Effectiveness of protocol for the isolation of Wharton’s Jelly stem cells in large-scale applications

The Wharton’s Jelly (WJ) of the umbilical cord (UC) is an excellent source of mesenchymal stem cells (MSCs) with a range of potential therapeutic applications. The present study was conducted to demonstrate the efficiency of the protocols used by Biogenea-Cellgenea Ltd. for isolation and expansion of WJ MSCs from donors across Greece. Umbilical cord samples were collected from 599 females following childbirth and processed for WJ MSC isolation. Stem cells were expanded using DMEM-based media and cell counts and overall viability figures derived using Trypan blue exclusion. To investigate the application of isolation and expansion protocols on samples received 1, 2, 3, 4 and 5 d after their collection, ten fresh samples were processed at these time intervals and evaluated. The cellular yield of most WJ samples was 1.1–5.0?×?10⁶ cells at 21–30 d after processing. As culture time increased, cell counts decreased. Statistical analysis of mean cell counts showed a significant reduction after 21 d. Finally, we demonstrate for the first time that it is possible to obtain satisfactory cell numbers from samples processed 1, 2, 3, 4 and even 5 d after collection. We have derived favourable data on the protocols used at Biogenea-Cellgenea Ltd. to isolate and culture MSCs from the WJ. Protocol choice is crucial when handling large numbers of samples on a daily basis and should be made to ensure the best possible outcome.     

Isolation,expansion and characterisation of mesenchymal stem cells from human bone marrow,adipose tissue,umbilical cord blood and matrix: a comparative study

The multipotent and immunosuppressive capacities of mesenchymal stem cells (MSCs) attract several scientists worldwide towards translational research focusing on treatment of diseases including liver failure. Though MSC’s have been isolated from different sources, researchers do not concur on the best source for expansion and clinical translation. In this study, we have compared the isolation, proliferation and expansion of MSCs from umbilical cord blood (UCB), Wharton’s Jelly (WJ), bone marrow (BM) and adipose tissue (AT). MSCs were isolated by density gradient separation from UCB, BM and AT and by both enzymatic and explant method for WJ. The MSCs are characterized by their ability to adhere to plastic, expression of positive (CD105, CD73, CD90, CD29, CD44) and negative (CD45, CD14, CD34) markers by flow cytometry and also by their in vitro adipogenic, osteogenic and chondrogenic differentiation. This comprehensive study clearly shows that WJ is better than UCB both in terms of rapidity, yield and ease of procedure. AT and BM are autologous sources for MSC’s but the specimen collection involves cumbersome and painful procedures and an invasive approach. However being autologous, they are safe and probable candidates for therapeutic future applications.

Electronic supplementary material

The online version of this article (doi:10.1007/s10616-014-9718-z) contains supplementary material, which is available to authorized users.     

Comparative Characterization of Cells from the Various Compartments of the Human Umbilical Cord Shows that the Wharton’s Jelly Compartment Provides the Best Source of Clinically Utilizable Mesenchymal Stem Cells

The human umbilical cord (UC) is an attractive source of mesenchymal stem cells (MSCs) with unique advantages over other MSC sources. They have been isolated from different compartments of the UC but there has been no rigorous comparison to identify the compartment with the best clinical utility. We compared the histology, fresh and cultured cell numbers, morphology, proliferation, viability, stemness characteristics and differentiation potential of cells from the amnion (AM), subamnion (SA), perivascular (PV), Wharton’s jelly (WJ) and mixed cord (MC) of five UCs. The WJ occupied the largest area in the UC from which 4.61 ± 0.57 x 10⁶ /cm fresh cells could be isolated without culture compared to AM, SA, PV and MC that required culture. The WJ and PV had significantly lesser CD40+ non-stem cell contaminants (26-27%) compared to SA, AM and MC (51-70%). Cells from all compartments were proliferative, expressed the typical MSC-CD, HLA, and ESC markers, telomerase, had normal karyotypes and differentiated into adipocyte, chondrocyte and osteocyte lineages. The cells from WJ showed significantly greater CD24+ and CD108+ numbers and fluorescence intensities that discriminate between MSCs and non-stem cell mesenchymal cells, were negative for the fibroblast-specific and activating-proteins (FSP, FAP) and showed greater osteogenic and chondrogenic differentiation potential compared to AM, SA, PV and MC. Cells from the WJ offer the best clinical utility as (i) they have less non-stem cell contaminants (ii) can be generated in large numbers with minimal culture avoiding changes in phenotype, (iii) their derivation is quick and easy to standardize, (iv) they are rich in stemness characteristics and (v) have high differentiation potential. Our results show that when isolating MSCs from the UC, the WJ should be the preferred compartment, and a standardized method of derivation must be used so as to make meaningful comparisons of data between research groups.     

