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Laboratory of Human Stem Cells, Institute of Experimental Cardiology, National Cardiology Research Center of the Russian Ministry of Health, Moscow, Russia
Laboratory of Human Stem Cells, Institute of Experimental Cardiology, National Cardiology Research Center, 3rd Cherepkovskaya Str. 15-A, Moscow 121552, Russian Federation. Telephone: 7-095-414-6949; Fax: 7-095-149-2652
Mesenchymal stem cells (MSCs) have the capability for renewal and differentiation into various lineages of mesenchymal tissues. These features of MSCs attract a lot of attention from investigators in the context of cell-based therapies of several human diseases. Despite the fact that bone marrow represents the main available source of MSCs, the use of bone marrow-derived cells is not always acceptable due to the high degree of viral infection and the significant drop in cell number and proliferative/differentiation capacity with age. Thus, the search for possible alternative MSC sources remains to be validated. Umbilical cord blood is a rich source of hematopoietic stem/progenitor cells and does not contain mesenchymal progenitors. However, MSCs circulate in the blood of preterm fetuses and may be successfully isolated and expanded. Where these cells home at the end of gestation is not clear. In this investigation, we have made an attempt to isolate MSCs from the subendothelial layer of umbilical cord vein using two standard methodological approaches: the routine isolation of human umbilical vein endothelial cell protocol and culture of isolated cells under conditions appropriate for bone-marrow-derived MSCs. Our results suggest that cord vasculature contains a high number of MSC-like elements forming colonies of fibroblastoid cells that may be successfully expanded in culture. These MSC-like cells contain no endothelium- or leukocyte-specific antigens but express alpha-smooth muscle actin and several mesenchymal cell markers. Therefore, umbilical cord/placenta stroma could be regarded as an alternative source of MSCs for experimental and clinical needs.
Mesenchymal stem cells (MSCs) comprise a rare population of multipotent progenitors capable of supporting hematopoiesis and differentiating into three (osteogenic, adipogenic, and chondrogenic) or more (myogenic, cardiomyogenic, etc.) lineages [1–3]. Due to this ability, confirmed by the results of either in vitro experiments [4–7] or in vivo studies [8–10], MSCs appear to be an attractive tool in the context of tissue engineering and cell-based therapy. Currently, bone marrow represents the main source of MSCs for both experimental and clinical studies [2, 3]. However, the number of bone marrow MSCs significantly decreases with age , which makes the search for adequate alternative sources of these cells for autologous and allogenic use necessary. In this connection, most attention should be paid to tissues containing cells with higher proliferative potency, capability of differentiation, and lower risk of viral contamination.
Umbilical cord blood (UCB) is a rich source of hemopoietic stem/progenitor cells useful for clinical applications [12, 13]. The data concerning the presence of MSCs in UCB are controversial. On the one hand, these cells could not be isolated or successfully cultured from term UCB . At the same time, results obtained by Campagnoli et al.  and Erices et al.  suggest that MSCs are present in several fetal organs and circulate in the blood of preterm fetuses simultaneously with hematopoietic precursors. Thus, the fact that in the middle of gestation UCB is enriched in pluripotential MSCs seems to be validated. The questions arise: where do these cells home after they leave circulation and is the excess of MSCs possibly deposed in placenta/umbilical cord stroma, including that of blood vessels?
With these questions in mind, we have made an attempt to establish MSC cultures from the subendothelial layer of the human umbilical cord vein using two standard approaches: routine human umbilical vein endothelial cell (HUVEC) isolation and the culture of isolated cell populations under conditions appropriate for bone-marrow-derived mesenchymal progenitors.
Obtained results suggest that the population of MSC-like cells is present within the umbilical vein endothelial/subendothelial layer and may be expanded in culture. These cells display a fibroblast-like morphology, express mesenchymal markers, and are able to differentiate into, at least, adipocytes and osteoblasts.
