MSC Growth Research Articles

Polyethylene glycol precipitation is an efficient method to obtain extracellular vesicle-depleted fetal bovine serum.

Mesenchymal stromal/stem cell derived-extracellular vesicles (MSC-EVs) have gained interest as drug delivery nanoparticles, having immunoregulatory and potentiating tissue repair property. To maintain growth of MSCs and obtain pure MSC-derived EVs, the culture media should contain fetal bovine serum (FBS) devoid of EVs, as the presence of FBS EVs confounds the properties of MSC-EVs. Therefore, we tested three methods: 18h ultracentrifugation (UC) and ultrafiltration (UF), which are common FBS EV depletion methods in current MSC-EV research, and polyethylene glycol (PEG) precipitation to obtain three EV depleted FBS (EVdFBS) batches, and compared them to FBS and commercial (Com) EVdFBS on human adipose stem cell (hADSC) growth, differentiation, enrichment of EVs in hADSC supernatant and their biological function on collagen metabolism. Our comparative study showed UC and UF vary in terms of depletion efficiency and do not completely deplete EVs and affects the growth-promoting quality of FBS. Specifically, FBS EV depletion was comparable between PEG (95.6%) and UF (96.6%) but less by UC (82%), as compared to FBS. FBS protein loss was markedly different among PEG (47%), UF (87%), and UC (51%), implying the ratio of EV depletion over protein loss was PEG (2.03), UF (1.11), and UC (1.61). A significant decrease of TGFβ/Smad signaling, involving in MSC growth and physiology, was observed by UF. After 96 hours of exposure to 5% FBS or 5% four different EVdFBS cell growth media, the osteogenesis ability of hADSCs was not impaired but slightly lower mRNA expression level of Col2a observed in EVdFBS media during chondrogenesis. In consistent with low confluency of hADSCs observed by optical microscope, cell proliferation in response to 5% UF EVdFBS media was inhibited significantly. Importantly, more and purer ADSCs EVs were obtained from ADSCs cultured in 5% PEG EVdFBS media, and they retained bioactive as they upregulated the expression of Col1a1, TIMP1 of human knee synovial fibroblast. Taken together, this study showed that PEG precipitation is the most efficient method to obtain EV depleted FBS for growth of MSCs, and to obtain MSC EVs with minimal FBS EV contamination.

LINC01638 sustains human mesenchymal stem cell self-renewal and competency for osteogenic cell fate

The skeleton forms from multipotent human mesenchymal stem cells (hMSCs) competent to commit to specific lineages. Long noncoding RNAs (lncRNAs) have been identified as key epigenetic regulators of tissue development. However, regulation of osteogenesis by lncRNAs as mediators of commitment to the bone phenotype is largely unexplored. We focused on LINC01638, which is highly expressed in hMSCs and has been studied in cancers, but not in regulating osteogenesis. We demonstrated that LINC01638 promotes initiation of the osteoblast phenotype. Our findings reveal that LINC01638 is present at low levels during the induction of osteoblast differentiation. CRISPRi knockdown of LINC01638 in MSCs prevents osteogenesis and alkaline phosphatase expression, inhibiting osteoblast differentiation. This resulted in decreased MSC growth rate, accompanied by double-strand breaks, DNA damage, and cell senescence. Transcriptome profiling of control and LINC01638-depleted hMSCs identified > 2000 differentially expressed mRNAs related to cell cycle, cell division, spindle formation, DNA repair, and osteogenesis. Using ChIRP-qPCR, molecular mechanisms of chromatin interactions revealed the LINC01638 locus (Chr 22) includes many lncRNAs and bone-related genes. These novel findings identify the obligatory role for LINC01638 to sustain MSC pluripotency regulating osteoblast commitment and growth, as well as for physiological remodeling of bone tissue.

The roles of growth factors and hormones in the regulation of muscle satellite cells for cultured meat production.