Cryopreservation of hMSCs seeded silk nanofibers based tissue engineered constructs

Long term cryopreservation of tissue engineering constructs is of paramount importance to meet off-the shelf requirements for medical applications. In the present study, the effect of cryopreservation using natural osmolytes such as trehalose and ectoin with and without conventional Me₂SO on the cryopreservation of tissue engineered constructs (TECs) was evaluated. MSCs derived from umbilical cord were seeded on electrospun nanofibrous silk fibroin scaffolds and cultured to develop TECs. TECs were subjected to controlled rate freezing using nine different freezing solutions. Among these, freezing medium consisting of natural osmolytes like trehalose (40 mM), ectoin (40 mM), catalase (100 μg) as antioxidant and Me₂SO (2.5%) was found to be the most effective. Optimality of the chosen cryoprotectants was confirmed by cell viability (PI live/dead staining), cell proliferation (MTT assay), microstructure analysis (SEM), membrane integrity (confocal microscopy) and in vitro osteogenic differentiation (ALP assay, RT-PCR and histology) study carried out with post-thaw cryopreserved TECs. The mechanical integrity of the cryopreserved scaffold was found to be unaltered.     

Isolation of mesenchymal stem cells from equine umbilical cord blood

Background  

There are no published studies on stem cells from equine cord blood although commercial storage of equine cord blood for future autologous stem cell transplantations is available. Mesenchymal stem cells (MSC) have been isolated from fresh umbilical cord blood of humans collected non-invasively at the time of birth and from sheep cord blood collected invasively by a surgical intrauterine approach. Mesenchymal stem cells isolation percentage from frozen-thawed human cord blood is low and the future isolation percentage of MSCs from cryopreserved equine cord blood is therefore expectedly low. The hypothesis of this study was that equine MSCs could be isolated from fresh whole equine cord blood.     

Comparison of immunomodulatory properties of mesenchymal stem cells derived from adult human tissues

Mesenchymal stem cells (MSCs), which evoke only minimal immune reactivity, may have anti-inflammatory and immunomodulatory effects. In this study, we conducted a comparative analysis of the immunomodulatory properties of MSCs derived from adult human tissues including bone marrow (BM), adipose tissues (AT), umbilical cord blood (CB), and cord Wharton’s jelly (WJ). Using a multiple cytokine detection assay, we showed that there were no significant differences in levels of secreted factors from non-stimulated MSCs. We compared the immunosuppressive effect of BM-MSCs, AT-MSCs, CB-MSCs, and WJ-MSCs on phytohemagglutinin-induced T-cell proliferation. AT-MSCs, CB-MSCs, and WJ-MSCs effectively suppressed mitogen-induced T-cell proliferation as effectively as did BM-MSCs. Levels of interferon (IFN)-γ and tumor necrosis factor (TNF)-α secreted from activated T-cells increased over time, but these levels were significantly reduced when cocultured with each type of MSCs. In addition, the expression of hepatocyte growth factor, IL-10, transforming growth factor-β₁, cyclooxygenase (COX)-1, and COX-2 were unchanged in MSCs treated with IFN-γ and/or TNF-α, while indoleamine 2,3-dioxygenase (IDO) expression increased. IFN-γ and/or TNF-α produced by activated T-cells were correlated with induction of IDO expression by MSCs, which, in turn, suppressed T-cell proliferation. These findings suggest that MSCs derived from AT, CB, or WJ could be substituted for BM-MSCs for treatment of allogeneic conflicts.     

