Optimization of Culture Conditions for the Expansion of Umbilical Cord-Derived Mesenchymal Stem or Stromal Cell-Like Cells Using Xeno-Free Culture Conditions

Abstract

First isolated from bone marrow, mesenchymal stem or stromal cells (MSC) were shown to be present in several postnatal and extraembryonic tissues as well as in a large variety of fetal tissues (e.g., fatty tissue, dental pulp, placenta, umbilical cord blood, and tissue). In this study, an optimized protocol for the expansion of MSC-like cells from whole umbilical cord tissue under xeno-free culture conditions is proposed. Different fetal calf sera and human serum (HS) were compared with regard to cell proliferation and MSC marker stability in long-term expansion experiments, and HS was shown to support optimal growth conditions. Additionally, the optimal concentration of HS during the cultivation was determined. With regard to cell proliferative potential, apoptosis, colony-forming unit fibroblast frequency, and cell senescence, our findings suggest that an efficient expansion of the cells is carried out best in media supplemented with 10% HS. Under our given xeno-free culture conditions, MSC-like cells were found to display in vitro immunoprivileged and immunomodulatory properties, which were assessed by co-culture and transwell culture experiments with carboxyfluorescein diacetate succinimidyl ester-labeled peripheral blood mononuclear cells. These findings may be of great value for the establishment of biotechnological protocols for the delivery of sufficient cell numbers of high quality for regenerative medicine purposes.

The Nontoxic Cell Cycle Modulator Indirubin Augments Transduction of Adeno-Associated Viral Vectors and Zinc-Finger Nuclease-Mediated Gene Targeting

“…Umbilical cordderived mesenchymal stromal cells (UC-MSCs) were isolated as previously described (Hatlapatka et al, 2011) and cultured in a-MEM Glutamax (Invitrogen) supplemented with 10% FCS selected for MSC (Stem Cell Technologies), 100 IU/ml penicillin, and 10 mg/ml streptomycin (PAA Laboratories GmbH). The polyclonal target cells HeLa.696, HT1080.696, U2OS.696, and UC-MSC.696 were generated by lentiviral transduction as previously described (Cornu and Cathomen, 2007;Gellhaus et al, 2010).…”

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

“…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).…”

“…Thus, they display immune-privilege properties (52)(53)(54)(55), have been shown to be well tolerated in allogeneic transplantation experiments (56,57), and can be differentiated into cells of mesodermal origin such as bone, cartilage, and adipose tissue (58). Moreover, hUC-MSCs cultured in neural induction medium develop neural morphologies and express neural markers such as Nestin, glial fibrillary acidic protein, and NeuN (5,46,59,60), suggesting that they have great potential for cell-based therapies in the context of central nervous system diseases.…”

“…To avoid the disadvantages of enzymatic digestion, which has been shown to potentially alter cell proliferation and function, some groups have developed UC explant culture approaches that have been shown to be simple, reproducible, and efficient (63)(64)(65)(66). However, considering the heterogeneity of the processes used for cell isolation and expansion, and in view of their use for clinical applications, some groups have tried to develop optimized and standardized methods based on the use of xeno-and serum-free culture media (52,67,68) or by taking advantage of the plastic-adherence properties of MSCs that allow cell expansion without UC dissection or enzymatic digestion (69). In addition, in view of their clinical use in both the autologous and allogeneic settings, hUC-MSCs are likely to be cryopreserved and expanded after an initial thawing step.…”

Embryonic stem cells conditioned medium enhances Wharton’s jelly-derived mesenchymal stem cells expansion under hypoxic condition

“…At present, serumfree medium is usually selected for clinical grade expansion of stem cells to prevent prion or xenogeneic proteins transmissions. Although 10 % human serum has shown to support cell proliferation of umbilical cord-derived MSCs under xeno-free culture condition, this medium remains including serum with serves as undesirable source of pathogen contamination (Hatlapatka et al 2011). Human platelet lysate (hPL) has been shown to support MSCs expansion (Capelli et al 2007;Xia et al 2011;Ben Azouna et al 2012).…”