Materials and Methods
Cell Culture Media, Plastics, and Chemicals
Medium 199 with Earle's salts, Dulbecco's modified Eagle's medium with low glucose (DMEM-LG), Dulbecco's phosphate-buffered saline (PBS), Earle's balanced salt solution (EBSS), penicillin-streptomycin, L-glutamine, sodium pyruvate, and trypsin-EDTA were obtained from GIBCO Invitrogen Corp. (Paisley, Scotland, UK; http://www.invitrogen.com). Fetal bovine serum ([FBS] preselected for the growth of human mesenchymal cells) was obtained from StemCell Technologies (Vancouver, Canada; http://www.stemcell.com). Collagenase type IV, bovine serum albumin (BSA), and Triton X-100 were acquired from Sigma Chemical Co. (St. Louis, MO; http://www.sigmaaldrich.com). Cell culture plastic was from Corning Inc. (Corning, NY; http://www.corning.com) and Sigma-Aldrich.
Isolation and Culture of MSC-Like Cells
Umbilical cords (n = 26; gestational ages, 39-40 weeks) were collected and processed within 6-12 hours after normal deliveries. The cord vein was canulated on both sides and washed out with EBSS. The vessel was filled with 0.1% collagenase in Medium 199 supplemented with antibiotics and incubated at 37°C for 15 minutes. The vein was then washed with EBSS and, after gentle “massage” of the cord, the suspension of endothelial and subendothelial cells was collected. The cells were centrifuged for 10 minutes at 600 g and resuspended in DMEM-LG supplemented with 20 mM HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, and 10% FBS. After counting, cell suspension was seeded in noncoated 75-cm2 culture flasks with a density of approximately 103 cells/cm2. Cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2, with a change of culture medium every other day.
Approximately 2 weeks later, when well-developed colonies of fibroblast-like cells appeared, cultures were washed with EBSS, harvested with 0.05% trypsin-0.02% EDTA, and passaged (without dilution) into a new flask for further expansion or onto 8-chamber Permanox slides for histochemical staining.
Immunophenotyping of Cultured Cells
Part of primary cultures in flasks, as well as cells cultured on chamber slides, were washed with EBSS and fixed for 15 minutes with 4% paraformaldehyde in PBS containing 0.1% Triton X-100 or 1% paraformaldehyde in PBS for visualization of intracellular and surface antigens, respectively. After several washes with PBS and PBS-1% BSA, cells were incubated for 1 hour with the following cell-specific antibodies: human von Willebrand factor (vWF); alpha-smooth muscle actin (ASMA); smooth muscle myosin (MySM); fibronectin (all from Sigma-Aldrich); CD34; E-selectin; ICAM-1; VCAM-1 (Becton Dickinson GmbH; Heidelberg, Germany; http://www.bd.com); monocyte-macrophage antigens (CD14, CD45, CD68; Biomeda; Foster City, CA; http://www.biomeda.com); PECAM-1 (clone VM64); and collagens I, II, and IV (kindly provided, respectively, by Dr. A. Mazurov and Dr. S. Domogatsky, Cardiology Research Center, Moscow). Steps of staining were performed using biotinylated anti-mouse or anti-rabbit IgG and extravidin-peroxidase complex (Extravidin staining kit, Sigma-Aldrich). Finally, the preparations were counterstained with hematoxylin and embedded in glycerol-gelatin.
The differentiation of MSC-like cells was assessed in the first- and second-passage cultures. Cells were cultured either in an adipogenic (0.5 μM isobutyl-methylxantine, 1 μM dexamethasone, 10 μM insulin, and 200 μM indomethacin) or osteogenic (0.1 μM dexamethasone, 10 μM β-glycerophosphate, and 50 μM ascorbate-phosphate; all from Sigma-Aldrich) medium . Two weeks later, intracellular lipid accumulation was visualized using Oil Red O staining; the examination for alkaline phosphatase activity (Alkaline phosphatase staining kit, Sigma-Aldrich) was used to assess osteogenic differentiation.
