Isolation and Characterization of Mesenchymal Stem Cells From the Sub-Amniotic Human Umbilical Cord Lining Membrane

Abstract

The use of human stem cells (SCs) is a promising novel approach for the treatment of many diseases and injuries. Umbilical cord and amniotic membrane represent good sources for SCs, because they are abundant sources and there are less ethical issues unlike embryonic SCs. We aimed to isolate and characterize adult SCs from the subamnion region of the umbilical cord/amniotic membrane. Because mesenchymal stem cells (MSCs) are thought to show less immunogenicity, we first focused on the characterization of MSCs. Significant expression of typical SC-specific markers, such as SSEA-4, Oct-4, and Nanog was observed. Subamniotic MSCs did not lose the expression of Oct-4 and Nanog after freeze-thawing. Cell surface expression of MSC markers (CD73 and CD105) was confirmed by flow cytometry, and cells also differentiated into adipogenic, osteogenic, and chondrogenic lineages. On the other hand, typical embryonic SC-specific markers were not expressed and the cells also did not grow in soft agar. Thus, the subamniotic MSCs are distinct from embryonic SCs and do not show tumorigenicity in vitro. The cord lining membrane (subamniotic) MSCs isolated by our method maintain typical characteristics of MSCs in vitro, but also showed several specific features.

Concise Review: Wharton's Jelly: The Rich, but Enigmatic, Source of Mesenchymal Stromal Cells

“…The amnion consists solely of epithelial cells, and thus not MSCs 58, but others have also noted stem cell characteristics in this population 59, although caution should be expressed since Miki et al 59 referenced an extraction technique first described by Akle et al 60 where they stated that “ The amniotic membrane thus obtained consisted of an epithelial monolayer on a basement membrane with an underlying collagen matrix containing a few fibroblasts .”(another example of how harvesting procedures can affect the interpretation of experimental outcomes). Nevertheless, the sub‐amnion is mesenchymal and does contain MSCs 13, 18, 57, 61. Thus, there is evidence, at least in the umbilical cord, that MSCs can be isolated from nonperivascular regions.…”

Transplantation of umbilical cord–derived mesenchymal stem cells as a novel strategy to protect the central nervous system: technical aspects, preclinical studies, and clinical perspectives

“…In this context, the UC is gaining increasing attention because it contains high amounts of MSCs that have been shown to be isolatable from 100% of the samples tested by several groups (27,42,45,46). Depending on the derivation protocol used, and the part of the UC from which they were isolated, various types of hUC-MSCs have been reported: UC matrix stem cells, UC perivascular cells, UC stromal cells, Wharton's jelly stem cells, cord-lining membrane MSCs, and UC-lining stem cells (5,20,30,(47)(48)(49). Even though the MSCs isolated from these various compartments have not yet been compared with each other, they have been shown to resemble BM-MSCs while retaining some specific characteristics: they express mesenchymal markers and, furthermore, constitutively express early embryonic transcription factors such as Nanog, Oct-4, and Sox-2 (50,51); they tend to express lower levels of human leukocyte antigen-DR than their BM counterparts (20,27); their growth behavior, including population doubling time and capacity to be maintained in culture over a long period, has been shown to be better than that of BM-MSCs (27,45,52); the frequency of colony-forming units-fibroblasts in the UC has been demonstrated to be higher than in the BM, ranging from 1:333 to 1:1,609 (vs. 1:10,000-1:36,000 in the BM) depending on the laboratory and the isolation procedures used (20,27).…”

Cord lining progenitor cells: potential in vitro adipogenesis model

“…Previous data have shown that they are multipotent progenitor cells. 11,12 Morphology and Oil Red O staining The morphology of CLMCs that were subjected to differentiation was observed to change from spindle-like fibroblastic cells at day 0 ( Figure 1) to irregular, cuboidal-shaped cells that were adipocytes-like at day 20 ( Figure 2). The intensity of Oil Red O staining in all four treatment groups gradually increased from day 1 to day 20, indicating accumulation of lipid droplets.…”