SEARCH

SEARCH BY CITATION

Keywords:

  • Cardiomyogenesis human mesenchymal stem cell;
  • Menstrual blood endometrial gland;
  • Cell sheet technology cardiac precursors

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Summary
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

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 recipient's 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.

Disclosure of potential conflicts of interest is found at the end of this article.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Summary
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

Author contributions: N.H.: conception and design, collection and assembly of data, data analysis and interpretation, final approval of manuscript; N.N.: conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; S.M.: conception and design, administrative support, collection and assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; S. Kira and Y.I.: collection and assembly of data, final approval of manuscript; K.S., C.C., T.K., S. Kyo, and T.S.: provision of study material, final approval of manuscript; T.U.: provision of study material, collection and assembly of data, final approval of manuscript; T.M.: collection and assembly of data, data analysis and interpretation, final approval of manuscript; K.M.: collection and assembly of data, final approval of manuscript; T.O.: administrative support, provision of study material, final approval of manuscript; M.S.: administrative support, final approval of manuscript; S.O.: financial support, administrative support, final approval of manuscript; A.U.: financial support, administrative support, manuscript writing, final approval of manuscript.

Marrow-derived mesenchymal stem cells (MSCs) are a potential cellular source for stem cell-based therapy, since they have the ability to differentiate into cardiomyocytes [1, 2], use of MSCs presents no ethical problems, and autologous MSCs have been injected into ischemic hearts clinically [3]. Direct injection of MSCs into the heart has been shown to be feasible in vivo [4, [5], [6]7], but with limited effect. The reason for this may be the extremely low rate of cardiomyogenesis exhibited by marrow-derived MSCs [2], with cardiac function improvement due to grafted MSC-induced neovascularization [7, 8] and an antiapoptotic effect on infarcted cardiomyocytes [9, 10]. To further improve prospects of restoring cardiac function, a search was initiated for another source of cells having high cardiomyogenic potential.

Our previous study showed that umbilical cord blood-derived mesenchymal stem cells (UCBMSCs) [11] and placental chorionic plate cells (PCPCs) [12] have a phenotype of mesenchymal cells and have higher cardiomyogenic differentiation ability in vitro. Since these materials are deemed medical waste and can be obtained without any ethical problems, they may be a suitable stem cell source for cardiac regenerative therapy. But the population of UCBMSCs in umbilical cord blood is scant [13] and there is also a problem in establishing PCPCs, since placental tissue contains a lot of maternal decidua-derived mesenchymal cells that could contaminate PCPCs. Therefore, it is difficult to obtain enough of these cells without using a limiting dilution method and/or massive ex vivo propagation, which may cause instability of the genome [14]. Consequently, material that contains a large amount of mesenchymal cells during the first few passages should be a highly suitable source of stem cells.

A previous paper suggests that endometrium contains an MSC-like population [15] and menstrual blood-derived mesenchymal (MMCs) cells have a pluripotent differentiation ability in vitro [16]. The data presented here demonstrate that human menstrual blood-derived mesenchymal cells and uterine endometrial gland-derived mesenchymal cells (EMCs) have a strong potential for cardiomyogenic transdifferentiation in vitro and in vivo. Moreover, large amounts of MMCs could be obtained from the first passage of menstrual blood culture, and MMCs have been shown to restore impaired cardiac function through marked cardiomyogenesis in vivo.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Summary
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

Isolation of MMCs and EMCs

After informed consent was obtained, mesenchymal cells from approximately 10 ml of menstrual blood of six women (20–30 years old) were collected on the first day of menstruation. The samples were suspended in Dulbecco's modified Eagle's medium (DMEM) high glucose supplemented with 10% FBS, and split into two 10-cm dishes. The estimated adherent cell number at the start of culture was approximately 1 × 107. The growth curve and phase-contrast microscopic view are shown in supplemental online Fig. 1. The results for MMCs obtained from six women were the same. A human endometrial tissue sample was also taken from a 52-year-old woman undergoing hysterectomy [17]. Individual endometrial glands were isolated under a microscope and then seeded. After the retroviral transfection of HPV16E6, E7, and hTERT [2], endometrial cell strains were generated by the limiting dilution method. Two strains exhibiting rapid cell division cycles were designated EMC100 and EMC214 (Fig. 3B and 3D, respectively). EMC100 and EMC214 showed adherent spindle shape morphology that proliferated for more than 250 population doublings without changing cardiomyogenic differentiation ability.

Isolation of Marrow-Derived Mesenchymal Stem Cells

Bone marrow-derived mesenchymal stem cells (BMMSCs) were obtained from a 41-year-old male as described previously [2].

