Naoko Hida

Novel Cardiac Precursor‐Like Cells from Human Menstrual Blood‐Derived Mesenchymal Cells

Stem cell therapy can help repair damaged heart tissue. Yet many of the suitable cells currently identified for human use are difficult to obtain and involve invasive procedures. In our search for novel stem cells with a higher cardiomyogenic potential than those available from bone marrow, we discovered that potent cardiac precursor‐like cells can be harvested from human menstrual blood. This represents a new, noninvasive, and potent source of cardiac stem cell therapeutic material. We demonstrate that menstrual blood‐derived mesenchymal cells (MMCs) began beating spontaneously after induction, exhibiting cardiomyocyte‐specific action potentials. Cardiac troponin‐I‐positive cardiomyocytes accounted for 27%–32% of the MMCs in vitro. The MMCs proliferated, on average, 28 generations without affecting cardiomyogenic transdifferentiation ability, and expressed mRNA of GATA‐4 before cardiomyogenic induction. Hypothesizing that the majority of cardiomyogenic cells in MMCs originated from detached uterine endometrial glands, we established monoclonal endometrial gland‐derived mesenchymal cells (EMCs), 76%–97% of which transdifferentiated into cardiac cells in vitro. Both EMCs and MMCs were positive for CD29, CD105 and negative for CD34, CD45. EMCs engrafted onto a recipients heart using a novel 3‐dimensional EMC cell sheet manipulation transdifferentiated into cardiac tissue layer in vivo. Transplanted MMCs also significantly restored impaired cardiac function, decreasing the myocardial infarction (MI) area in the nude rat model, with tissue of MMC‐derived cardiomyocytes observed in the MI area in vivo. Thus, MMCs appear to be a potential novel, easily accessible source of material for cardiac stem cell‐based therapy.

Xenografted Human Amniotic Membrane–Derived Mesenchymal Stem Cells Are Immunologically Tolerated and Transdifferentiated Into Cardiomyocytes

Rationale: Amniotic membrane is known to have the ability to transdifferentiate into multiple organs and is expected to stimulate a reduced immunologic reaction. Objective: Determine whether human amniotic membrane–derived mesenchymal cells (hAMCs) can be an ideal allograftable stem cell source for cardiac regenerative medicine. Methods and Results: We established hAMCs. After cardiomyogenic induction in vitro, hAMCs beat spontaneously, and the calculated cardiomyogenic transdifferentiation efficiency was 33%. Transplantation of hAMCs 2 weeks after myocardial infarction improved impaired left ventricular fractional shortening measured by echocardiogram (34±2% [n=8] to 39±2% [n=11]; P<0.05) and decreased myocardial fibrosis area (18±1% [n=9] to 13±1% [n=10]; P<0.05), significantly. Furthermore hAMCs transplanted into the infarcted myocardium of Wistar rats were transdifferentiated into cardiomyocytes in situ and survived for more than 4 weeks after the transplantation without using any immunosuppressant. Immunologic tolerance was caused by the hAMC-derived HLA-G expression, lack of MHC expression of hAMCs, and activation of FOXP3-positive regulatory T cells. Administration of IL-10 or progesterone, which is known to play an important role in feto-maternal tolerance during pregnancy, markedly increased HLA-G expression in hAMCs in vitro and, surprisingly, also increased cardiomyogenic transdifferentiation efficiency in vitro and in vivo. Conclusions: Because hAMCs have a high ability to transdifferentiate into cardiomyocytes and to acquire immunologic tolerance in vivo, they can be a promising cellular source for allograftable stem cells for cardiac regenerative medicine.

The Significant Cardiomyogenic Potential of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells In Vitro

We tested the cardiomyogenic potential of the human umbilical cord blood‐derived mesenchymal stem cells (UCBMSCs). Both the number and function of stem cells may be depressed in senile patients with severe coronary risk factors. Therefore, stem cells obtained from such patients may not function well. For this reason, UCBMSCs are potentially a new cell source for stem cell‐based therapy, since such cells can be obtained from younger populations and are being routinely utilized for clinical patients. The human UCBMSCs (5 × 103 per cm2) were cocultured with fetal murine cardiomyocytes ([CM] 1 × 105 per cm2). On day 5 of cocultivation, approximately half of the green fluorescent protein (GFP)‐labeled UCBMSCs contracted rhythmically and synchronously, suggesting the presence of electrical communication between the UCBMSCs. The fractional shortening of the contracted UCBMSCs was 6.5% ± 0.7% (n = 20). The UCBMSC‐derived cardiomyocytes stained positive for cardiac troponin‐I (clear striation +) and connexin 43 (diffuse dot‐like staining at the margin of the cell) by the immunocytochemical method. Cardiac troponin‐I positive cardiomyocytes accounted for 45% ± 3% of GFP‐labeled UCBMSCs. The cardiomyocyte‐specific long action potential duration (186 ± 12 milliseconds) was recorded with a glass microelectrode from the GFP‐labeled UCBMSCs. CM were observed in UCBMSCs, which were cocultivated in the same dish with mouse cardiomyocytes separated by a collagen membrane. Cell fusion, therefore, was not a major cause of CM in the UCBMSCs. Approximately half of the human UCBMSCs were successfully transdifferentiated into cardiomyocytes in vitro. UCBMSCs can be a promising cellular source for cardiac stem cell‐based therapy.

