Mesenchymal stromal cell‐derived factors promote the colonization of collagen 3D scaffolds with human skin cells

The development of stem cell technology in combination with advances in biomaterials has opened new ways of producing engineered tissue substitutes. In this study, we investigated whether the therapeutic potential of an acellular porous scaffold made of type I collagen can be improved by the addition of a powerful trophic agent in the form of mesenchymal stromal cells conditioned medium (MSC‐CM) in order to be used as an acellular scaffold for skin wound healing treatment. Our experiments showed that MSC‐CM sustained the adherence of keratinocytes and fibroblasts as well as the proliferation of keratinocytes. Moreover, MSC‐CM had chemoattractant properties for keratinocytes and endothelial cells, attributable to the content of trophic and pro‐angiogenic factors. Also, for the dermal fibroblasts cultured on collagen scaffold in the presence of MSC‐CM versus serum control, the ratio between collagen III and I mRNAs increased by 2‐fold. Furthermore, the gene expression for α‐smooth muscle actin, tissue inhibitor of metalloproteinase‐1 and 2 and matrix metalloproteinase‐14 was significantly increased by approximately 2‐fold. In conclusion, factors existing in MSC‐CM improve the colonization of collagen 3D scaffolds, by sustaining the adherence and proliferation of keratinocytes and by inducing a pro‐healing phenotype in fibroblasts.     

Differentiation and regeneration potential of mesenchymal progenitor cells derived from traumatized muscle tissue

Mesenchymal stem cell (MSC) therapy is a promising approach to promote tissue regeneration by either differentiating the MSCs into the desired cell type or by using their trophic functions to promote endogenous tissue repair. These strategies of regenerative medicine are limited by the availability of MSCs at the point of clinical care. Our laboratory has recently identified multipotent mesenchymal progenitor cells (MPCs) in traumatically injured muscle tissue, and the objective of this study was to compare these cells to a typical population of bone marrow derived MSCs. Our hypothesis was that the MPCs exhibit multilineage differentiation and expression of trophic properties that make functionally them equivalent to bone marrow derived MSCs for tissue regeneration therapies. Quantitative evaluation of their proliferation, metabolic activity, expression of characteristic cell-surface markers and baseline gene expression profile demonstrate substantial similarity between the two cell types. The MPCs were capable of differentiation into osteoblasts, adipocytes and chondrocytes, but they appeared to demonstrate limited lineage commitment compared to the bone marrow derived MSCs. The MPCs also exhibited trophic (i.e. immunoregulatory and pro-angiogenic) properties that were comparable to those of MSCs. These results suggest that the traumatized muscle derived MPCs may not be a direct substitute for bone marrow derived MSCs. However, because of their availability and abundance, particularly following orthopaedic injuries when traumatized muscle is available to harvest autologous cells, MPCs are a promising cell source for regenerative medicine therapies designed to take advantage of their trophic properties.     

Mixed effects of long-term frozen storage on cord tissue stem cells

Background aimsCord tissue (CT) storage is promoted as an opportunity to preserve a source of mesenchymal stromal cells (MSCs) for future use. We analyzed maximal MSC yields from fresh and frozen CT including functional capacity after long-term cryopreservation as a means of assessing potential utility.MethodsCT was evaluated immediately upon harvest or frozen and banked for 5 years before analysis. Upon thawing, cell viability and yield were determined, as were growth characteristics and the ability to differentiate into various tissues. After thawing, enzymatic digestion of CT to release MSCs resulted in poor cell recoveries and few viable cells, requiring explant cultures to recover sufficient cell numbers for analysis. Upon expansion of surviving cells, fluorescence-activated cell sorter analysis showed the cells to be MSCs based on phenotype (CD34–, 45–, 44+, 90+, 105+) and function (ability to form adipocytes and osteoblasts). Frozen CT, however, exhibited decreased plating efficiency, increased doubling times but near equivalent maximum cell expansion, compared with fresh CT.ConclusionsPoor cell yields and recoveries, along with slower growth characteristics, make frozen CT a less-than-optimal choice for MSC banking, despite good functional recovery. In addition, because the amount of fresh CT available at birth is limited and total MSC yields are low, even fresh CT-MSC requires extensive in vitro expansion before clinical use, which limits it application.     