Coculture with Murine Fetal Cardiomyocytes

MMCs, EMCs, and BMMSCs were infected with enhanced green fluorescent protein (EGFP) expressing adenovirus [2]. Fetal cardiomyocytes were obtained from hearts of day-17 mouse fetuses, as previously described [2]. The isolated cardiomyocytes were replated at 5 × 104/cm2 on top of a floating athelocollagen membrane (CM-6, 40-μm thickness; Koken, Tokyo, http://www.kokenmpc.co.jp/english/products/collagen/cell_culture/cm-6_24/index.html) that is permeable for only small molecules (less than 5,000 MW). The next day, the athelocollagen membrane was plated upside down on the culture dish. Harvested EGFP-labeled MMCs and EMCs were then seeded upon the athelocollagen surface (bottom surface) at 7 × 103/cm2 (Fig. 1M). In several experiments (Figs. 1G–1L, 2, 3E, 3H, 3K–3M, 4, supplemental online Fig. 2, examination of chromosome chimeras), we did not use the athelocollagen membrane for the coculture system.

Immunocytochemistry and Immunohistochemistry

A laser confocal microscope (FV1000; Olympus, Tokyo, http://www.olympus-global.com) was used for immunocytochemical analysis. Samples were stained with mouse monoclonal anti-cardiac troponin-I antibody (4T21 Lot 98/10-T21-C2; HyTest, Euro, Finland, http://www.hytest.fi/) or with mouse monoclonal anti-sarcomeric α-actinin antibody (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), or anti-connexin 43 antibody (Sigma-Aldrich) diluted 1:300 overnight at 4°C, then stained with TRITC-conjugated anti-mouse antibody (Sigma-Aldrich), TRITC-conjugated anti-rabbit antibody (Sigma-Aldrich), and Cy5-conjugated anti-mouse IgG (Chemicon, Temecula, CA, http://www.chemicon.com) diluted 1:100, containing 4′-6-diamidino-2-phenylindole (DAPI; Wako Chemical, Osaka, Japan, http://www.wako-chem.co.jp/english) at 1:300 for 30 minutes at 25°C–28°C. See also supplemental online data 1 for detail of method.

Functional Analysis

The method of action potential (AP) recording was as previously described [2] but with slight modification. A fluorescence inverted microscope (IX-70; Olympus) was used for AP recording. The microscope was equipped with a recording chamber and a noiseless heating plate (Microwarm Plate; Kitazato Supply, Fujinomiya, Shizuoka, Japan, http://www.kitazato-supply.com). A 10-mM volume of HEPES (Sigma-Aldrich) was added to the culture medium to stabilize the pH of the perfusate at 7.5. Standard glass microelectrodes having a direct current resistance of 15–25 MΩ when filled with pipette solution were used. Alexa 568 compound was dissolved to a concentration of 0.5 mM in 2 M of KCl solution in order to completely dissolve the Alexa 568 in the pipette solution. The electrodes were positioned with a motor-driven micromanipulator (PCS-5000; Burleigh Instrument, Inc., New York) under optical control. Spontaneously beating EGFP-positive cells were selected as targets, and after the APs of the target cells had been recorded, the dye was injected by iontophoresis (−7 nA for 10–20 seconds). The extent of dye transfer was monitored under a fluorescence microscope, and digital images were recorded with a digital photo camera (EOS-digital; Canon, Tokyo, http://www.canon.com) mounted on the microscope. The recording pipette was connected to a patch-clamp amplifier (MEZ-8300; Nihon Kohden, Tokyo, http://www.nihonkohden.com). The amplified signal was filtered with a 4-pole Bessel filter (NF-3625; NF electronic instrument; NF Corp., Tokyo, http://www.nfcorp.co.jp/english/index.html) set at 2 kHz, then digitized with an A/D converter with a sampling frequency of 10 kHz (Digidata 1,322A; Molecular Devices Corp., Union City, CA, http://www.moleculardevices.com). Pacemaker potential was defined by the slowly depolarizing membrane potential at phase IV of the AP.

Alexa 568 was injected into cells via recording microelectrodes to stain the cells and confirm that the AP was generated by EGFP-positive cells (Fig. 1G–1I, 3E, 3H). Since the dye did not diffuse into the EGFP-negative murine cardiomyocytes, there were no tight cell-to-cell heterologous connections (i.e., gap junctions), at least in the in vitro condition. In some experiments, Alexa 568 diffused into the EGFP-positive satellite EMCs and MMCs, suggesting that a homologous cell-to-cell connection had been established at least 1 week after cocultivation. The measured parameters of the APs were averaged and are shown in Figure 1K.

The fluorescent image of the beating MMCs and EMCs was monitored using a CCD camera (Ikegami Tsushin Co., Ltd, http://www.ikegami.co.jp) and was stored using digital video. The video images (National Television Standards Committee format, 29.97 frame/second) of contraction of EMCs and MMCs were stored in a personal computer as MPEG-2 format files, then analyzed later. Both edges of the EGFP-positive EMCs and MMCs along the line (Figs. 1L, 3K) were automatically detected, and the distance between both edges was measured from each video frame using an image edge-detection program using Igor Pro 4 (Wavemetrics Inc., Lake Oswego, OR) [11].

Calculation of Induction Rate

The MMCs and EMCs were exposed to 3 μM 5-azacytidine (5-azaC; Sigma-Aldrich) for 24 hours to induce cell differentiation, or were left untreated. The 5-azaC-treated and nontreated MMCs or EMCs, cultivated with or without murine fetal cardiomyocytes, were enzymatically dissociated and stained, then observed by confocal laser microscope (supplemental online data 2 for detail of method). The cardiomyogenic induction rate (average of 10 separate experiments) was calculated as the fraction of cardiac troponin-I-positive cells in the EGFP-positive cells.