Cultured meat is a potential sustainable food generated by the in vitro myogenesis of muscle satellite (stem) cells (MSCs). The self-renewal and differentiation properties of MSCs are of primary interest for cultured meat production. MSC proliferation and differentiation are influenced by a variety of growth factors such as insulin-like growth factors (IGF-1 and IGF-2), transforming growth factor beta (TGF-β), fibroblast growth factors (FGF-2 and FGF-21), platelet-derived growth factor (PDGF) and hepatocyte growth factor (HGF) and by hormones like insulin, testosterone, glucocorticoids, and thyroid hormones. In this review, we investigated the roles of growth factors and hormones during cultured meat production because these factors provide signals for MSC growth and structural stability. The aim of this article is to provide the important idea about different growth factors such as FGF (enhance the cell proliferation and differentiation), IGF-1 (increase the number of myoblasts), PDGF (myoblast proliferation), TGF-β1 (muscle repair) and hormones such as insulin (cell survival and growth), testosterone (muscle fiber size), dexamethasone (myoblast proliferation and differentiation), and thyroid hormones (amount and diameter of muscle fibers and determine the usual pattern of fiber distributions) as media components during myogenesis for cultured meat production.

Sonic Hedgehog Induces Mesenchymal Stromal Cell Senescence-Associated Secretory Phenotype and Chondrocyte Apoptosis in Human Osteoarthritic Cartilage.

Hedgehog (HH) signaling plays a critical role in osteoarthritis (OA) pathogenesis, but the molecular mechanism remains to be elucidated. We show here that Sonic Hedgehog (SHH) gene expression is initiated in human normal cartilage stromal cells (NCSC) and increased in OA cartilage mesenchymal stromal cells (OA-MSCs) during aging. Manifesting a reciprocal cellular distribution pattern, the SHH receptors PTCH1 and SMO and transcription factors GLI2 and GLI3 are expressed by chondrocytes (OAC) in OA cartilage. SHH autocrine treatment of osteoarthritis MSC stimulates proliferation, chondrogenesis, hypertrophy, and replicative senescence with elevated SASP gene expression including IL1B, IL6, CXCL1, and CXCL8. SHH paracrine treatment of OAC suppresses COL2A1, stimulates MMP13, and induces chondrocyte apoptosis. The OA-MSC conditioned medium recapitulates the stimulatory effects of SHH on OAC catabolism and apoptosis. SHH knock-down in OA-MSC not only inhibits catabolic and senescence marker expression in OA-MSC, but also abolishes the effect of the OA-MSC conditioned medium on OAC catabolism and apoptosis. We propose that SHH is a key mediator between OA-MSC and OA chondrocytes interaction in human OA cartilage via two mechanisms: (1) SHH mediates MSC growth and aging by activating not only its proliferation and chondrogenesis, but also low-grade inflammation and replicative senescence, and (2) SHH mediates OA-MSC-induced OAC catabolism and apoptosis by creating a pro-inflammatory microenvironment favoring tissue degeneration during OA pathogenesis.

Abstract PO-001: Heterogeneous overexpression of HES6 promotes the growth of uterine leiomyomas

Abstract Introduction: Uterine leiomyomas are a common cause of infertility, pelvic pain and irregular vaginal bleeding. Robust evidence suggests that leiomyomas arise from a distinct population of myometrial-specific stem cells (mSC). However, the mechanisms responsible for driving the mal-differentiation of mSC and subsequent leiomyoma growth remain poorly understood. Here, we explore the role of HES6, a basic helix-loop-helix (bHLH) co-factor critical for promoting myogenic differentiation, in uterine leiomyomas. Methods: After obtaining IRB approval, real time PCR (qPCR) and Western blot were used to evaluate patterns of HES6 expression using RNA and protein prepared from matched specimens of myometrium, leiomyoma and uterine leiomyosarcoma (uLMS). Primary cultures were generated as previously described. HES6-specific siRNAs were used to target gene expression in vitro. Standardized assays were used to measure proliferation (CellTiter-Glo, Promega) and apoptosis (Caspase 3/7 assay). A commercially available antibody specific for HES6 was used to localize HES6 expression in formalin-fixed, paraffin-embedded (FFPE) tissue specimens by immunohistochemistry (IHC). Patterns of gene expression and splicing were profiled by Next Generation Sequencing. Student’s t-test was used to assess statistical significance. Results: We found that, on average, levels of HES6 transcript were >15-fold higher when specimens of leiomyomas were compared to adjacent normal myometrium (p<0.01). No relationship between expression of HES6 in myometrium or leiomyoma, menstrual phase (n=58), menopausal status (n=12) or clinical treatment of pre- or postmenopausal women with medroxyprogesterone (Pre: n=12; Post: n=6) was observed. When matched FFPE specimens were evaluated by IHC, we found that HES6 expression was selectively limited to organized subsets of cells within large leiomyomas as well as the blood vessels surrounding these tumors. Levels of HES6 were >4-fold lower in specimens of uLMS (n=6) than proliferative or secretory phase myometrium (p<0.01). In vitro, knockdown of HES6 expression significantly reduced rates of proliferation and enhanced rates of apoptosis across multiple primary cultures derived from matched specimens of myometrium and leiomyomas (n=10, p<0.05). Of the 123 gene products who expression was significantly altered (p<0.05) in response to targeting HES6 expression, levels of the mitochrondrial solute transporter SLC25A22 was most dramatically disrupted. Conclusions: Overexpression of HES6 is a reliable feature important for promoting the growth of benign but not malignant uterine smooth muscle tumors within metabolically defined subsets of uterine smooth muscle cells. We are currently working to determine how this highly novel pattern of intratumoral heterogeneity promotes leiomyoma growth and its hierarchical relationship to early events in the mal-differentiation of mSC. Citation Format: Joie Z. Guner, Venkata A. Jonnalagadda, Ninad A. Patel, Ramya P. Masand, Cecilia Valdes, Williams E. Gibbons, Matthew L. Anderson. Heterogeneous overexpression of HES6 promotes the growth of uterine leiomyomas [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-001.