Human mesenchymal stromal cells can uptake and release ciprofloxacin,acquiring in vitro anti-bacterial activity

Background aimsTraditional antibiotic therapy is based on the oral or systemic injection of antibiotics that are often unable to stop a deep infection (eg, osteomyelitis). We studied whether or not bone marrow stromal cells (BM-MSCs) are able to uptake and release ciprofloxacin (CPX), a fluoroquinolone considered the drug of choice for the treatment of chronic osteomyelitis because of its favorable penetration into poorly vascularized sites of infection.MethodsHuman bone marrow stromal cells (BM-MSCs) were primed with CPX (BM-MSCsCPX) according to a methodology previously standardized in our laboratory for paclitaxel (PTX). The anti-microbial activity of CPX released from BM-MSCs cells (BM-MSCsCPX-CM) or supernatant from cell lysate (BM-MSCsCPX-LYS) was evaluated by agar dilution and microdilution methods on three bacterial strains (Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa). To investigate whether or not primed cells (BM-MSCsCPX) were able to directly act on the bacterial growth, co-colture was performed by mixing E. coli suspension to an increasing number of BM-MSCsCPX. The anti-bacterial activity was determined as number of BM-MSCsCPX that completely inhibited bacterial growth.ResultsThe results demonstrated that BM-MSCsCPX are able to uptake and then release CPX in the conditioned medium. The loaded antibiotic maintains its active form throughout the process as tested on bacteria.ConclusionsOur findings suggest that CPX-loaded MSCs may represent an important device for carrying and delivering CPX (and perhaps other antibiotics) into infected deep microenvironments; they could be used for local application and by systemic infusion when their homing capacity into the bone is cleared.     

Nonadhesive populations in cultures of mesenchymal stromal cells from hematopoietic organs in mouse and rat

The study of adhesive properties of multipotent mesenchymal stromal cells evaluated from fibroblast colony-forming units in the bone marrow of adult mice and rats in populations of cells attached and unattached to plastic substrate after 2 h to 7 days in culture demonstrated both similarities and differences. The increase in the fibroblast colony-forming units in the adhesive population peaked on day 7 of in vitro culture in both cases; however, nearly no fibroblast colony-forming units were observed in the nonadhesive population from the mouse bone marrow in this period. Conversely, the number of colonies from the rat bone marrow nonadhesive population on day 7 of culture considerably increased, and this nonadhesive population in long-term culture became the source for subsequent nonadhesive subpopulations containing fibroblast colony-forming units. After 7 days of in vitro culture, the suspension of cells isolated from the liver of 17-day-old rat fetuses also contained a fraction of unattached fibroblast colony-forming units. In the nonadhesive subpopulations from the bone marrow and fetal liver, fibroblast colony-forming units were observed up to day 48 and 30, respectively. Stromal cell precursors of nonadhesive subpopulations from the rat bone marrow featured a period of colony formation reduced to 7 days (i.e., they were formed 1.5-2 times faster compared to the primary culture). The total number of fibroblast colony-forming units from all nonadhesive subpopulations was roughly 6 and 7.4 times that of the adhesive population of the primary culture from the bone marrow and fetal liver, respectively. Considering that the mammalian bone marrow remains the preferred source of mesenchymal stromal cells, using nonadhesive subpopulations in the presented culture system can considerably increase the yield of stromal precursor cells     

Plasminogen activator inhibitor-1 and tenascin-C secreted by equine mesenchymal stromal cells stimulate dermal fibroblast migration in vitro and contribute to wound healing in vivo

Background

Impaired cutaneous wound healing is common in humans, and treatments are often ineffective. Based on the significant emotional and economic burden of impaired wound healing, innovative therapies are needed. The potential of mesenchymal stromal cell (MSC)–secreted factors to treat cutaneous wounds is an active area of research that is in need of refinement before effective clinical trials can be initiated. The aims of the present study were to (i) study which MSC-secreted factors stimulate dermal fibroblast (DF) migration in vitro and (ii) evaluate the potential of these factors to promote wound healing in vivo.

Osteogenic differentiation of multipotent mesenchymal stromal cells isolated from human bone marrow and subcutaneous adipose tissue

Cellular populations with phenotypes similar to multipotent mesenchymal stromal cells were isolated from two different sources, including human bone marrow (BM) and subcutaneous adipose tissue (SAT). Comparative analysis of the efficiency of differentiation in the direction of osteogenesis has revealed morphological changes confirmed by staining with Alizarin red and von Kossa in bone marrow cells at the 14th day and in adipose tissue cells at the 28th day of cultivation in the medium with inductors. Analysis of expression of the osteopontin, osteocalcin, and bone sialoprotein genes in RT-PCR reactions has detected essential differences in the potential of these cells to differentiate into bone tissue cells. Cells isolated from BM of both the control and experimental groups were positive for octeopontin (OP) on the 14th day. Unlike these cells, in cells isolated from SAT in medium without an inductor, no product of OP gene expression was identified. In the cells subjected to differentiation, OP appeared at day 14. In the BM cells, octeocalcin (OC) was found at the 14th day, while the bone sialoprotein (BS) was found at the 21st day of cultivation in induction medium. In cells isolated from SAT, OC, and BS were not detected, even at the 28th day after the beginning of induction.     