Examination of Chromosomes of MMCs or EMCs and Murine Cell Chimeras

To rule out cell fusion-dependent cardiomyogenesis, chromosomes from MMCs or EMCs cocultivated without separation by the athelocollagen membrane from murine cardiomyocytes for 1 week were stained using a human chromosome-specific probe and a mouse chromosome-specific probe (Chromosome Science Labo, Hokkaido, Japan, http://www.chromoscience.jp/en/probe/page01/page01e.html) and spectral karyotyping with fluorescent in situ hybridization chromosome painting technique (Applied Spectral Imaging, Vista, CA, http://www.spectral-imaging.com), according to the manufacturer's protocol.

RNA Extraction and RT-PCR

Reverse transcriptase polymerase chain reaction (RT-PCR) was done as described previously [2]. Primers for the following genes were used: cardiac transcription factors—Csx/Nkx-2.5 and GATA4; cardiac hormones—atrial natriuretic peptide and brain natriuretic peptide; cardiac structural proteins—cardiac troponin I, cardiac troponin T, myosin light chain-2a, myosin light chain-2v, and cardiac-actin; and ion channel—cyclic nucleotide-gated potassium channel 2 (supplemental online Table 1). The internal control was 18S rRNA. PCR primers were prepared such that they would amplify the human but not the mouse genes.

Flow Cytometric Analysis

The cells were analyzed using an EPICS ALTRA analyzer (Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com). Antibodies (anti-human CD10, CD13, CD14, CD24, CD29, CD31, CD34, CD44, CD45, CD54, CD55, CD59, CD71, CD73, CDw90, CD105, CD106, CD117, CD133, CD140a, CD166, CD309, HLA-ABC, and HLA-DR) [12] were purchased from Beckman Coulter, Immunotech (Luminy, France, http://www.beckmancoulter.com/products/pr_immunology.asp), Cytotech (Hellebaek, Denmark, http://www.cytotech.dk/index.html), Santa Cruz Biotechnology Inc. (Santa Cruz, CA, http://www.scbt.com), RDI (Research Diagnostics, Inc., Concord, MA, http://www.researchd.com), and Pharmingen Pharmaceutical, Inc. (San Diego, http://www.bdbiosciences.com/index_us.shtml).

In Vivo Cardiomyogenic Differentiation of EMCs

EGFP-labeled EMC tissue graft, made by a novel 3-dimensional cell sheet manipulation, was transplanted into male F344 nude rats (Clea, Tokyo, http://www.clea-japan.com/) (8 weeks of age). EMC100s and EMC214s (2 × 105/cm2) were plated onto fibrin polymer-coated culture dishes. Four days after plating, EMCs were detached as previously described [18], and transplanted onto the surface of the recipient heart (Fig. 5A) [19]. At 2 weeks after transplantation, immunohistochemical analysis was performed. EGFP-labeled EMC tissue graft on the fibrin polymer-coated culture dish did not show cardiomyogenic differentiation in vitro.

MMC Transplantation in Myocardial Infarction Model In Vivo

Recipient male F344 nude rats (Clea) (6 weeks of age) were anesthetized with 2% isoflurane gas. After left thoracotomy, the left ventricle was exposed and left anterior coronary artery was ligated by 6–0 silk suture. The complete occlusion of the coronary artery was confirmed by the cyanotic color and dyskinetic motion of the left ventricular anterior wall. In some rats, we did not ligate the coronary artery (Sham). The chest was closed and animals survived for 2 weeks to create complete myocardial infarction.

Two weeks after the first operation, rats with myocardial infarction were randomized for the control myocardial infarction (MI) group, the MI+BMMSC group, and the MI+MMC group, and were blinded immediately before the cell injection. Echocardiograms were performed on the anesthetized (2% isoflurane) rats. Data were collected three times and averaged. Immediately before transplantation, ∼1–2 × 106 of EGFP-positive MMC or BMMSC suspension was drawn up into a 50-μl Hamilton syringe (Hamilton Co., Reno, NV, http://www.hamiltoncompany.com/main_usa.asp) with a 31-gauge needle. A 10-μl portion of the cell suspension was injected into the center and margin of the infarcted myocardium (MI+MMC, Fig. 7A). In the control MI group, culture medium or ∼1–2 × 106 of murine cardiac fibroblast was injected. Immediately before cell transplantation, 2-dimensional and M-mode echocardiographic (8.5 MHz linear transducer, EnVisor C; Phillips Medical System, Andover, MA, http://www.medical.philips.com/index.html) images were obtained to assess left ventricular (LV) end-diastolic dimension and LV end-systolic dimension at the mid-papillary muscle level.