Expression of CD44, CD29 and osteocalcin differentiation factors in the alveolar bone during past fixation of a free gingival graft

Aim: To study the localization of stem stromal cells on the surface of a free gingival graft.
 Material and methods: We studied the immunolocalization of stem cell markers (CD29, CD44 and osteocalcin) the mucosa plate and bone tissue of the lower jaw of outbred miniature pigs with bone fixation of a free gingival graft.
 Results: In the third month after the operation, a new bone tissue is formed in the contact area, characterized by formed bone plates, osteons, and a system of Havers and Volkman channels. When studying the immunolocalization of CD29, CD44, and osteocalcin, we detected a heterogeneous distribution. CD29and CD44-immunopositive cells prevail at the border of the newly formed bone and its own gingival plate, as well as in the cavity of the Havers canals. Osteocalcin-positive cells are localized mainly in the layer of common plates, on the surface of the bone plates of osteons surrounding the Havers channel and in its cavity, which are in close proximity to the graft.
 Conclusion: Bone fixation of SDT remodels the alveolar bone, showing signs of a reparative process. The presence of expression of CD29 and CD44 in bone tissue indicates the activation of mesenchymal cambial cells, which differentiate into osteocalcin-containing osteoblasts. The inducing role of a free gingival graft in the formation of a new bone is determined by the ability of its cells to synthesize trophic factors stimulating the proliferation and differentiation of MSC and vascular growth. Thus, the model on miniature pigs is a useful tool for studying this process.

The Coronavirus Pandemic (SARS-CoV-2): New Problems Demand New Solutions, the Alternative of Mesenchymal (Stem) Stromal Cells.

Mesenchymal (stem) stromal cells (MSC) can be a therapeutic alternative for COVID-19 considering their anti-inflammatory, regenerative, angiogenic, and even antimicrobial capacity. Preliminary data point to therapeutic interest of MSC for patients with COVID-19, and their effect seems based on the MSC’s ability to curb the cytokine storm caused by COVID-19. In fact, promising clinical studies using MSC to treat COVID-19, are currently underway. For this reason, now is the time to firmly consider new approaches to MSC research that addresses key issues, like selecting the most optimal type of MSC for each indication, assuming the heterogeneity of the donor-dependent MSC and the biological niche where MSC are located.

Phenotypic instability of mesenchymal stromal cells isolated and expanded in different growth media