Co-transplantation of mesenchymal stromal cells and cord blood cells in treatment of diabetes

Background aimsStem cells provide a promising source for treatment of type 1 diabetes, but the treatment strategy and mechanism remain unclear. The aims of this study were to investigate whether co-transplantation of umbilical cord-derived mesenchymal stromal cells (UC-MSCs) and cord blood mononuclear cells (CB-MNCs) could reverse hyperglycemia in type 1 diabetic mice and to determine the appropriate ratio for co-transplantation. The treatment mechanism was also studied.MethodsA simple and efficient isolation method was developed to generate qualified UC-MSCs. UC-MSCs and CB-MNCs were then transplanted into type 1 diabetic mice at different ratios (UC-MSCs to CB-MNCs = 1:1, 1:4, 1:10) to observe the change in blood glucose concentration. Histology, immunohistochemistry, and human Alu polymerase chain reaction assay were performed to evaluate for the presence of donor-derived cells and the repair of endogenous islets. We also induced UC-MSCs into islet-like cells under specific culture conditions to determine their differentiate potential in vitro.ResultsCo-transplantation of UC-MSCs and CB-MNCs at a ratio of 1:4 effectively reversed hyperglycemia in diabetic mice. The detection of human Alu sequence indicated that the engraftment of donor-derived cells had homed into the recipient's pancreas and kidney. Although neither human insulin nor human nuclei antigen was detected in the regenerated pancreas, UC-MSCs could differentiate into insulin-secreted cells in vitro.ConclusionsCo-transplantation of UC-MSCs and CB-MNCs at a ratio of 1:4 could efficiently reverse hyperglycemia and repair pancreatic tissue.     

Banking of perinatal mesenchymal stem/stromal cells for stem cell-based personalized medicine over lifetime: Matters arising

Mesenchymal stromal/stem cells (MSCs) are currently applied in regenerative medicine and tissue engineering. Numerous clinical studies have indicated that MSCs from different tissue sources can provide therapeutic benefits for patients. MSCs derived from either human adult or perinatal tissues have their own unique advantages in their medical practices. Usually, clinical studies are conducted by using of cultured MSCs after thawing or short-term cryopreserved-then-thawed MSCs prior to administration for the treatment of a wide range of diseases and medical disorders. Currently, cryogenically banking perinatal MSCs for potential personalized medicine for later use in lifetime has raised growing interest in China as well as in many other countries. Meanwhile, this has led to questions regarding the availability, stability, consistency, multipotency, and therapeutic efficiency of the potential perinatal MSC-derived therapeutic products after long-term cryostorage. This opinion review does not minimize any therapeutic benefit of perinatal MSCs in many diseases after short-term cryopreservation. This article mainly describes what is known about banking perinatal MSCs in China and, importantly, it is to recognize the limitation and uncertainty of the perinatal MSCs stored in cryobanks for stem cell medical treatments in whole life. This article also provides several recommendations for banking of perinatal MSCs for potentially future personalized medicine, albeit it is impossible to anticipate whether the donor will benefit from banked MSCs during her/his lifetime.     

Autophagy and mitochondrial remodelling in mouse mesenchymal stromal cells challenged with Staphylococcus epidermidis

The bone marrow stroma constitutes the marrow‐blood barrier, which sustains immunochemical homoeostasis and protection of the haematopoietic tissue in sequelae of systemic bacterial infections. Under these conditions, the bone marrow stromal cells affected by circulating bacterial pathogens shall elicit the adaptive stress‐response mechanisms to maintain integrity of the barrier. The objective of this communication was to demonstrate (i) that in vitro challenge of mesenchymal stromal cells, i.e. colony‐forming unit fibroblasts (CFU‐F), with Staphylococcus epidermidis can activate the autophagy pathway to execute antibacterial defence response, and (ii) that homoeostatic shift because of the bacteria‐induced stress includes the mitochondrial remodelling and sequestration of compromised organelles via mitophagy. Implication of Drp1 and PINK1–PARK2‐dependent mechanisms in the mitophagy turnover of the aberrant mitochondria in mesenchymal stromal cells is investigated and discussed.     