Two weeks after the transplantation, a similar echocardiogram was performed again; then after opening the abdomen, a blood sample was drawn from the abdominal great vein; then the left diaphragm was dissected to insert a 22-gauge manometer line into the left ventricle, which was connected to the transducer (model TP-400T; Nihon Kohden) to monitor left ventricular pressure. The electrocardiogram and measured pressure were digitized by PowerLabo (ADInstruments, Milford, MA, http://www.adinstruments.com) at the sample frequency of 10 KHz and stored in a personal computer (Macintosh iBook G4; Apple, Cupertino, CA, http://www.apple.com).

Tissue samples were obtained by fixing and slicing along the short axis of the left ventricle, for every 1-mm depth of the ventricle. After Masson's trichrome staining, digital images of samples were collected using a light microscope (IX-70; Olympus). The images were digitized and analyzed using an Igor Pro 4 (Wavemetrics Inc.). The pixel area of blue color (fibrosis area) was defined as the infarcted area, and the pixel area of red color was defined as “survived” myocardium. The data on each pixel area from each slice were collated and the percentage fibrosis area was calculated as follows: % Fibrosis = 100 × (Pixel area of blue color)/(Pixel area of blue color and red color).

Statistical Analysis

All data are shown as the mean value ± SE. The difference among mean values was determined with analysis of variance. The posthoc test (Bonferroni) was used when three or more groups were compared. Student's t test was used when two values were compared. Statistical significance was set at p < .05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Summary
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

Cardiomyogenic Transdifferentiation of MMCs

To exclude cell fusion-dependent cardiomyogenesis [20], EGFP-labeled MMCs were cocultured in the same dish with mouse cardiomyocytes, separated by a 40-μm high-density athelocollagen membrane (Fig. 1M). The two cell types were never in direct contact. On day 5 after cocultivation commenced, approximately half of the MMCs were beating strongly in a synchronized manner (supplemental online Video 1). Immunocytochemistry revealed that the MMCs were stained positive by the anti-cardiac troponin-I antibody (Fig. 1C–1E). Clear striations of red fluorescence of troponin-I in the differentiated MMCs (Fig. 1D, 1E) were observed. Troponin-I and EGFP staining appeared alternately in a striated manner, suggesting troponin-I expressed in the EGFP-positive cell (Fig. 1E, 1F). Clear striations were observed with red fluorescence of α-actinin in the differentiated MMCs (Fig. 2B) and diffuse dot-like staining pattern of connexin 43 around the margin of each EGFP-positive cardiomyocyte (Fig. 2C–2F), suggesting that these human transdifferentiated cardiomyocytes have tight electrical coupling with each other. APs were recorded from spontaneously beating MMCs. The APs obtained from MMCs showed clear cardiomyocyte-specific sustained plateaus and slowly depolarizing resting membrane potentials—so-called “pacemaker potentials” (Fig. 1J, 1K)—and were, therefore, determined to be APs of cardiomyocytes, not of smooth muscle cells, nerve cells, or skeletal muscle cells. The fractional shortening (% FS) of the MMCs was analyzed (Fig. 1L) using a cell edge detection program. The EGFP-positive cells contracted simultaneously within the whole visual field. The % FS was 5.9 ± 0.5% (n = 19).

thumbnail image

Figure Figure 1.. Cardiomyogenic differentiation of menstrual blood-derived mesenchymal cells (MMCs) in vitro. (A): Phase-contrast microscopic view of MMC (bar denotes 100 μm), regarded as being PD1, or day 2. (B): The representative growth curves of MMCs as a function of time after the culture. The growth curves from all three donors are linear over at least 25 population doublings. (C–F): Laser confocal microscopic view of immunocytochemistry of differentiated MMCs with anti-cardiac troponin-I (Trop-I) antibody. Enhanced green fluorescent protein (EGFP)-positive (green) human MMCs expressed Trop-I (red). Scale bar denotes 20 μm. (D): Expansion of area within the white box in (C). Clear striation pattern of Trop-I is observed. Trop-I and EGFP images along the yellow line are shown in (E, F). (E, F): Trop-I and EGFP staining was observed alternately in striated manner, suggesting Trop-I is expressed in the EGFP-positive cell. (G–I): EGFP-labeled MMCs were injected with Alexa 568 solution (red) through a microelectrode to confirm that the recorded signal was obtained from MMCs. (J): Representative action potential traces are shown (horizontal line denotes 500 ms). The vertical line denotes 50 mV, and dotted horizontal line denotes 0 mV. (K): Action potential parameters. (L): A representative still image (left panel) and detected fractional shortening (% FS) along the white line obtained from sites a, b, and c are shown in right panel. (M): Experimental schema. Abbreviations: ADP, action potential duration; BCL, basic cycle length; DAPI, 4′-6-diamidino-2-phenylindole; MDP, maximum diastolic potential.

Download figure to PowerPoint

thumbnail image

Figure Figure 2.. Immunocytochemical analysis of menstrual blood-derived mesenchymal cells (MMCs) and EMC214s stained with anti-sarcomeric α-actinin and connexin 43. (A–L): Laser confocal microscopic view of immunocytochemistry of differentiated MMCs and EMC214s with anti-sarcomeric α-actinin (α-actinin) and connexin 43 (Cx43) antibody. (A–F, G–L): Enhanced green fluorescent protein (EGFP)-positive (E, K; green) human MMCs and EMC214s express α-actinin (B, H; red) and Cx43 (C, I; cyan). Nuclei are stained with 4′-6-diamidino-2-phenylindole (DAPI) (A, G; blue). Clear striation patterns of α-actinin and diffuse Cx43 dot-like staining around the margin of the MMCs and EMC214s were observed. Scale bars in the figure denote 50 μm.