Initially, primary cultures were presented mostly by clusters of endothelial cells (ECs) with typical endothelial morphology. However, in contrast to parallel cultures growing in standard endothelial conditions (Medium 199 with 10% FBS), cells growing in DMEM-10% FBS did not spread, migrate, or proliferate. As a result, the endothelial islands remained compact. As soon as 1 week after cultivation, numerous fibroblast-like cells could be observed between ECs (Fig. 1A). Subsequently, they formed colonies, then expanded, and by the third week, a homogeneous layer of fibroblastoid (MSC-like) cells occupied the whole plastic surface (Figs. 1B–1D).
In contrast to neighboring ECs, MSC-like cells were vWF and PECAM-1 negative (Figs. 2A and 2B). Further characterization studies revealed that these cells did not contain MySM, did not express monocyte/macrophage antigens, were ASMA positive, and synthesized and deposited fibronectin and type I (but not type II or IV) collagen into the extracellular matrix.
The first-passage cultures (Figs. 1E and 1F) obtained by reseeding of the primary colonies presented as a practically homogeneous population of CD34− fibroblast-like cells expressing ASMA (Fig. 2C), fibronectin (Fig. 2D), type I collagen (Fig. 2E), and VCAM-1 (Fig. 2F); numerous cells were moderately ICAM-1 positive (not shown). The content of vWF- and PECAM-1-positive (endothelial) cells did not exceed 0.5%-1% of the total cell number (Figs. 2G and 2H).
Further characterization studies performed on MSC-like cells revealed their potential to differentiate into adipocytes and osteoblasts. Adipogenic differentiation was apparent after 1 week of incubation with adipogenic supplementation. By the end of the second week, almost all cells contained numerous Oil-Red-O-positive lipid droplets (Fig. 3A). Similarly, most of the MSC-like cells became alkaline-phosphatase-positive when the regular culture medium was replaced by osteogenic medium (Fig. 3B). Nontreated control cultures did not show spontaneous adipocyte or osteoblast formation even after 3-4 weeks of cultivation (Fig. 3C).
The results obtained suggest that MSC-like cells are present in the subendothelial layer of the human umbilical cord vein and can be successfully isolated, cultured, and expanded using routine technical approaches. Initially, the resulting primary cultures consist of two main cell types: vWF- and PECAM-1-positive endothelial cells and ASMA-positive MSC-like cells. However, described culture conditions seem to be inappropriate for EC growth; cells spread weakly and practically do not proliferate. Even after the first passage, the cultures became homogeneous and practically free of contaminating endothelium. Some primary isolates contained a limited number of smooth muscle cells (SMCs), but these cells could be easily differentiated at the earliest stages of culturing by typical morphology and strong positive staining for MySM. Consequently, cultures containing contaminating SMCs were discarded. Thus, as a result of short enzymatic digestion, it is possible to obtain cell populations positively differing from similar cell populations isolated from other sources alternative to bone marrow, particularly from processed lipoaspirate . Usually, only one contaminating cell type is present (ECs) that essentially does not affect the final outcome. Other cellular components of the venous wall or Wharton's jelly (SMCs, fibroblasts) are absent in the cell suspensions due to the short time of incubation with collagenase, leaving deeper layers intact. Pericytes, which are present in processed lipoaspirate and bone marrow aspirates as a component of the microvascular tree, are by definition absent in large vessels .
The results of morphological studies and immunophenotyping of cultured MSC-like cells from human umbilical cord vein suggest that these cells closely resemble cultured MSCs obtained from bone marrow and other sources [2, 3, 15–17]. Fibroblastoid morphology, absence of endothelial and leukocyte-associated markers, and expression of ASMA and cell adhesion molecules typical for myelosupportive stroma support the thought that they are mesenchymal progenitors. Currently, we will not speculate about the pluripotency of umbilical-cord-derived MSC-like cells; this aspect of their biology is under investigation. However, the preliminary results are promising; the predominant number of cells accumulates lipids or expresses alkaline phosphatase when exposed to proper culture conditions , i.e., displays at least bidirectional differentiation potential. If the multilineage differentiation capability of these cells is documented, the umbilical cord/placenta vessels may serve as a rich source of MSCs for experimental and clinical needs.
Authors thank Dr. Andrey G. Pronin (Division of Gynecology, 26th Maternity Hospital, Moscow) for help with umbilical cord collections.