DNA methylation pattern changes upon long-term culture and aging of human mesenchymal stromal cells

Within 2–3 months of in vitro culture-expansion, mesenchymal stromal cells (MSC) undergo replicative senescence characterized by cell enlargement, loss of differentiation potential and ultimate growth arrest. In this study, we have analyzed DNA methylation changes upon long-term culture of MSC by using the HumanMethylation27 BeadChip microarray assessing 27 578 unique CpG sites. Furthermore, we have compared MSC from young and elderly donors. Overall, methylation patterns were maintained throughout both long-term culture and aging but highly significant differences were observed at specific CpG sites. Many of these differences were observed in homeobox genes and genes involved in cell differentiation. Methylation changes were verified by pyrosequencing after bisulfite conversion and compared to gene expression data. Notably, methylation changes in MSC were overlapping in long-term culture and aging in vivo . This supports the notion that replicative senescence and aging represent developmental processes that are regulated by specific epigenetic modifications.     

Cell-cycle phases and genetic profile of bone marrow-derived mesenchymal stromal cells expanded in vitro from healthy donors

Human mesenchymal stromal cells (MSCs) expanded in vitro for cell therapy approaches need to be carefully investigated for genetic stability, by employing both molecular and conventional karyotyping. Reliability of cytogenetic analysis may be hampered in some MSC samples by the difficulty of obtaining an adequate number of metaphases. In an attempt to overcome this problem, a methodology apt to evaluate the cell‐cycle structure on synchronous MSCs was optimised. Results obtained in five independent experiments by comparing cell‐cycle analysis of synchronous and asynchronous MSC populations evaluated at early and late culture passages documented that in synchronous MSCs, 30% of cells entered G2/M phase after about 27–28 h of culture, while in asynchronous MSCs only 8% of cells in G2/M phase could be observed at the same time point. Cytogenetic analysis on synchronous MSCs allowed us to obtain 20–25 valuable metaphases/slide, whereas only 0–4 metaphases/slide were detectable in asynchronous preparations. J. Cell. Biochem. 112: 1817–1821, 2011. © 2011 Wiley‐Liss, Inc.     

Functional potentials of human hematopoietic progenitor cells are maintained by mesenchymal stromal cells and not impaired by plerixafor

Background aimsMesenchymal stromal cells (MSCs) resemble an essential component of the bone marrow niche for maintenance of stemness of hematopoietic progenitor cells (HPCs). Perturbation of the C-X-C chemokine receptor type 4 (CXCR4)/stromal cell-derived factor-1α (SDF-1α) axis by plerixafor (AMD3100) mobilizes HPCs from their niche; however, little is known about how plerixafor affects interaction of HPCs and MSCs in vitro.MethodsWe monitored cell division kinetics, surface expression of CD34 and CXCR4, migration behavior and colony-forming frequency of HPCs on co-culture with MSCs either with or without exposure to plerixafor.ResultsCo-culture with MSCs significantly accelerated cell division kinetics of HPCs. Despite this, the proportion of CD34⁺ cells was significantly increased on co-culture, whereas the expression of CXCR4 was reduced. In addition, co-culture with MSCs led to significantly higher colony-forming capacity and enhanced migration rate of HPCs compared with mono-culture conditions. The composition of MSC sub-populations—and conversely their hematopoiesis supportive functions—may be influenced by culture conditions. We compared the stromal function of MSCs isolated with three different culture media. Overall, the supporting potentials of these MSC preparations were quite similar. Perturbation by the CXCR4-antagonist plerixafor reduced the cell division kinetics of HPCs on co-culture with MSCs. However, the progenitor cell potential of the HPCs as reflected by colony-forming capacity was not affected by plerixafor.ConclusionsThese results support the notion that the CXCR4/SDF-1α axis is critical for HPC-MSC interaction with regard to migration, adhesion and regulation of proliferation but not for maintenance of primitive progenitor cells.     