Download figure to PowerPoint

The percentage of cardiac troponin-I-positive cells was calculated to determine the cardiomyogenic transdifferentiation rate. Whereas MMCs without cocultivation did not show any troponin-I expression (supplemental online Figs. 1A–1D, 2A, 2B), 27%–32% of MMCs became positive for cardiac troponin-I antibody as a result of the cocultivation (Figs. 1C–1F, 4A, supplemental online Fig. 2C, 2D). A cytosine analog, 5-azaC, has a remarkable effect on cell transdifferentiation and has been shown to induce transdifferentiation BMMSCs into cardiomyocytes in mice by nonspecific demethylation of the genome [1]. Cardiomyogenic transdifferentiation was observed in the cocultivated MMCs without any 5-azaC pretreatment, meaning that 5-azaC was not essential for cardiomyogenic transdifferentiation. Nuclear fusion between the cocultivated MMCs and murine cardiomyocytes without separation of the athelocollagen membrane was observed in only 0.16% (3/1846).

Cardiomyogenic Transdifferentiation of EMCs

We hypothesized that the origin of cardiomyogenic cells in the MMCs was the endometrial gland, since MMCs have a high content of detached endometrial glands, whereas circulating blood-derived endothelial progenitor cells [21] or marrow-derived MSCs [2] do not have such high cardiomyogenic differentiation ability. We consequently established a line of EMCs (Fig. 3B, 3D) with a lifespan prolonged by a cell cycle-mediated gene to ensure a supply of cells for analysis. Almost all EMCs beat strongly in a synchronized manner (supplemental online Video 1), and 76.4%–96.5% became positive for cardiac troponin-I antibody as a result of cocultivation (Figs. 3A, 3C, 4B, 4C, supplemental online Fig. 2E–2L). EMCs were also positive for sarcomeric α-actinin and connexin 43 (Fig. 2G–2L). APs were recorded from EMCs. The APs obtained from EMCs showed clear cardiomyocyte-specific sustained plateaus and, in some cells, pacemaker potentials (Fig. 3E–3J). The EGFP-positive EMCs contracted simultaneously within the whole visual field (Fig. 3L, 3M). Nuclear fusion between the cocultivated EMC100s or EMC214s and murine cardiomyocytes without separation of the athelocollagen membrane was observed in only 0.57% (6/1058) or 0.28% (5/1758), respectively.

thumbnail image

Figure Figure 3.. Cardiomyogenic differentiation of endometrial gland-derived mesenchymal cells (EMCs) in vitro. (A, C): Immunocytochemistry of differentiated EMC100s (A) and EMC214s (C) with anti-cardiac troponin-I (Trop-I) antibody. The cells were stained with 4′-6-diamidino-2-phenylindole (DAPI; blue), and anti-cardiac troponin-I antibody (red). Enhanced green fluorescent protein (EGFP)-positive (green) human EMCs expressed Trop-I (red). Please note clear striation staining pattern of Trop-I (A, C) in EMCs. Scale bar denotes 20 μm. (B, D): Phase-contrast images of EMC100s (B) and EMC214s (D) before the cardiomyogenic induction. (E, H): EGFP-labeled EMC100s and EMC214s (green) were injected with Alexa 568 solution (red) through a microelectrode (E, H), and a recorded signal was obtained from the cells. Representative action potential traces are shown (F, G: EMC100; I, J: EMC214). Action potential of E is expanded in the inset (the vertical line denotes 100 ms). The vertical line denotes 50 mV and dotted horizontal line denotes 0 mV levels. (K–M): A representative still image (K) and detected fractional shortening (% FS) along the white line obtained from sites a, b, c, and d in (L) are shown in (M). (M): The measured % FS was averaged and is shown.

Download figure to PowerPoint

thumbnail image

Figure Figure 4.. Cardiomyogenic transdifferentiation rates and expression of cardiomyocyte-specific genes and cell surface markers of menstrual blood-derived mesenchymal cells (MMCs) and endometrial gland-derived mesenchymal cells (EMCs). (A–D): Cardiomyogenic transdifferentiation rates of MMCs, EMCs, and bone marrow-derived mesenchymal stem cells (BMMSCs). The character in each column denotes pretreatment with 5-azacytidine (5-azaC) or the lack of treatment (non). (E): Reverse transcriptase polymerase chain reaction (PCR) was performed with PCR primers with specificity for human genes encoding cardiac proteins but not for the corresponding murine genes (supplemental online Table 1). Human heart and mouse heart cells were used as a positive control and negative control, respectively. Most human cardiac genes were constitutively expressed in the default state of MMCs and EMCs. (F): Summary of flow cytometric analysis of MMCs and EMCs with fluorescein isothiocyanate–coupled antibodies against human surface antigens. Abbreviations: DW, distilled water; EGFP, enhanced green fluorescent protein; hANP, human atrial natriuretic peptide; hBNP, human brain natriuretic peptide; HCN2, cyclic nucleotide-gated potassium channel 2; MLC2a, myosin light chain 2a; MLC2V, myosin light chain 2v; TnI, Trop-I, cardiac troponin I; TnT, cardiac troponin T.