Background & Aim Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are an attractive candidate for use in regenerative therapies and are being widely investigated for the treatment of a broad spectrum of diseases. Despite this, the efficacy of treatment is unproven and interpretation of trial data is difficult, in part due to the heterogeneity of MSC preparations as a result of variable manufacturing processes. MSCs are currently isolated in basal media containing fetal bovine serum (FBS), platelet lysate or other supplements but little is understood about the impact of changes in media composition on the biology, therapeutic action or potency of MSCs. The aim of this study was to analyse the influence of widely used culture media on the growth parameters, differentiation potential and surface marker expression of human BM-MSCs. Methods, Results & Conclusion Whole bone marrow obtained from the iliac crest of a 24-year-old healthy donor was plated in a standard basal medium with 5 different supplements: (1) a proprietary serum-free supplement, (2) 5% human platelet lysate, (3) 10% FBS, screened and proven to support MSC isolation and expansion (4) 10% screened FBS and 1 ng/ml recombinant human basic fibroblast growth factor (FGF2) and (5) 10% unscreened FBS and 1 ng/ml FGF2. The impact of media composition on growth kinetics, morphology, differentiation propensity and surface immunophenotype was assessed. The latter involved the BD Lyoplate flow cytometry screening panel of 242 monoclonal antibodies. MSC growth kinetics were highly influenced by expansion medium, with the greatest proliferation rate observed in MSCs cultured in the serum-free supplemented medium. Cell morphology varied greatly between cell preparations. Adipogenesis and osteogenesis were maintained in all culture conditions, but the degree of differentiation varied between conditions. Chondrogenesis was most starkly effected by changes in expansion medium and was only observed in serum-containing media. Analysis of surface marker expression revealed significant variation in response to culture conditions, with 70 of the 242 markers differentially expressed between cell preparations. These data suggest that changes in the composition of the culture media – for example switching batches of FBS – have profound effects on key biological attributes of BM-MSCs and on their surface marker expression profiles. This demonstrates the critical need for harmonisation of media formulation to aid interpretation of clinical results.

Lipofilling. The review of the technique. Modern opportunities and prospects for dermal scar correction

Since of the invention lipofilling, there was a significant change in ideas of the role used autologous fat tissue (Auto Fat) from natural filler of defects to the product for stimulating regeneration. There was a considerable evolution in technology of liposuction and Auto Fat processing, material and methodical providing of the procedure was improved. Opening and studying role of the stromal-vascular fraction (SVF) of the adipose tissue, in particular mesenchymal stem cells of the adipose tissue (MSC FT), promoted considerable extension of indications while using this method in aesthetic medicine. During experience accumulation of lipofilling application new concepts of application of Auto Fat in combination with MSC FT, platelet-enriched plasma (PRP) and growth factors were developed for scar correction. Understanding of a key role recipient area in succesful engrafting Auto Fat on scars resulted in development of transcutaneous aponeurotomy in a combination with a lipofilling (riggotomy). The procedure of a nanolipofilling which can be considered an attempt of the isolated use of lipofilling for regeneration stimulation and remodeling of cicatricial tissue was developed. However, despite the achieved success, carrying out further researches which would allow to confirm this method efficacy from the point of view evidence-based medicine and develop clear clinical guidelines about its application for treatment of dermal scars would be necessary.

Impact of Vancomycin Treatment on Human Mesenchymal Stromal Cells During Osteogenic Differentiation

Category: Basic Sciences/Biologics Introduction/Purpose: Vancomycin is often delivered locally for surgical site infection prophylaxis. Recent reports of possible osteotoxicity have led to uncertainty concerning vancomycin’s safety in the setting of arthrodesis and bone healing. Bone formation during arthrodesis takes place as recruited human mesenchymal stromal cells (hMSCs) proliferate and differentiate into mature osteoblasts. The purpose of this research was to determine the impact of vancomycin treatment on hMSCs during osteogenic differentiation. Methods: Human MSCs were cultured in MSC growth media to an appropriate confluence. Cells were cultured for 24 hours to facilitate adherence, after which the media was aspirated and replaced with osteogenic differentiation media (Lonza, Switzerland). Osteogenic differentiation media was supplemented with vancomycin powder to yield solutions with concentrations of 0, 50, 500 & 5000 µg/mL. Fresh vancomycin powder was added with every media change. MSCs viability and proliferation were assessed via live/dead staining with 1 µM calcein-AM and 0.5 µM ethidium homodimer-1 (EthD-1) after 1, 3, and 7 days of differentiation and vancomycin treatment. Mineralization of differentiated cells was assessed via staining with 40 mM alizarin red (ARS; pH 4.1) after 21 days. Semi-quantification of the degree of mineralization was performed by measuring absorbance values at 405 nm using a microplate reader. Microscopy was used for qualitative evaluation. Results: Cell viability decreased with increasing vancomycin concentrations. Impairment of hMSC proliferation was also observed with increasing concentrations of vancomycin. MSCs treated with 5000 µg/mL vancomycin demonstrated significantly less cell growth compared to all other treatment groups (P=0.0001). Absorbance measurements from each well stained with alizarin red was used for semi-quantification of the degree of mineralization. As vancomycin concentrations were increased, absorbance levels decreased (Figure). This reduction in mineralization was also demonstrated qualitatively; with alizarin red less apparent in the wells with increasing vancomycin concentrations (Figure). Conclusion: Local vancomycin is utilized for prevention of infection, often in procedures that necessitate the formation of new bone. Bone healing requires migration, proliferation and differentiation of hMSCs. This work demonstrates impaired viability and function of hMSCs following vancomycin as well as decreased osteoblastic mineralization. Future work will require in vivo studies aimed at determining relative nonunion rates in the setting of vancomycin prophylaxis. Still, the results of this study suggest that vancomycin may be toxic to hMSCs and caution should be exercised by providers when considering vancomycin in foot and ankle patients requiring bony healing following fracture or arthrodesis.

