Menstrual Blood-derived Mesenchymal Cells Research Articles

Human Menstrual Blood-Derived Mesenchymal Cells as New Human Feeder Layer System for Human Embryonic Stem Cells.

Human embryonic stem cells (hESCs) in general require coculture with feeder layers in order to remain undifferentiated. However, the use of animal-derived feeder layers is incompatible with the clinical setting. The objective of this work was to investigate whether human menstrual blood-derived mesenchymal cells (MBMCs) can substitute mouse embryonic fibroblasts (MEFs) as a feeder layer for H9-hESCs. Both feeder cell types were isolated and cultured in DMEM F-12 and high glucose DMEM, respectively. After three passages, they were inactivated with mitomycin C. To test MBMC feeder layer capacity, hESCs were grown over MBMCs and MEFs under standard conditions. hESC growth, proliferation, survival, and maintenance of the undifferentiated state were evaluated. hESCs grown over MBMCs preserved their undifferentiated state presenting standard morphology, expressing alkaline phosphatase, transcription factors OCT3/4, SOX2, and NANOG by RT-PCR and SSEA-4 and OCT3/4 by immunofluorescence assays. It is noteworthy that none of the feeder cells expressed these proteins. The average colony size of the hESCs on MBMCs was higher when compared to MEFs (p < 0.05; mean ± SD, n = 3). Growth factor analysis revealed amplification of the transcripts for FGF-2, BMP4, TGF-β, VEGF, and PEDF by RT-PCR in MBMCs and MEFs before and after inactivation. Furthermore, similar embryoid body formation, size, and morphology were observed in both feeder layers. In addition, EBs expressed marker genes for the three germ layers cultured on both feeder cells. In conclusion, MBMCs are able to maintain hESCs in an undifferentiated state with comparable efficiency to MEFs. Therefore, MBMCs are a suitable alternative to animal-derived feeder layers for growing hESCs.

Evidences for the involvement of cell surface glycans in stem cell pluripotency and differentiation

Induced pluripotent stem (iPS) cells are somatic cells that have been reprogrammed to a pluripotent state via the introduction of defined transcription factors. Although iPS is a potentially valuable resource for regenerative medicine and drug development, several issues regarding their pluripotency, differentiation propensity and potential for tumorigenesis remain to be elucidated. Analysis of cell surface glycans has arisen as an interesting tool for the characterization of iPS. An appropriate characterization of glycan surface molecules of human embryonic stem (hES) cells and iPS cells might generate crucial data to highlight their role in the acquisition and maintenance of pluripotency. In this study, we characterized the surface glycans of iPS generated from menstrual blood-derived mesenchymal cells (iPS-MBMC). We demonstrated that, upon spontaneous differentiation, iPS-MBMC present high amounts of terminal β-galactopyranoside residues, pointing to an important role of terminal-linked sialic acids in pluripotency maintenance. The removal of sialic acids by neuraminidase induces iPS-MBMC and hES cells differentiation, prompting an ectoderm commitment. Exposed β-galactopyranose residues might be recognized by carbohydrate-binding molecules found on the cell surface, which could modulate intercellular or intracellular interactions. Together, our results point for the first time to the involvement of the presence of terminal sialic acid in the maintenance of embryonic stem cell pluripotency and, therefore, the modulation of sialic acid biosynthesis emerges as a mechanism that may govern stem cell differentiation.

Human Menstrual Blood-Derived Mesenchymal Cells as a Cell Source of Rapid and Efficient Nuclear Reprogramming

Induced pluripotent stem cells (iPSCs) were originally generated by forced ectopic expression of four transcription factors genes-OCT4, KLF4, SOX2, and c-MYC-in fibroblasts. However, the efficiency of iPSCs obtention is extremely low, and reprogramming takes about 20 days. We reasoned that adult cells showing basal expression of core embryonic stem (ES) cell regulator genes could be a better cell source for reprogramming. Menstrual blood-derived mesenchymal cells (MBMCs) are multipotent cells that show detectable levels of some of the core ES cells regulators. The aim of this study was to determine whether reprogramming efficiency could be increased by using MBMCs as a cell source to generate iPSCs. MBMCs were transduced with recombinant retroviruses expressing the coding regions of OCT4, SOX2, and KLF4 genes. Cells with high nucleus/cytoplasm ratio can be detected about 5 days of posttransduction, and colonies of typical ES-like cells begun to appear after 7 days. At day 15, colonies were picked up and expanded for characterization. Most of the clones were morphologically identical to ES cells and positive at the mRNA and protein levels for all pluripotency markers tested. The clones are capable of forming embryoid bodies and to differentiate in vitro into cells of the three germ cell layers. Our results show that the reprogramming was faster and with efficiency around 2-5%, even in the absence of ectopic expression of c-MYC. To date, this is the first study showing MBMCs as a cell source for nuclear reprogramming.

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.

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 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.