Download figure to PowerPoint

Expression of Cardiomyocyte-Specific Genes and Surface Markers of EMCs and MMCs

The RT-PCR was performed with primers that hybridized with human cardiomyocyte-specific genes but not with the murine orthologs. Differentiated MMCs and EMCs expressed cardiac-specific genes (Fig. 4D). Interestingly, most of the analyzed genes were expressed in the cells before the induction of transdifferentiation by cocultivation.

There is no difference between surface markers of the MMCs and EMCs. Both cells were positive for CD29 (integrin β1), CD59, and negative for CD14, CD34, CD45, CD309 (Flk-1), etc. (Fig. 4E, supplemental online Fig. 3A–3C).

Cardiomyogenic Effects In Vivo

An EGFP-labeled EMC tissue graft made by a novel 3-dimensional cell sheet manipulation [18] was transplanted into male F344 nude rats to ensure in vivo cardiomyogenic transdifferentiation ability. The EGFP-positive cell layer (green) was observed at the epicardial surface of the host heart (Fig. 5B–5D). Whole EMCs throughout the layer expressed a clear striation staining pattern of sarcomeric α-actinin (Fig. 5B–5G), suggesting extremely high cardiomyogenic transdifferentiation ability of EMCs in situ.

thumbnail image

Figure Figure 5.. In vivo cardiomyogenesis of endometrium-derived mesenchymal cells (EMCs) in cell sheet tissue graft on host heart. (A): Macroscopic view of enhanced green fluorescent protein (EGFP)-labeled EMC tissue graft (sheet) on the epicardial surface of the recipient's heart. (B–D): Two weeks after transplantation, immunohistochemistry revealed survival of EMC tissue layer (green) on the recipient heart. Scale bar denotes 100 μm. (C): Engrafted EMCs stained positive with anti-sarcomeric α-actinin (red; α-actinin). (E–G): The area in the white box in (B) is shown in greater detail in (E–G). (F): The clear striation pattern of α-actinin staining was observed throughout the entire layer of engrafted EMCs, suggesting extremely high cardiomyogenic potential of EMCs in situ. Scale bar denotes 20 μm.

Download figure to PowerPoint

MMCs or BMMSCs were transplanted into the nude rats with MI in vivo. Echocardiography showed that the left ventricular fractional shortening (% LVFS) in the MI+MMC group was significantly greater than it in the MI+BMMSC group at 2 weeks after transplantation (Fig. 6A–6I, supplemental online Fig. 4). The MI area was digitized and every 1-mm depth of tissue section stained with Masson's trichrome (Fig. 6J–6O); averaged data are shown in Figure 6P. The MI area was significantly lower in the MI+MMC group than in the MI+BMMSC group. The EGFP-positive mass of MMCs observed in the MI area expressed a clear striation staining pattern of cardiac troponin-I (Fig. 7) and sarcomeric α-actinin (supplemental online Fig. 5), suggesting an extremely high in situ cardiomyogenic transdifferentiation ability of MMCs, which contributed to improvement in cardiac function.

thumbnail image

Figure Figure 6.. The effect of menstrual blood-derived mesenchymal cell (MMC) transplantation on cardiac function. (A–D): Representative M-mode echocardiographic images. The contraction of the left ventricular (LV) anterior wall was improved by transplantation of MMCs (white arrows). The symbol of and number in each group is depicted at the bottom left of each image. (E–I): Measured LV parameters are averaged and shown at 2 weeks and 4 weeks after the myocardial infarction (MI). The significant improvement of (F) LV end-systolic diameter (LVESd) and (E) % fractional shortening (% LVFS) were observed. The diameter of (H) anterior left ventricular wall thickness (AW), and (I) posterior left ventricular wall thickness (PW). There is no statistical significance. (J–O): Representative Masson's trichrome stain images (J, L, N) and digitized images (K, M, O) of control MI group, MI+bone marrow-derived mesenchymal stem cell (BMMSC), and MI+MMC group are shown. (P): The calculated % fibrosis areas are summed and averaged. The MMC transplantation showed significant reduction of % fibrosis area. Abbreviations: Endo, endocardium; Epi, epicardium; NS, not significant.

