Optimization of human umbilical cord blood-derived mesenchymal stem cell isolation and culture methods in serum- and xeno-free conditions

Background Although umbilical cord blood (UCB) is identified as a source of mesenchymal stem cells (MSCs) with various advantages, the success in cell isolation is volatile. Therefore, it is necessary to optimize methods of cord blood-derived MSC (UCB-MSC) isolation and culture. In this study, we evaluated the efficiency of UCB-MSC isolation and expansion using different commercially available serum- and xeno-free media and investigated the capacity of autologous serum and plasma as a supplement to support cell proliferation. Additionally, we defined the presence of multilineage-differentiating stress-enduring (Muse) cells in the UCB-MSC population. Functions of UCB-MSC in in vitro angiogenesis processes and anti-cancer were also verified. Methods Mononuclear cells were isolated using density gradient separation and cultured in four commercial media kits, as well as four surface coating solutions. UCB-MSCs were characterized and tested on tube formation assay, and co-cultured with SK-MEL cells in a transwell system. Results The results showed that only StemMACS™ MSC Expansion Media is more appropriate to isolate and culture UCB-MSCs. The cells exhibited a high cell proliferation rate, CFU forming capability, MSC surface marker expression, trilineage differentiate potential, and chromosome stability. In addition, the culture conditions with autologous serum coating and autologous plasma supplement enhanced cell growth and colony forming. This cell population contained Muse cells at rate of 0.3%. Moreover, UCB-MSCs could induce the tube formation of human umbilical vein endothelial cells and inhibit more than 50% of SK-MEL cell growth. Conclusions UCB-MSCs could be high-yield isolated and expanded under serum- and xeno-free conditions by using the StemMACS™ MSC Expansion Media kit. Autologous serum coating and plasma supplement enhanced cell proliferation. These UCB-MSCs had effected the tube formation process and an anti-cancer impact.

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. Graphical abstract

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.

Improved Models of Human Endometrial Organoids Based on Hydrogels from Decellularized Endometrium

Organoids are three-dimensional (3D) multicellular tissue models that mimic their corresponding in vivo tissue. Successful efforts have derived organoids from primary tissues such as intestine, liver, and pancreas. For human uterine endometrium, the recent generation of 3D structures from primary endometrial cells is inspiring new studies of this important tissue using precise preclinical models. To improve on these 3D models, we decellularized pig endometrium containing tissue-specific extracellular matrix and generated a hydrogel (EndoECM). Next, we derived three lines of human endometrial organoids and cultured them in optimal and suboptimal culture expansion media with or without EndoECM (0.01 mg/mL) as a soluble additive. We characterized the resultant organoids to verify their epithelial origin, long-term chromosomal stability, and stemness properties. Lastly, we determined their proliferation potential under different culture conditions using proliferation rates and immunohistochemical methods. Our results demonstrate the importance of a bioactive environment for the maintenance and proliferation of human endometrial organoids.

Expression of CD146 and Regenerative Cytokines by Human Placenta-Derived Mesenchymal Stromal Cells upon Expansion in Different GMP-Compliant Media

Mesenchymal stromal cells (MSCs) have been successfully employed in clinical applications. In most studies, autologous MSCs from the bone marrow (bmMSCs) were used, and others employed autologous adipose tissue-derived stromal cells (ADSCs). Recently, clinical feasibility studies provided evidence that MSCs from human term placenta (pMSCs) can be used for homologous therapy facilitating access to regenerative cells in emergency situations, when autologous cells are not available or not suitable. We therefore investigated the expression of MSC stemness marker CD146 and the expression of neuro- and myoregenerative cytokines by human pMSCs after expansion in three different media compliant with good manufacturing protocols (GMP) in comparison to pMSCs expanded in a commercial MSC expansion media. To replace xenobiotic serum in the GMP-compliant media employed in this study, either human serum, human serum plus platelet lysate (PLL), or human plasma plus PLL was used. We report that enrichment of media with PLL accelerates pMSC proliferation but reduces the expression of the stemness marker CD146 significantly, while PLL deprivation enhanced the CD146 expression. In contrast, the reduced expression of CD146 by PLL deprivation was not observed on bmMSCs. The expression of the cytokines investigated was not modulated significantly by PLL. We conclude that accelerated expansion of pMSCs in GMP-compliant media enriched by PLL reduces the expression of stemness marker CD146, but does not influence the expression of neuro- and myoregenerative cytokines.

Long-term expansion and differentiation of adult murine epidermal stem cells in 3D organoid cultures

Mammalian epidermal stem cells maintain homeostasis of the skin epidermis and contribute to its regeneration throughout adult life. While 2D mouse epidermal stem cell cultures have been established decades ago, a long-term, feeder cell- and serum-free culture system recapitulating murine epidermal architecture has not been available. Here we describe an epidermal organoid culture system that allows long-term, genetically stable expansion of adult epidermal stem cells. Our epidermal expansion media combines atypically high calcium concentrations, activation of cAMP, FGF, and R-spondin signaling with inhibition of bone morphogenetic protein (BMP) signaling. Organoids are established robustly from adult mouse skin and expand over at least 6 mo, while maintaining the basal-apical organization of the mouse interfollicular epidermis. The system represents a powerful tool to study epidermal homeostasis and disease in vitro.