Can the life span of human marrow stromal cells be prolonged by bmi‐1, E6, E7, and/or telomerase without affecting cardiomyogenic differentiation?

Cell transplantation has recently been challenged to improve cardiac function of severe heart failure. Human mesenchymal stem cells (hMSCs) are multipotent cells that can be isolated from adult marrow stroma, but because of their limited life span, it is difficult to study them further. To overcome this problem, we attempted to prolong the life span of hMSCs and investigate whether the hMSCs modified with cell‐cycle‐associated genes can differentiate into cardiomyocytes in vitro.

Treatment of human mesenchymal stem cells with angiotensin receptor blocker improved efficiency of cardiomyogenic transdifferentiation and improved cardiac function via angiogenesis.

To improve the modest efficacy of mesenchymal stem cell (MSC) transplantation, the treatment of human MSCs with angiotensin receptor blockers (ARBs) was investigated. MSCs were cultured with or without the medium containing 3 μmol/l of ARBs before cardiomyogenic induction. After cardiomyogenic induction in vitro, cardiomyogenic transdifferentiation efficiency (CTE) was calculated by immunocytochemistry using anticardiac troponin‐I antibody. In the nude rat chronic myocardial infarction model, we injected MSCs pretreated with candesartan (A‐BM; n = 18) or injected MSCs without pretreatment of candesartan (BM; n = 25), each having survived for 2 weeks. The left ventricular function, as measured by echocardiogram, was compared with cardiomyogenic transdifferentiation in vivo, as determined by immunohistochemistry. Pretreatment with ARBs significantly increased the CTE in vitro (10.1 ± 0.8 n = 12 vs. 4.6 ± 0.3% n = 25, p < .05). Transplantation of candesartan‐pretreated MSCs significantly improved the change in left ventricular ejection fraction (BM; −7.2 ± 2.0 vs. A‐BM; 3.3 ± 2.3%). Immunohistochemistry revealed significant improvement of cardiomyogenic transdifferentiation in A‐BM in vivo (BM; 0 ± 0 vs. A‐BM; 0.014 ± 0.006%). Transplantation of ARB‐pretreated MSCs significantly improved cardiac function and can be a promising cardiac stem cell source from which to expect cardiomyogenesis. STEM CELLS 2011;29:1405–1414

Pretreatment of Human Mesenchymal Stem Cells with Pioglitazone Improved Efficiency of Cardiomyogenic Transdifferentiation and Cardiac Function

The efficacy of transplantation of default human marrow‐derived mesenchymal stem cells (MSCs) was modest. In this study, our challenge was to improve the efficacy of MSC transplantation in vivo by pretreatment of MSCs with pioglitazone. MSCs were cultured with or without medium containing 1 μM of pioglitazone before cardiomyogenic induction. After cardiomyogenic induction in vitro, cardiomyogenic transdifferentiation efficiency (CTE) was calculated by immunocytochemistry using anti‐cardiac troponin‐I antibody. For the in vivo experiments, myocardial infarction (MI) at the anterior left ventricle was made in nude rats. Two weeks after MI, MSCs pretreated with pioglitazone (p‐BM; n = 30) or without pioglitazone (BM; n = 17) were injected, and then survived for 2 weeks. We compared left ventricular function by echocardiogram and immunohistochemistry to observe cardiomyogenic transdifferentiation in vivo. Pretreatment with pioglitazone significantly increased the CTE in vitro (1.9% ± 0.2% n = 47 vs. 39.5% ± 4.7% n = 13, p < .05). Transplantation of pioglitazone pretreated MSCs significantly improved change in left ventricular % fractional shortening (BM; −4.8% ± 2.1%, vs. p‐BM; 5.2% ± 1.5%). Immunohistochemistry revealed significant improvement of cardiomyogenic transdifferentiation in p‐BM in vivo (BM; 0% ± 0% n = 5, vs. p‐BM; 0.077% ± 0.041% n = 5). Transplantation of pioglitazone‐pretreated MSCs significantly improved cardiac function and can be a promising cardiac stem cell source to expect cardiomyogenesis. STEM CELLS 2011; 29:357–366

Serum‐independent Cardiomyogenic Transdifferentiation in Human Endometrium‐derived Mesenchymal Cells