Download figure to PowerPoint

thumbnail image

Figure Figure 7.. Cardiomyogenesis of engrafted menstrual blood-derived mesenchymal cells (MMCs) in vivo. (A): Macroscopic view of the recipient's heart immediately after enhanced green fluorescent protein (EGFP)-labeled MMC transplantation (white arrows) into the myocardial infarction area of the recipient's heart. (B–L): Two weeks after transplantation, immunohistochemistry revealed survival of the MMC tissue layer (green) on the treated heart. (B–D): Engrafted MMCs stained positive with anti-cardiac troponin-I (red; Trop-1). Scale bar denotes 100 μm. (E–H, I–L): The area in the white box in (D) was observed in higher resolution (E–H) and the white box in (H) was also observed in higher resolution (I–L). (K): The clear striation pattern of Trop-1 staining was observed throughout the whole layer of engrafted MMCs, suggesting extremely high cardiomyogenic potential of MMCs in situ. Scale bar denotes 20 μm. Abbreviations: DAPI, 4′-6-diamidino-2-phenylindole; Endo, endocardium; Epi, epicardium.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Summary
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

Mechanisms of Highly Cardiomyogenic Transdifferentiation Ability of MMCs and EMCs

The gene expression pattern of MMCs and EMCs before cardiomyogenic transdifferentiation is quite different from that of marrow-derived MSCs [2]. GATA-4 expression in the MMCs and EMCs, and Csx/Nkx 2.5 expression in EMCs with the ability of self-renewal suggest that MMCs and EMCs both have cardiogenic potential and may be termed “cardiac precursor cells” due to their biological features. Cardiac mRNA but not cardiac protein (i.e., troponin-I) was expressed at the default state in the present study, suggesting that both genetic and epigenetic factors may be essential to cause physiologically functioning cardiomyogenic differentiation in MMCs and EMCs. The mechanism of the drastic improvement in the transdifferentiation rate of MMCs and EMCs may be attributable to the default characteristics (expression level of cardiomyocyte-specific mRNA) of MMCs and EMCs in culture compared to marrow-derived MSCs. Highest cardiomyogenic transdifferentiation efficiency was observed in EMC214s (96.5%), EMC100s (76.4%), UCBMSCs (44.9%) [11], MMCs (33.2%), PCPCs (15.1%) [12], and BMMSCs (0.3%, Fig. 4D) [2] in that order. In the practical point of view, EMCs and UCBMSCs are difficult to obtain in enough numbers during the first few passages. MMCs are, therefore, the most suitable cellular source for cardiac stem cell therapy, having a high cardiomyogenic transdifferentiation efficiency. MMCs, EMCs, UCBMSC, and PCPCs are derived from the organ that is related to the pregnancy, therefore the high cardiomyogenic transdifferentiation ability of mesenchymal cells may be caused by a pregnancy-related environmental condition.

Origin of the MMCs and EMCs

Cell surface marker analysis revealed that MMCs are neither encirculating endothelial progenitor cells [22] nor macrophages, but are mesenchymal phenotype cells. We speculated that MMCs may originate in uterine endometrial glands since a lot of detached endometrial glands were observed in menstrual blood and EMCs have the same surface marker as the MMCs, as well as an extremely high cardiomyogenic potential (76.4%–96.5% and 33.2%, respectively). As has been reported, MSCs cannot be detected in circulating blood and all tissues have MSC reservoirs localized in the perivascular niche [23], so EMCs and MMCs do not seem to originate from BMMSCs.

Clinical Contribution

In the present study, MMC transplantation improved impaired cardiac function in vivo. Since MMCs were transplanted at 2 weeks after coronary occlusion, when myocardial necrosis had been completed, the improvement of cardiac function is not due only to transplanted MMC-induced neovascularization [7, 8] or an antiapoptotic [9] effect on infarcted cardiomyocytes. Since they display high cardiomyogenic transdifferentiation ability in vitro and massive cardiomyogenic transdifferentiation in vivo, MMC-derived cardiomyocytes may play a role in the improvement of cardiac function in the present study. Myocardial infarction is known to suppress contraction ability of cardiomyocytes even at normal zone by left ventricular remodeling. Therefore MMC-derived paracrine factors may also play an important role in recovery of % LVFS by prevention of development of LV remodeling.

Neovascularization and the antiapoptotic effect are important for improving cardiac function to some extent. However, the feasible effect is dependent on the number of residual host cardiomyocytes in the infarcted myocardium. To achieve further improvement of cardiac function, a stem cell source that can be expected to exhibit powerful cardiomyogenic transdifferentiation in situ is required. MMCs can be transdifferentiated into cardiomyocytes in situ on the recipient heart, suggesting that they are a promising source for cardiac stem cell-based therapy material, significantly more efficient for cardiomyogenesis than BMMSCs.

MMCs can be readily obtained in a noninvasive manner from young female volunteers, and stored. It should therefore be possible to obtain MMCs of all the HLA types, possibly enabling the establishment of an MMC bank system to facilitate cardiac stem cell-based therapy.