Optimization and scale-up of Wharton's jelly-derived mesenchymal stem cells for clinical applications

MSCs are promising candidates for stem cell therapy and regenerative medicine. Umbilical cord is the easiest obtainable biological source of MSCs and the Wharton's jelly of the umbilical cord is a rich source of fetus-derived stem cells. However, the use of MSCs for therapeutic application is based on their subsequent large-scale in vitro expansion. A fast and efficient protocol for generation of large quantities of MSCs is required to meet the clinical demand and biomedical research needs. Here we have optimized conditions for scaling up of WJ-MSCs. Low seeding density along with basic fibroblast growth factor (bFGF) supplementation in the growth medium, which is DMEM-KO, resulted in propagation of more than 1 x 10(8) cells within a time period of 15 days from a single umbilical cord. The upscaled WJ-MSCs retained their differentiation potential and immunosuppressive capacity. They expressed the typical hMSC surface antigens and the addition of bFGF in the culture medium did not affect the expression levels of HLA-DR and CD 44. A normal karyotype was confirmed in the large-scale expanded WJ-MSCs. Hence, in this study we attempted rapid clinical-scale expansion of WJ-MSCs which would allow these fetus-derived stem cells to be used for various allogeneic cell-based transplantations and tissue engineering.     

Quantitative analysis of composite umbilical cord tissue health using a standardized explant approach and an assay of metabolic activity

Background

Umbilical cord (UC) tissue can be collected in a noninvasive procedure and is enriched in progenitor cells with potential therapeutic value. Mesenchymal stromal cells (MSCs) can be reliably harvested from fresh or cryopreserved UC tissue by explant outgrowth with no apparent impact on functionality. A number of stem cell banks offer cryopreservation of UC tissue, alongside cord blood, for future cell-based applications. In this setting, measuring and monitoring UC quality is critical.

Comparison of vitrification and slow cooling for umbilical tissues

The tissue cryopreservation maintains the cellular metabolism in a quiescence state and makes the conservation possible for an indefinite period of time. The choice of an appropriate cryopreservation protocol is essential for maintenance of cryopreserved tissue banks. This study evaluated 10 samples of umbilical cord, from which small fragments of tissue (Wharton’s jelly and cord lining membrane) were subjected to two protocols of cryopreservation: slow cooling and vitrification. The samples were frozen for a period of time ranging from 5 to 78 days. The efficiency of cryopreservation was evaluated by testing cell viability, histological analysis, cell culture, cytogenetic analysis and comparison with the results of the fresh samples. The results showed that the slow cooling protocol was more efficient than the vitrification for cryopreservation of umbilical cord tissue, because it has caused fewer changes in the structure of tissue (edema and degeneration of the epithelium) and, despite the significant decrease cell viability compared to fresh samples, the ability of cell proliferation in vitro was preserved in most samples. In conclusion, this study showed that it is possible to cryopreserve small fragments of tissue from the umbilical cord and, to obtain viable cells capable of proliferation in vitro after thawing, contributing to the creation of a frozen tissue bank.     

Osteogenic differentiation of GFP-labeled human umbilical cord blood derived mesenchymal stem cells after cryopreservation

The osteogenic capacity of human umbilical cord blood derived mesenchymal stem cells (UCB-MSCs) has been demonstrated both in vitro and in vivo. Therefore, cell labeling and storage are becoming necessary for researching the potential therapeutic use of UCB-MSCs for bone tissue engineering. The aim of this study was to determine the effect of cryopreservation on the osteogenic differentiation of green fluorescent protein (GFP)-marked UCB-MSCs in vitro. MSCs were isolated from full-term human UCB, expanded, transfected with the GFP gene, and then cryopreserved in liquid nitrogen for 4 weeks. After thawing, cell surface antigen markers and osteogenic potential were analyzed, and the luminescence of these cells was observed by fluorescence microscopy. The results demonstrate that cryopreservation has no effect on the cell phenotype, GFP expression or osteogenic differentiation of UCB-MSCs, showing that cryopreserved GFP-labeled UCB-MSCs might be applied for bone tissue engineering.     