Media with high concentrations of serum are commonly used to induce cardiomyogenic transdifferentiation in mesenchymal stem cells; however, serum contains numerous unknown growth factors and interferes with definition of specific cardiomyogenic transdifferentiation factors secreted from feeder cells. In the present study, we determined whether the transdifferentiation of human mesenchymal cells can be observed in a FBS-free medium. The efficiency of transdifferentiation was observed in 10% FBS-containing standard medium (10%FBS) and in FBS-free medium containing insulin and thyroxin (FBS-free). In the present study, we used human uterine endometrium-derived mesenchymal cells (EMC100, EMC214) and menstrual blood-derived mesenchymal cells (MMCs). After cardiomyogenic transdifferentiation, the efficiency and physiological properties of cardiomyogenesis (fractional shortening of the cell [%FS] and action potential [AP]) were evaluated. The efficiency of transdifferentiation in EMC100 and in MMCs increased 36%* and 163%* (*P < 0.05), respectively. The %FS in EMCs increased to 103%*. AP-duration more than 250 ms with a marked plateau was only observed in FBS-free (3/19), and not in 10% FBS (0/41). The cardiomyogenic transdifferentiation of human mesenchymal cells can be observed in the FBS-free medium. Phenotypes of generated cardiomyocytes were significantly more physiological in FBS-free than in 10% FBS.

Epigenetic mark sequence of the H19 gene in human sperm.

We have investigated the epigenetic mark in the human H19 gene. The H19 promoter is methylation-free in human sperm, but it is methylated in the paternally derived allele of most adult tissues. Consequently, the H19 gene is exclusively transcribed from the maternal allele. It was demonstrated that the differentially methylated region (DMR) located 2 kb upstream from mouse H19 is essential for the imprinting of H19. A 39 bp sequence in DMR has a high degree of similarity between humans, mice and rats. The highly conserved 15 bp core region of the consensus sequence contains four methylatable sites, and thus has been proposed as a potential imprinting mark region. In this study, fine epigenetic sequencing analysis was performed on the sperm DNA in comparison with other adult organs. Interestingly, the conserved sequence of the potential mark region was methylated in almost all the sperm genomes analyzed. Furthermore, the single dinucleotide CpG, whose methylation affects the accessibility of the element to CTCF, was methylated in the conserved core in the human sperm. These results suggest that the human core sequences may act as an imprinting center in the reciprocal monoallelic expression of H19.

Magnetic resonance monitoring of superparamagnetic iron oxide (SPIO)-labeled stem cells transplanted into the inner ear

In the field of regenerative medicine, cell transplantation or cell-based therapies for inner ear defects are considered to be promising candidates for a therapeutic strategy. In this paper, we report on a study that examined the use of magnetic resonance imaging (MRI) to monitor stem cells transplanted into the cochlea labeled with superparamagnetic iron oxide (SPIO), a contrast agent commonly used with MRI. First, we demonstrated in vitro that stem cells efficiently took up SPIO particles. This was confirmed by Prussian blue staining and TEM. In MRI studies, T2 relaxation times of SPIO-labeled cells decreased in a dose-dependent manner. Next, we transplanted SPIO-labeled cells directly into the cochlea in vivo and then performed MRI 1h, 2 weeks, and 4 weeks after transplantation. The images were evaluated objectively by measuring signal intensity (SI). SI within the ears receiving transplants was significantly lower (P<0.05) than that of control sides at the 1-h assessment. This novel method will be helpful for evaluating stem cell therapies, which represents a new strategy for inner ear regeneration. To the best of our knowledge, this study is the first to demonstrate that local transplantation of labeled stem cells into the inner ear can be visualized in vivo via MRI.

CD34 and CD49f Double-Positive and Lineage Marker-Negative Cells Isolated from Human Myometrium Exhibit Stem Cell-Like Properties Involved in Pregnancy-Induced Uterine Remodeling

ABSTRACT Repeated and dramatic pregnancy-induced uterine enlargement and remodeling throughout reproductive life suggests the existence of uterine smooth muscle stem/progenitor cells. The aim of this study was to isolate and characterize stem/progenitor-like cells from human myometrium through identification of specific surface markers. We here identify CD49f and CD34 as markers to permit selection of the stem/progenitor cell-like population from human myometrium and show that human CD45– CD31– glycophorin A– and CD49f+ CD34+ myometrial cells exhibit stem cell-like properties. These include side population phenotypes, an undifferentiated status, high colony-forming ability, multilineage differentiation into smooth muscle cells, osteoblasts, adipocytes, and chondrocytes, and in vivo myometrial tissue reconstitution following xenotransplantation. Furthermore, CD45– CD31– glycophorin A– and CD49f+ CD34+ myometrial cells proliferate under hypoxic conditions in vitro and, compared with the untreated nonpregnant myometrium, show greater expansion in the estrogen-treated nonpregnant myometrium and further in the pregnant myometrium in mice upon xenotransplantation. These results suggest that the newly identified myometrial stem/progenitor-like cells influenced by hypoxia and sex steroids may participate in pregnancy-induced uterine enlargement and remodeling, providing novel insights into human myometrial physiology.