Human Adipose-Derived Mesenchymal Stromal Cells Exhibit High HLA-DR Levels and Altered Cellular Characteristics under a Xeno-free and Serum-free Condition.

BACKGROUND: We have observed an increased expression of negative markers in some clinical-grade, xeno- and serum-free cultured adipose-derived mesenchymal stem/stromal cell (ADMSC) samples. It gave rise to concern that xeno- and serum-free conditions might have unexpected effects on human ADMSCs. This study aims to test this hypothesis for two xeno- and serum-free media, PowerStem MSC1 media (PS) and StemMACS MSC Expansion Media (SM), that support the in vitro expansion of ADMSCs. METHODS: We investigated the expression of negative markers in 42 clinical-grade ADMSC samples expanded in PS. Next, we cultured ADMSCs from seven donors in PS and SM and examined their growth and colony-forming ability, surface marker expression, differentiation, cell cycle and senescence, as well as genetic stability of two passages representing an early and late passage for therapeutic MSCs. RESULTS: 15 of 42 clinical-grade PS-expanded ADMSC samples showed an increased expression of negative markers ranging from 2.73% to 34.24%, which positively correlated with the age of donors. This rise of negative markers was related to an upregulation of Human Leukocyte Antigen - DR (HLA-DR). In addition, the PS-cultured cells presented decreased growth ability, lower frequencies of cells in S/G2/M phases, and increased ß-galactosidase activity in passage 7 suggesting their senescent feature compared to those grown in SM. Although MSCs of both PS and SM cultures were capable of multilineage differentiation, the PS-cultured cells demonstrated chromosomal abnormalities in passage 7 compared to the normal karyotype of their SM counterparts. CONCLUSIONS: These findings suggest that the SM media is more suitable for the expansion of therapeutic ADMSCs than PS. The study also hints a change of ADMSC features at more advanced passages and with increased donor's age. Thus, it emphasizes the necessity to cover these aspects in the quality control of therapeutic MSC products.

Standardized xeno- and serum-free culture platform enables large-scale expansion of high-quality mesenchymal stem/stromal cells from perinatal and adult tissue sources.

BACKGROUND AIMS: Mesenchymal stem/stromal cells (MSCs) are of interest for the treatment of graft-versus-host disease, autoimmune diseases, osteoarthritis and neurological and cardiovascular diseases. Increasing numbers of clinical trials emphasize the need for standardized manufacturing of these cells. However, many challenges related to diverse isolation and expansion protocols and differences in cell tissue sources exist. As a result, the cell products used in numerous trials vary greatly in characteristics and potency. METHODS: The authors have established a standardized culture platform using xeno- and serum-free commercial media for expansion of MSCs derived from umbilical cord (UC), bone marrow and adipose-derived (AD) and examined their functional characteristics. RESULTS: MSCs from the tested sources stably expanded in vitro and retained their biomarker expression and normal karyotype at early and later passages and after cryopreservation. MSCs were capable of colony formation and successfully differentiated into osteogenic, adipogenic and chondrogenic lineages. Pilot expansion of UC-MSCs and AD-MSCs to clinical scale revealed that the cells met the required quality standard for therapeutic applications. CONCLUSIONS: The authors' data suggest that xeno- and serum-free culture conditions are suitable for large-scale expansion and enable comparative study of MSCs of different origins. This is of importance for therapeutic purposes, especially because of the numerous variations in pre-clinical and clinical protocols for MSC-based products.

Influences of Xeno-Free Media on Mesenchymal Stem Cell Expansion for Clinical Application.