Isolation of three important types of stem cells from the same samples of banked umbilical cord blood

It is known that umbilical cord blood (UCB) is a rich source of stem cells with practical and ethical advantages. Three important types of stem cells which can be harvested from umbilical cord blood and used in disease treatment are hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs). Since these stem cells have shown enormous potential in regenerative medicine, numerous umbilical cord blood banks have been established. In this study, we examined the ability of banked UCB collected to produce three types of stem cells from the same samples with characteristics of HSCs, MSCs and EPCs. We were able to obtain homogeneous plastic rapidly-adherent cells (with characteristics of MSCs), slowly-adherent (with characteristics of EPCs) and non-adherent cells (with characteristics of HSCs) from the mononuclear cell fractions of cryopreserved UCB. Using a protocol of 48?h supernatant transferring, we successfully isolated MSCs which expressed CD13, CD44 and CD90 while CD34, CD45 and CD133 negative, had typical fibroblast-like shape, and was able to differentiate into adipocytes; EPCs which were CD34, and CD90 positive, CD13, CD44, CD45 and CD133 negative, adherent with cobble-like shape; HSCs which formed colonies when cultured in MethoCult medium.     

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 aimsMesenchymal 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.MethodsThe 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.ResultsMSCs 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.ConclusionsThe 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.     

Hypoxic Preconditioning Increases Survival and Pro-Angiogenic Capacity of Human Cord Blood Mesenchymal Stromal Cells In Vitro

Hypoxic preconditioning was shown to improve the therapeutic efficacy of bone marrow-derived multipotent mesenchymal stromal cells (MSCs) upon transplantation in ischemic tissue. Given the interest in clinical applications of umbilical cord blood-derived MSCs, we developed a specific hypoxic preconditioning protocol and investigated its anti-apoptotic and pro-angiogenic effects on cord blood MSCs undergoing simulated ischemia in vitro by subjecting them to hypoxia and nutrient deprivation with or without preceding hypoxic preconditioning. Cell number, metabolic activity, surface marker expression, chromosomal stability, apoptosis (caspases-3/7 activity) and necrosis were determined, and phosphorylation, mRNA expression and protein secretion of selected apoptosis and angiogenesis-regulating factors were quantified. Then, human umbilical vein endothelial cells (HUVEC) were subjected to simulated ischemia in co-culture with hypoxically preconditioned or naïve cord blood MSCs, and HUVEC proliferation was measured. Migration, proliferation and nitric oxide production of HUVECs were determined in presence of cord blood MSC-conditioned medium. Cord blood MSCs proved least sensitive to simulated ischemia when they were preconditioned for 24 h, while their basic behavior, immunophenotype and karyotype in culture remained unchanged. Here, “post-ischemic” cell number and metabolic activity were enhanced and caspase-3/7 activity and lactate dehydrogenase release were reduced as compared to non-preconditioned cells. Phosphorylation of AKT and BAD, mRNA expression of BCL-XL, BAG1 and VEGF, and VEGF protein secretion were higher in preconditioned cells. Hypoxically preconditioned cord blood MSCs enhanced HUVEC proliferation and migration, while nitric oxide production remained unchanged. We conclude that hypoxic preconditioning protects cord blood MSCs by activation of anti-apoptotic signaling mechanisms and enhances their angiogenic potential. Hence, hypoxic preconditioning might be a translationally relevant strategy to increase the tolerance of cord blood MSCs to ischemia and improve their therapeutic efficacy in clinical applications.     

Characterizing human mesenchymal stromal cells’ immune-modulatory potency using targeted lipidomic profiling of sphingolipids

Cell therapies are expected to increase over the next decade owing to increasing demand for clinical applications. Mesenchymal stromal cells (MSCs) have been explored to treat a number of diseases, with some successes in early clinical trials. Despite early successes, poor MSC characterization results in lessened therapeutic capacity once in vivo. Here, we characterized MSCs derived from bone marrow (BM), adipose tissue and umbilical cord tissue for sphingolipids (SLs), a class of bioactive lipids, using liquid chromatography/tandem mass spectrometry. We found that ceramide levels differed based on the donor's sex in BM-MSCs. We detected fatty acyl chain variants in MSCs from all three sources. Linear discriminant analysis revealed that MSCs separated based on tissue source. Principal component analysis showed that interferon-γ–primed and unstimulated MSCs separated according to their SL signature. Lastly, we detected higher ceramide levels in low indoleamine 2,3-dioxygenase MSCs, indicating that sphingomyelinase or ceramidase enzymatic activity may be involved in their immune potency.     