137 - Targeted optimization of chemically-defined medium for msc growth in bioreactors

137 - Targeted optimization of chemically-defined medium for msc growth in bioreactors

A chitosan/dextran-based hydrogel as a delivery vehicle of human bone-marrow derived mesenchymal stem cells

A chitosan/dextran-based (CD) injectable, surgical hydrogel has been developed and shown to be an effective post-operative aid in prevention of scar tissue formation in vivo. The CD hydrogel’s effectiveness in a surgical setting prompted an investigation into its capacity as a potential delivery vehicle for bone marrow derived mesenchymal stem cells (BM-MSCs) for regenerative wound healing applications. By housing BM-MSCs within a biocompatible, injectable, hydrogel matrix, viability and protection in cultivation, as well as direct delivery to the damaged site in the host tissue may be achieved. In vitro BM-MSC cell viability in the presence of CD hydrogel was determined by LIVE/DEAD® fluoresence staining. Flow cytometry studies revealed expression of a conventional BM-MSC surface marker profile. A colony forming cell assay showed a slight statistically significant decrease in the number of colonies grown in CD hydrogel as compared to control cells. In addition, BM-MSCs in the CD hydrogel were able to successfully differentiate into adipocytes and osteocytes. In summary, the CD hydrogel supports MSC growth and differentiation; and therefore, may be used as a potential stem cell delivery vehicle for regenerative medicine and tissue engineering applications.

Potency testing of mesenchymal stromal cell growth expanded in human platelet lysate from different human tissues.

BackgroundMesenchymal stromal cells (MSCs) have been largely investigated, in the past decade, as potential therapeutic strategies for various acute and chronic pathological conditions. MSCs isolated from different sources, such as bone marrow (BM), umbilical cord tissue (UCT) and adipose tissue (AT), share many biological features, although they may show some differences on cumulative yield, proliferative ability and differentiation potential. The standardization of MSCs growth and their functional amplification is a mandatory objective of cell therapies. The aim of this study was to evaluate the cumulative yield and the ex vivo amplification potential of MSCs obtained from various sources and different subjects, using defined culture conditions with a standardized platelet lysate (PL) as growth stimulus.MethodsMSCs isolated from BM, UCT and AT and expanded in human PL were compared in terms of cumulative yield and growth potential per gram of starting tissue. MSCs morphology, phenotype, differentiation potential, and immunomodulatory properties were also investigated to evaluate their biological characteristics.ResultsThe use of standardized PL-based culture conditions resulted in a very low variability of MSC growth. Our data showed that AT has the greater capacity to generate MSC per gram of initial tissue, compared to BM and UCT. However, UCT-MSCs replicated faster than AT-MSCs and BM-MSCs, revealing a greater proliferation capacity of this source irrespective of its lower MSC yield. All MSCs exhibited the typical MSC phenotype and the ability to differentiate into all mesodermal lineages, while BM-MSCs showed the most prominent immunosuppressive effect in vitro.ConclusionsThe adoption of standardized culture conditions may help researchers and clinicians to reveal particular characteristics and inter-individual variability of MSCs sourced from different tissues. These data will be beneficial to set the standards for tissue collection and MSCs clinical-scale expansion both for cell banking and for cell-based therapy settings.