Mesenchymal stem/stromal cells (MSCs) are multipotent somatic stem/progenitor cells that can be isolated from various tissues and have attracted increasing attention from the scientific community. This is due to MSCs showing great potential for incurable disease treatment, and most applications of MSCs involve tissue degeneration and treatment of immune- and inflammation-mediated diseases. Conventional MSC cultures contain fetal bovine serum (FBS), which is a common supplement for cell development but is also a risk factor for exposure to animal-derived pathogens. To avoid the risks resulting from the xenogeneic origin and animal-derived pathogens of FBS, xeno-free media have been developed and commercialized to satisfy MSC expansion demands for human clinical applications. This review summarized and provided an overview of xeno-free media that are currently used for MSC expansion. Additionally, we discussed the influences of different xeno-free media on MSC biology with particular regard to cell morphology, surface marker expression, proliferation, differentiation and immunomodulation. The xeno-free media can be serum-free and xeno-free media or media supplemented with some human-originating substances, such as human serum, human platelet lysates, human umbilical cord serum/plasma, or human plasma-derived supplements for cell culture medium. These media have capacity to maintain a spindle-shaped morphology, the expression of typical surface markers, and the capacity of multipotent differentiation and immunomodulation of MSCs. Xeno-free media showed potential for safe use for human clinical treatment. However, the influences of these xeno-free media on MSCs are various and any xeno-free medium should be examined prior to being used for MSC cultures.

Differential Wound Healing Capacity of Mesenchymal Stem Cell-Derived Exosomes Originated From Bone Marrow, Adipose Tissue and Umbilical Cord Under Serum- and Xeno-Free Condition.

Exosomes are nano-scale and closed membrane vesicles which are promising for therapeutic applications due to exosome-enclosed therapeutic molecules such as DNA, small RNAs, proteins and lipids. Recently, it has been demonstrated that mesenchymal stem cell (MSC)-derived exosomes have capacity to regulate many biological events associated with wound healing process, such as cell proliferation, cell migration and blood vessel formation. This study investigated the regenerative potentials for cutaneous tissue, in regard to growth factors associated with wound healing and skin cell proliferation and migration, by exosomes released from primary MSCs originated from bone marrow (BM), adipose tissue (AD), and umbilical cord (UC) under serum- and xeno-free condition. We found crucial wound healing-mediated growth factors, such as vascular endothelial growth factor A (VEGF-A), fibroblast growth factor 2 (FGF-2), hepatocyte growth factor (HGF), and platelet-derived growth factor BB (PDGF-BB) in exosomes derived from all three MSC sources. However, expression levels of these growth factors in exosomes were influenced by MSC origins, especially transforming growth factor beta (TGF-ß) was only detected in UCMSC-derived exosomes. All exosomes released by three MSCs sources induced keratinocyte and fibroblast proliferation and migration; and, the induction of cell migration is a dependent manner with the higher dose of exosomes was used (20 µg), the faster migration rate was observed. Additionally, the influences of exosomes on cell proliferation and migration was associated with exosome origins and also target cells of exosomes that the greatest induction of primary dermal fibroblasts belongs to BMMSC-derived exosomes and keratinocytes belongs to UCMSC-derived exosomes. Data from this study indicated that BMMSCs and UCMSCs under clinical condition secreted exosomes are promising to develop into therapeutic products for wound healing treatment.

Induction of Antitumor Immunity by Exosomes Isolated from Cryopreserved Cord Blood Monocyte-Derived Dendritic Cells.

(1) Background: Dendritic cell (DC) vaccination has shown outstanding achievements in cancer treatment, although it still has some adverse side effects. Vaccination with DC-derived exosomes has been thought to overcome the side effects of the parental DCs. (2) Method: We performed the experiments to check the ability of cryopreserved umbilical cord blood mononuclear cell-derived DCs (cryo CBMDCs) and their exosomes to prime allogeneic T cell proliferation and allogeneic peripheral blood mononuclear cell (alloPBMCs) cytotoxicity against A549 lung cancer cells. (3) Results: We found that both lung tumor cell lysate-pulsed DCs and their exosomes could induce allogeneic T cell proliferation. Moreover, alloPBMCs primed with tumor cell lysate-pulsed DCs and their exosomes have a greater cytotoxic activity against A549 cells compared to unprimed cells and cells primed with unpulsed DCs and their exosomes. (4) Conclusion: Tumor cell lysate-pulsed DCs and their exosomes should be considered to develop into a novel immunotherapeutic strategy-e.g., vaccines-for patients with lung cancer. Our results also suggested that cryo umbilical cord blood mononuclear cells source, which is a readily and available source, is effective for generation of allogeneic DCs and their exosomes will be material for vaccinating against cancer.