action Potentials in iPSC Models of Inherited Cardiac Arrhythmia

Summary Long-QT syndrome mutations can cause syncope and sudden death by prolonging the cardiac action potential (AP). Ion channels affected by mutations are various, and the influences of cellular calcium cycling on LQTS cardiac events are unknown. To better understand LQTS arrhythmias, we performed current-clamp and intracellular calcium ([Ca2+]i) measurements on cardiomyocytes differentiated from patient-derived induced pluripotent stem cells (iPS-CM). In myocytes carrying an LQT2 mutation (HERG-A422T), APs and [Ca2+]i transients were prolonged in parallel. APs were abbreviated by nifedipine exposure and further lengthened upon releasing intracellularly stored Ca2+. Validating this model, control iPS-CM treated with HERG-blocking drugs recapitulated the LQT2 phenotype. In LQT3 iPS-CM, expressing NaV1.5-N406K, APs and [Ca2+]i transients were markedly prolonged. AP prolongation was sensitive to tetrodotoxin and to inhibiting Na+-Ca2+ exchange. These results suggest that LQTS mutations act partly on cytosolic Ca2+ cycling, potentially providing a basis for functionally targeted interventions regardless of the specific mutation site.

Generation of cardiac and endothelial cells from neonatal mouse testis-derived multipotent germline stem cells

Multipotent germline stem (mGS) cells have been established from neonatal mouse testes. Here, we compared mGS, embryonic stem (ES), and embryonic germ (EG) cells with regard to their ability to differentiate into mesodermal cells, namely, cardiomyocytes and endothelial cells. The in situ morphological appearances of undifferentiated mGS, ES, and EG cells were similar, and 4 days after being induced to differentiate, approximately 30%–40% of each cell type differentiated into Flk1+ cells. The sorted Flk1+ cells differentiated efficiently into cardiomyocytes and endothelial cells. By day 10 after differentiation induction, the three cell types generated equal number of endothelial colonies. However, by day 13 after differentiation induction, the Flk1+ mGS cells generated more contractile colonies than did the Flk1+ ES cells, whereas the Flk1+ EG cells generated equivalent numbers as the Flk1+ mGS cells. Reverse transcriptase polymerase chain reaction (RT‐PCR) analysis of differentiation markers such as Rex1, FGF‐5, GATA‐4, Brachyury, and Flk1 revealed that mGS cells expressed these markers more slowly during days 0–4 after differentiation induction than did ES cells, but that this mGS cell pattern was similar to that of the EG cells. RT‐PCR analysis also revealed that the three differentiation cell types expressed various cardiac markers. Moreover, immunohistochemical analysis revealed that the contractile colonies derived from Flk1+ mGS cells express mature cardiac cell‐specific markers. In conclusion, mGS cells are phenotypically similar to ES and EG cells and have a similar potential to differentiate into cardiomyocytes and endothelial cells.

Long‐Term Culture of Postnatal Mouse Hepatic Stem/Progenitor Cells and Their Relative Developmental Hierarchy

Few studies on the long‐term culture of postnatal mouse hepatic stem/progenitor cells have been reported. We successfully adapted a serum‐free culture system that we employed previously to expand fetal mouse hepatic stem/progenitor cells and maintained them in culture over long periods. The expanded postnatal cells contained immature α‐fetoprotein‐positive cells along with hepatocytic and cholangiocytic lineage‐committed cells. These cells expressed CD49f but not CD45, CD34, Thy‐1, c‐kit, CD31, or flk‐1, and oncostatin M induced their differentiation. This heterogeneous population contained side population (SP) cells, which express the ATP‐binding cassette transporter ABCG2, and sca‐1+ cells. As mice aged, the frequency of SP and sca‐1+ cells decreased along with the ability of cultured cells to expand. Approximately 20%–40% of the SP cells expressed sca‐1, but only a few sca‐1+ cells were also SP cells. Analysis of colonies derived from single SP or sca‐1+ cells revealed that, although both cells had dual differentiation potential and self‐renewal ability, SP cells formed colonies more efficiently and gave rise to SP and sca‐1+ cells, whereas sca‐1+ cells generated only sca‐1+ progeny. Thus, SP cells are more characteristic of stem cells than are sca‐1+ cells. In regenerating livers, ABCG2+ cells and sca‐1+ cells were detected around or in the portal area (the putative hepatic stem cell niche). The expanded cells share many features of fetal hepatic stem/progenitor cells or oval cells and may be useful in determining the mechanisms whereby hepatic stem cells self‐renew and differentiate.