Fibrinolytic crosstalk with endothelial cells expands murine mesenchymal stromal cells

AbstractTissue plasminogen activator (tPA), aside from its vascular fibrinolytic action, exerts various effects within the body, ranging from synaptic plasticity to control of cell fate. Here, we observed that by activating plasminogen and matrix metalloproteinase-9, tPA expands murine bone marrow–derived CD45−TER119−Sca-1+PDGFRα+ mesenchymal stromal cells (PαS-MSCs) in vivo through a crosstalk between PαS-MSCs and endothelial cells. Mechanistically, tPA induces the release of Kit ligand from PαS-MSCs, which activates c-Kit+ endothelial cells to secrete MSC growth factors: platelet-derived growth factor-BB (PDGF-BB) and fibroblast growth factor 2 (FGF2). In synergy, FGF2 and PDGF-BB upregulate PDGFRα expression in PαS-MSCs, which ultimately leads to PαS-MSC expansion. These data show a novel mechanism by which the fibrinolytic system expands PαS-MSCs through a cytokine crosstalk between niche cells.

Effects of Exendin-4 on bone marrow mesenchymal stem cell proliferation, migration and apoptosis in vitro.

Mesenchymal stem cells (MSC) are regarded as an attractive source of therapeutic stem cells for myocardial infarction. However, their limited self-renewal capacity, low migration capacity and poor viability after transplantation hamper the clinical use of MSC; thus, a strategy to enhance the biological functions of MSC is required. Exendin-4 (Ex-4), a glucagon-like peptide-1 receptor agonist, exerts cell-protective effects on many types of cells. However, little information is available regarding the influence of Ex-4 on MSC. In our study, MSC were isolated from bone marrow and cultured in vitro. After treatment with Ex-4, MSC displayed a higher proliferative capacity, increased C-X-C motif receptor 4 (CXCR4) expression and an enhanced migration response. Moreover, in H2O2-induced apoptosis, Ex-4 preserved mitochondrial function through scavenging ROS and balancing the expression of anti- and pro-apoptotic proteins, leading to the inhibition of the mitochondria-dependent cell death pathways and increased cell survival. Moreover, higher phospho-Akt (p-Akt) expression was observed after Ex-4 intervention. However, blockade of the PI3K/Akt pathway with inhibitors suppressed the above cytoprotective effects of Ex-4, suggesting that the PI3K/Akt pathway is partly responsible for Ex-4-mediated MSC growth, mobilization and survival. These findings provide an attractive method of maximizing the effectiveness of MSC-based therapies in clinical applications.

The Stem Cell Potential and Multipotency of Human Adipose Tissue-Derived Stem Cells Vary by Cell Donor and Are Different from Those of Other Types of Stem Cells

Human adipose tissue-derived mesenchymal stem cells (AT-MSCs) from various sites are applied in tissue engineering and cell therapy. The condition of AT-MSCs depends on the donor's age, body mass index (BMI), and gender. AT-MSCs from 66 human donors were analyzed, and the cells were sorted according to donor age (10-19 years: n = 1; 20-29 years: n = 5; 30-39 years: n = 12; 40-49 years: n = 22; 50-59 years: n = 12; 60-69 years: n = 9, and 70 years or older: n = 5), BMI (under 25, 25-30, and over 30), and gender (19 males and 48 females). Additionally, AT-MSCs were compared to bone marrow MSCs and chorionic tissue-derived MSCs. We measured the MSC yield, growth rate, colony-forming units, multipotency, and surface antigens. AT-MSC proliferation was greater in cells isolated from individuals aged less than 30 years compared to the proliferation of AT-MSCs from those over 50 years old. BMI was correlated with osteogenic differentiation potency; increased BMI enhanced osteogenesis. Adipogenic differentiation was more strongly induced in cells isolated from donors aged less than 30 years compared to those isolated from other age groups. Also, a BMI above 30 was associated with enhanced adipogenic differentiation compared to cells isolated from individuals with a BMI below 25. Bone marrow MSCs were strongly induced to differentiate along both osteogenic and adipogenic lineages, whereas AT-MSCs predominantly differentiated into the chondrogenic lineage. Therefore, the type of regeneration required and variations among potential donors must be carefully considered when selecting MSCs for use in applied tissue engineering or cell therapy.