Thrombopoietin Affects the Behavior of Myelofibrosis Stem Cells

Recently the presence of myelofibrosis (MF) stem cells (MF-SC) in the spleens of MF patients which when transplanted into NOD/SCID/IL2R null (NSG) mice were capable of generating multiple hematopoietic lineages that belonged to the malignant clone has been observed. Although MF is characterized by marrow megakaryocyte (Mk) hyperplasia, limited numbers of human marrow Mks were, however, observed in these transplanted mice and evidence of both marrow and splenic fibrosis 9 months after the transplantation was lacking (Wang X, et al. J Clin Invest. 2012; 122:3888). Splenic MF CD34+ cells did retain the capacity to differentiate in vitro into CD41a+ and CD61+ Mks in the presence of thrombopoietin (TPO). We therefore investigated whether the administration of romiplostim, a novel human TPO peptide mimetic which lacks homology to endogenous TPO in MF humanized mice, might create appropriate environmental cues which affect the behavior of MF-SCs. Elevated levels of TPO (345±114ng/ml) were detected in MF plasmas (n=13) as compared to levels detected in normal plasma (10±4ng/ml, n=6, P=0.049), indicating the possibility that TPO affects MF-SCs and MF hematopoietic progenitor cells (HPC). Following the culture of CB (n=3) or PB MF CD34+ cells (n=4) for 1 and 2 wks in serum free expansion media (SFEM) supplemented with SCF (50ng/ml) + romiplostim (100ng/ml), the numbers of total cells, CD34+Lin- cells and assayable HPCs, including CFU-Mk, CFU-GM and BFU-E, CD41a+CD34-CD15- cells and CD15+CD34-CD41a- cells generated were greater (CB) or similar (MF) to the number generated in the cultures containing SCF + TPO (100ng/ml). Moreover, similar proportions of colonies (CFU-GM+BFU-E) generated in cultures supplemented with SCF+TPO or SCF+ romiplostim (100ng/ml, 1000ng/ml) were JAK2V617F-positive. These findings suggest that both romiplostim and TPO are capable of promoting MF-SC and HPC proliferation in vitro. To assess the effects of romiplostim on human (h) platelet production, CB CD34+ cells (5×105) were transplanted via the tail vein into eight- to nine-week-old sublethally irradiated (240 cGy) NSG mice. One week after transplantation, mice were treated with water or 10, 100 or 1000 µg/kg of romiplostim. Both the percentage and number of hCD41a+ platelets in the PB increased 4 days following the treatment with 10ug/kg romiplostim, and peaked at day 8 with a 2.58±0.29 fold increase in the percentage (P<0.05) and 3.51±1.32 fold increase in the absolute number (P<0.05) of hCD41a+ platelets being observed. The number of human platelets in the PB, were reduced to the levels detected in mice not receiving romiplostim by day 15. MF splenic CD34+ cells (5-10×105) were next transplanted into romiplostim (10ug/kg or 30ug/kg) treated or control NSG mice. Two months after the transplantation, a 1.83±0.62 fold increase in the number of hCD41a+ cells was observed in the PB of mice receiving splenic MF CD34+ cells and romiplostim (10ug/kg) as compared with mice not treated with romiplostim. Moreover, a 7.13±2.13 fold elevation in the percentage of hCD45+ cells was detected in the PB of mice receiving splenic MF CD34+ cells and romiplostim (10ug/kg) as compared with mice not receiving romiplostim. Moreover, enhanced hCD45+ cell chimerism was achieved in both the marrow (55.6%, 7.2%) and spleen (47.7%, 1.5%) of mice receiving splenic CD34+ cells from 2 MF patients and romiplostim as compared with mice not receiving romiplostim (marrow: 30.4%, 5.6%; spleen: 23.6%, 0.9%), respectively. The splenic MF CD34+ cells receiving or not receiving romiplostim had a similar differentiation patterns in the marrow and spleen, however, the numbers of hCD33+, hCD19+, hCD3+, hCD41a+ and hCD15+ cells detected in mice treated with romiplostim were dramatically greater than those detected in mice not receiving romiplostim. Greater numbers of hCD34+ cells were detected in the BM (7.1%; 3.5%) of recipient mice receiving romiplostim as that detected in mice not receiving romiplostim (6.6%; 0.6%). Furthermore, a similar degree of hCD45+ cell and hCD34+ cell chimerism was observed in both the marrow and spleen of mice receiving splenic MF CD34+ cells and romiplostim 4 months after the transplantation. These findings suggest that the administration of thrombopietin agonist has a profound effect on the behavior of MF-SCs and that the elevated levels of TPO documented in MF patients likely has important effects on the biology of MF-SCs. Disclosures Wang: The MPN Research Foundation (MPNRF) and the Leukemia & Lymphoma Society (LLS) : Research Funding.