Characterization of mesenchymal stem cells isolated from the human umbilical cord

Numerous papers have reported that mesenchymal stem cells (MSCs) can be isolated from various sources such as bone marrow, adipose tissue and others. Nonetheless it is an open question whether MSCs isolated from different sources represent a single cell lineage or if cells residing in different organs are separate members of a family of MSCs. Subendothelial tissue of the umbilical cord vein has been shown to be a promising source of MSCs. The aim of this study was to isolate and characterize cells derived from the subendothelial layer of umbilical cord veins as regards their clonogenicity and differentiation potential. The results from these experiments show that cells isolated from the umbilical cord vein displayed fibroblast-like morphology and grew into colonies. Immunophenotyping by flow cytometry revealed that the isolated cells were negative for the hematopoietic line markers HLA-DR and CD34 but were positive for CD29, CD90 and CD73. The isolated cells were also positive for survivin, Bcl-2, vimentin and endoglin, as confirmed by RT-PCR and immunofluorescence. These cells can be induced to differentiate into osteogenic and adipogenic cells, but a new finding is that these cells can be induced to differentiate into endothelial cells expressing CD31, vWF and KDR-2, and also form vessel-like structures in Matrigel. The differentiated cells stopped expressing survivin, thus showing a diminished proliferative potential. It can be assumed that the subendothelial layer of the umbilical cord vein contains a population of cells with the overall characteristics of MSCs, with the additional capability to transform into endothelial cells.     

Differential immunomodulation of human mesenchymal stromal cells from various sources in an inflammation mimetic milieu

Mesenchymal stromal cells (MSCs) are very advantageous in the field of regenerative medicine because of their immunomodulatory properties. However, reports show that these properties vary from source to source. Hence, understanding the source-dependent specificity of MSCs and their immunomodulatory abilities will enable optimal use of MSCs in cell-based therapies. Here, we studied human MSCs from three different sources, adipose tissue (AT), bone marrow (BM) and Wharton's jelly (WJ), with respect to phenotypic responses of human peripheral blood mononuclear immune cells (hPBMCs/MNCs) and the concurrent changes in cytokine expression in MSCs, under mitogen-stimulated co-culture conditions. We used cytometric analysis to study the immunoregulatory properties of MSCs on MNCs and cytokine profiling of MSCs using a customized PCR array and solid-phase sandwich enzyme-linked immunosorbent assay. Our results reveal differential modulation of immune cells as well as MSCs upon activation by the mitogen phytohemagglutinin, independently and in co-culture. Notably, we observed source-specific MSC-cytokine signatures under stimulated conditions. Our results show that AT-MSCs up-regulate VEGF, BM-MSCs up-regulate PTGS-2 and WJ-MSCs increase expression of IDO considerably compared with controls. This remarkable modulation in source-specific cytokine expression was also validated at a functional level by quantitative protein expression studies. In our hands, even though MSCs from AT, BM and WJ sources exhibit characteristic immunomodulatory properties, our results highlight that MSCs sourced from different tissues may exhibit unique cytokine signatures and thus may be suitable for specific regenerative applications.     

Consensus International Council for Commonality in Blood Banking Automation–International Society for Cell & Gene Therapy statement on standard nomenclature abbreviations for the tissue of origin of mesenchymal stromal cells

The Cellular Therapy Coding and Labeling Advisory Group of the International Council for Commonality in Blood Banking Automation and the International Society for Cell & Gene Therapy mesenchymal stromal cell (MSC) committee are providing specific recommendations on abbreviating tissue sources of culture-adapted MSCs. These recommendations include using abbreviations based on the ISBT 128 terminology model that specifies standard class names to distinguish cell types and tissue sources for culture-adapted MSCs. Thus, MSCs from bone marrow are MSC(M), MSCs from cord blood are MSC(CB), MSCs from adipose tissue are MSC(AT) and MSCs from Wharton's jelly are MSC(WJ). Additional recommendations include using these abbreviations through the full spectrum of pre-clinical, translational and clinical research for the development of culture-adapted MSC products. This does not apply to basic research focused on investigating the developmental origins, identity or functionalities of endogenous progenitor cells in different tissues. These recommendations will serve to harmonize nomenclature in describing research and development surrounding culture-adapted MSCs, many of which are destined for clinical and/or commercial translation. These recommendations will also serve to align research and development efforts on culture-adapted MSCs with other cell therapy products.