Modulation of the in vitro angiogenic potential of human mesenchymal stromal cells from different tissue sources

Mesenchymal stromal cells (MSCs) have been widely exploited for the treatment of several conditions due to their intrinsic regenerative and immunomodulatory properties. MSC have demonstrated to be particularly relevant for the treatment of ischemic diseases, where MSC-based therapies can stimulate angiogenesis and induce tissue regeneration. Regardless of the condition targeted, recent analyses of MSC-based clinical trials have demonstrated limited benefits indicating a need to improve the efficacy of this cell product. Preconditioning MSC ex vivo through microenvironment modulation was found to improve MSC survival rate and thus prolong their therapeutic effect. This workstudy aims at enhancing the in vitro angiogenic capacity of a potential MSC-based medicinal product by comparing different sources of MSC and culture conditions. MSC from three different sources (bone marrow [BM], adipose tissue [AT], and umbilical cord matrix [UCM]) were cultured with xenogeneic-/serum-free culture medium under static conditions and their angiogenic potential was studied. Results indicated a higher in vitro angiogenic capacity of UCM MSC, compared with cells derived from BM and AT. Physicochemical preconditioning of UCM MSC through a microcarrier-based culture platform and low oxygen concentration (2% O₂, compared with atmospheric air) increased the in vitro angiogenic potential of the cultured cells. Envisaging the clinical manufacturing of an allogeneic, off-the-shelf MSC-based product, preconditioned UCM MSC maintain the angiogenic gene expression profile upon cryopreservation and delivery processes in the conditions of our study. These results are expected to contribute to the development of MSC-based therapies in the context of angiogenesis.     

Human umbilical cord Wharton's jelly mesenchymal stem cells do not transform to tumor-associated fibroblasts in the presence of breast and ovarian cancer cells unlike bone marrow mesenchymal stem cells

Human bone marrow mesenchymal stem cells (hBMMSCs) were shown to transform into tumor-associated fibroblasts (TAFs) when in the vicinity of breast cancer tumors and played an important role in tumor enhancement and metastasis. In early human development MSCs migrating from the yolk sac and aorta-gonad-mesonephros (AGM) via the umbilical cord to the placenta and back to the fetal bone marrow were shown to get trapped in the gelatinous Wharton's jelly of the umbilical cord. The common origin of the Wharton's jelly MSCs and the finally homed hBMMSCs prompted us to evaluate whether hWJSCs are also involved in TAF transformation. hWJSCs and hBMMSCs were grown in the presence of breast and ovarian cancer cell conditioned medium (MDA-TCM, TOV-TCM) for 30 days. No changes were observed in the hWJSCs but the hBMMSCs transformed from short to thin long fibroblasts, their proliferation rates increased and CD marker expression decreased. The transformed hBMMSCs showed positive staining for the tumor-associated markers FSP, VEGF, EGF, and Tn-C. Real-time PCR and multiplex luminex bead analysis showed upregulation of TAF-related genes (FSP, FAP, Tn-C, Tsp-1, EGF, bFGF, IL-6, α-SMA, VEGF, and TGF-β) for hBMMSCs with low expression for hWJSCs. The luciferase assay showed that hWJSCs previously exposed to MDA-TCM or TOV-TCM had no stimulatory growth effect on luciferase-tagged MDA or TOV cells unlike hBMMSCs. The results confirmed that hWJSCs do not transform to the TAF phenotype and may therefore not be associated with enhanced growth of solid tumors making them a safe MSC for cell based therapies.     

Human platelet lysate stimulates high-passage and senescent human multipotent mesenchymal stromal cell growth and rejuvenation in vitro

Background aimsMultipotent mesenchymal stromal cells (MSCs) are clinically useful because of their immunomodulatory and regenerative properties, but MSC therapies are limited by the loss of self-renewal and cell plasticity associated with ex vivo expansion culture and, on transplantation, increased immunogenicity from xenogen exposure during culture. Recently, pooled human platelet lysate (hPL) has been used as a culture supplement to promote MSC growth; however, the effects of hPL on MSCs after fetal bovine serum (FBS) exposure remain unknown.MethodsMSCs were cultured in medium containing FBS or hPL for up to 16 passages, and cell size, doubling time and immunophenotype were determined. MSC senescence was assessed by means of a fluorometric assay for endogenous β-galactosidase expression. MSCs cultured with FBS for different numbers of passages were switched to hPL conditions to evaluate the ability of hPL to “rescue” the proliferative capacity of MSCs.ResultshPL culture resulted in more rapid cell proliferation at earlier passages (passage 5 or earlier) than remove FBS; by day 4, hPL (5%) yielded an MSC doubling time of 1.28 days compared with 1.52 days in 16% FBS. MSCs cultured first in FBS and switched to hPL proliferated more and demonstrated less β-galactosidase production and smaller cell sizes than remove MSCs continuously propagated in FBS.ConclusionshPL enables rapid expansion of MSCs without adversely affecting immunophenotype. hPL culture of aged and senescent MSCs demonstrated cellular rejuvenation, reflected by decreased doubling time and smaller cell size. These results suggest that expansion of MSCs in hPL after FBS exposure can enhance cell phenotype and proliferative capacity.