Cyclin-dependent kinase 9 forms a complex with GATA4 and is involved in the differentiation of mouse ES cells into cardiomyocytes

The treatment of ES cells with trichostatin A (TSA), an HDAC inhibitor, induces the acetylation of GATA4 as well as histones, and facilitates their differentiation into cardiomyocytes. Recently, we demonstrated that cyclin‐dependent kinase 9 (Cdk9), a core component of positive elongation factor‐b, is a novel GATA4‐binding partner. The present study examined whether Cdk9 forms a complex with GATA4 in mouse ES cells and is involved in their differentiation into cardiomyocytes. Mouse ES cells and Nkx2.5/GFP ES cells, in which green fluorescent protein (GFP) is expressed under the control of the cardiac‐specific Nkx2.5 promoter, were induced to differentiate on feeder‐free gelatin‐coated plates. Immunoprecipitation/Western blotting in nuclear extracts from mouse ES cells demonstrated that Cdk9 as well as cyclin T1 interact with GATA4 during myocardial differentiation. TSA treatment increased Nkx2.5/GFP‐positive cells and endogenous mRNA levels of Nkx2.5 and atrial natriuretic factor. To determine the role of Cdk9 in myocardial cell differentiation, we examined the effects of a dominant‐negative form of Cdk9 (DN‐Cdk9), which loses its kinase activity, and a Cdk9 kinase inhibitor, 5,6‐dichloro‐1‐β‐ribofuranosyl‐benzimidazole (DRB) on TSA‐induced myocardial cell differentiation. The introduction of the DN‐Cdk9 inhibited TSA‐induced increase in GFP expression in Nkx2.5/GFP ES cells. The administration of DRB into ES cells significantly inhibited TSA‐induced increase of endogenous Nkx2.5 mRNA levels in ES cells as well as GFP expression in Nkx2.5/GFP ES cells. These findings demonstrate that Cdk9 is involved in the differentiation of mouse ES cells into cardiomyocytes by interacting with GATA4. J. Cell. Physiol. 226: 248–254, 2010.

Bone marrow engraftment but limited expansion of hematopoietic cells from multipotent germline stem cells derived from neonatal mouse testis

OBJECTIVE Multipotent germline stem (mGS) cells derived from neonatal mouse testis, similar to embryonic stem (ES) cells, differentiate into various types of somatic cells in vitro and produce teratomas after inoculation into mice. In the present work, we examined mGS cells for hematopoietic progenitor potential in vitro and in vivo. MATERIALS AND METHODS mGS cells were differentiated on OP9 stromal cells and induced into Flk1(+) cells. Flk1(+) cells were sorted and replated on OP9 stromal cells with various cytokines and emerging hematopoietic cells were analyzed for lineage marker expression by fluorescein-activated cell sorting, progenitor activity by colony assay, and stem cell transplantation assay. RESULTS mGS cells, like ES cells, produce hematopoietic progenitors, including both primitive and definitive erythromyeloid, megakaryocyte, and B- and T-cell lineages via Flk1(+) progenitors. When transplanted into the bone marrow (BM) of nonobese diabetic/severe combined immunodeficient (NOD/SCID) gammac(null) mice directly, mGS-derived green fluorescent protein (GFP)-positive cells were detected 4 months later in the BM and spleen. GFP(+) donor cells were also identified in the Hoechst33342 side population, a feature of hematopoietic stem cells. However, these mGS-derived hematopoietic cells did not proliferate in vivo, even after exposure to hematopoietic stressors, such as 5-fluorouracil (5FU) injection or serial transplantation. CONCLUSION mGS cells produced multipotent hematopoietic progenitor cells with myeloid and lymphoid lineage potential in vitro and localized in the BM after intra-BM injection but, like ES cells, failed to expand or show stem cell repopulating ability in vivo.