Role of Established Cardiomyogenic EMC Cell Line for Determining Cardiomyogenic Factors

Several stem cell types are used for clinical patients. Of these, MSCs are reported to show cardiomyogenesis in vitro. Thus, the analysis of key mechanisms for cardiomyogenic differentiation in the human mesenchymal cell is extremely important in order to expand the efficacy of current cardiac stem cell therapy. However, it is very difficult to specify the key factor of cardiomyogenesis by in vivo experiment only. Establishment of EMCs and an in vitro cardiomyogenic differentiation assay system are essential. Stable and high cardiomyogenic transdifferentiation ability in our established system enables us to observe, with wide dynamic range, the effects of treatment for cardiomyogenesis. Moreover, the primary culture condition of murine cardiomyocytes usually fluctuates due to variations in environments, the skill of individual researchers, and institutional differences in isolation protocols. Our established EMCs may provide a good positive control for a cardiomyogenic assay system in vitro to check whether the feeder cell condition is suitable for cardiomyogenic assay. When feeder conditions are suitable, we can survey for possible cardiomyogenic assistant factors or appropriate culture conditions for human BMMSCs by applying various agents or modifying culture conditions systematically. Thus, by using our EMCs and cocultivation system, we may be able to expand the cardiomyogenic differentiation potential of marrow-derived MSCs. Consequently, we may be able to increase the efficacy of cardiac stem cell-based therapy dramatically.

Neither passive stretching of EMCs nor an application of the supernatant of murine cardiomyocyte culture medium to the EMCs alone caused cardiomyocyte differentiation. Taking these findings into account, the multiple environmental factors, including mechanical stretching and/or feeder cardiomyocyte-derived humoral factors, seem to contribute to cardiomyogenic transdifferentiation in human mesenchymal cells. Further experiments should be done.

Study Limitations

Cell fusion between the human cells (MMCs or EMCs) might be a major cause of EGFP-positive cardiomyocytes in the present study. However, EGFP-positive cardiomyocytes could be observed, even when human cells and murine cardiomyocytes were cocultured separately by the athelocollagen membrane that is permeable for only small molecules (less than 5,000 MW)—thus allowing no possible penetration of cells or organelles through the membrane (supplemental online Fig. 6). Furthermore, even if the cells were cocultured without the athelocollagen membrane, nuclear fusion between EMC100s, EMC214s, or MMCs and fetal murine cardiomyocytes was less than 1% in the present study. Moreover, transdifferentiated EMCs at the external layer of the cell sheet graft on the epicardial surface did not directly contact the host cardiomyocytes (Fig. 5). Taking these results into account, we concluded that the cell fusion did not play a major role in the observed significant cardiomyogenic potential of MMCs and EMCs in the present study.

Infarcted heart tissue may increase auto-fluorescence in some fixative conditions and such auto-fluorescence of host cardiomyocytes might be confused as EGFP-positive like cells. However, autofluorescence of the host myocardium adjacent to the infarcted area was not significant in our present condition (Figs. 5B, 6B, supplemental online Fig. 5B, 5F). Therefore, EGFP-positive tissue in the present study can be defined as of human cell origin and easily distinguished from the host heart by the EGFP fluorescent intensity.

The transfection of the cell cycle-mediated gene may increase cardiomyogenic differentiation to some extent. However, our previous study in human BMMSCs, [2] with the same combination of cell cycle-mediated gene transfection, did not show any increase in efficiency. Furthermore, non-gene-transfected MMCs have an extremely high cardiomyogenic efficiency compared to gene-transfected BMMSCs. Taking these results into account, we concluded that transfection of those genes does not play an essential role in causing such high cardiomyogenic differentiation efficiency in EMCs.

In comparison to previous papers, there was no observable effect of BMMSC transplantation on cardiac function in the present study. This discrepancy may be caused by different experimental conditions, that is, species difference between BMMSCs and the host animal [24], transplantation at acute myocardial infarction [25, [26]27], and usage of immunosuppressive agents, etc [24, [25], [26]27].

In the present study, we did not use a pressure-tipped catheter, therefore the LV dp/dt value may be underestimated.

Summary

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Summary
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

MMC transplantation decreased fibrosis area and restored the LV systolic function in the MI-model in vivo. Engrafted MMC transdifferentiated into cardiomyocyte within MI area. MMC can be a major cell source for stem cell therapy to achieve cardiomyogenesis.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Summary
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

The research of N.H. and N.N. was partially supported by a grant from the Ministry of Education, Science and Culture, Japan. A part of this work was undertaken at the Keio Integrated Medical Research Center. We thank M. Uchiyama, A. Furuta, K. Hayakawa, and K. Okamoto for help during the experiments. N.H. and N.N. contributed equally to this work. A part of this work was reported at the annual meeting of the American College of Cardiology 2005, 2006, and 2007.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Summary
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Summary
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information
FilenameFormatSizeDescription
SC-07-0826_Supplemental_Data.pdf52KSupplemental Data
SC-07-0826_Supplemental_Figure_1.tif1234KSupplemental Figure 1
SC-07-0826_Supplemental_Figure_2.tif939KSupplemental Figure 2
SC-07-0826_Supplemental_Figure_3.tif337KSupplemental Figure 3
SC-07-0826_Supplemental_Figure_4.tif95KSupplemental Figure 4
SC-07-0826_Supplemental_Figure_5.tif757KSupplemental Figure 5
SC-07-0826_Supplemental_Figure_6.tif1120KSupplemental Figure 6
SC-07-0826_Supplemental_Table.tif259KSupplemental Table
SC-07-0826_Supplemental_Movie.zip70668KSupplemental Movie

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.