FGF2 protects mouse mesenchymal stem cells from oxidative stress by modulating a twist2-p53 signaling axis

Although bone marrow is a low oxygen environment few studies have examined how oxidative stress alters MSC function. Previously, we reported that exposure of primary mouse MSCs to atmospheric oxygen rapidly induced mitochondrial ROS generation by a p53-dependent mechanism resulting in reduced cell growth and viability. In addition, we showed that long-term exposure to atmospheric oxygen selected for immortalized MSC clones with impaired p53 function but expansion of cells in 5% oxygen using a closed system generated large numbers of primary cells that retained a functional p53 protein. In this study we demonstrate that atmospheric oxygen impairs adipogenic and osteogenic differentiation of primary mouse MSCs and their ability to support hematopoiesis in vitro. Moreover, we demonstrate that FGF2 protects MSCs from the detrimental effects of oxygen via a p53 dependent mechanism. Specifically, culture of MSCs in FGF2-supplemented media blocked oxygen induced increases in mitochondrial ROS levels and p53 and BAX protein expression. This effect of FGF2 was strongly correlated with induction of Twist2 mRNA and protein expression. Transfection of MSCs with a Twist2-specific siRNA resulted in increased p53 protein expression and a concomitant increase in intracellular ROS levels. Biochemical studies demonstrated that Twist2 directly bound to p53 and enhanced its degradation. Moreover, knockdown of Twist2 but not Twist1 inhibited MSC growth and CFU-F activity and augmented adipogenic and osteogenic differentiation independent of p53. Additionally, sorting for oxidative stress resistant sub populations enriched for multi-potent progenitors that express high Twist2 and low p53 levels. These studies implicate Twist2 in self-maintenance of the progenitor pool, demonstrate a functional link between pathways that regulate MSC self-maintenance and oxidative stress, and provide a means to enrich progenitors that does not rely on non-specific surface markers.

Inactivated human platelet lysate with psoralen: a new perspective for mesenchymal stromal cell production in Good Manufacturing Practice conditions

Background aimsMesenchymal stromal cells (MSC) are ideal candidates for regenerative and immunomodulatory therapies. The use of xenogeneic protein–free Good Manufacturing Practice–compliant growth media is a prerequisite for clinical MSC isolation and expansion. Human platelet lysate (HPL) has been efficiently implemented into MSC clinical manufacturing as a substitute for fetal bovine serum (FBS). Because the use of human-derived blood materials alleviates immunologic risks but not the transmission of blood-borne viruses, the aim of our study was to test an even safer alternative than HPL to FBS: HPL subjected to pathogen inactivation by psoralen (iHPL). MethodsBone marrow samples were plated and expanded in α-minimum essential medium with 10% of three culture supplements: HPL, iHPL and FBS, at the same time. MSC morphology, growth and immunophenotype were analyzed at each passage. Karyotype, tumorigenicity and sterility were analyzed at the third passage. Statistical analyses were performed. ResultsThe MSCs cultivated in the three different culture conditions showed no significant differences in terms of fibroblast colony-forming unit number, immunophenotype or in their multipotent capacity. Conversely, the HPL/iHPL-MSCs were smaller, more numerous, had a higher proliferative potential and showed a higher Oct-3/4 and NANOG protein expression than did FBS-MSCs. Although HPL/iHPL-MSCs exhibit characteristics that may be attributable to a higher primitive stemness than FBS-MSCs, no tumorigenic mutations or karyotype modifications were observed. ConclusionsWe demonstrated that iHPL is safer than HPL and represents a good, Good Manufacturing Practice–compliant alternative to FBS for MSC clinical production that is even more advantageous in terms of cellular growth and stemness.

FGFR1 Signaling Stimulates Proliferation of Human Mesenchymal Stem Cells by Inhibiting the Cyclin-Dependent Kinase Inhibitors p21Waf1 and p27Kip1

Signaling through fibroblast growth factor receptor one (FGFR1) is a known inducer of proliferation in both embryonic and human adult mesenchymal stem cells (hMSCs) and positively regulates maintenance of stem cell viability. Leveraging the mitogenic potential of FGF2/FGFR1 signaling in stem cells for therapeutic applications necessitates a mechanistic understanding of how this receptor stimulates cell cycle progression. Using small interfering RNA (siRNA) depletion, antibody-inhibition, and small molecule inhibition, we establish that FGFR1 activity is rate limiting for self-renewal of hMSCs. We show that FGFR1 promotes stem cell proliferation through multiple mechanisms that unite to antagonize cyclin-dependent kinase (CDK) inhibitors. FGFR1 not only stimulates c-Myc to suppress transcription of the CDK inhibitors p21(Waf1) and p27(Kip1), thus promoting cell cycle progression but also increases the activity of protein kinase B (AKT) and the level of S-phase kinase-associated protein 2 (Skp2), resulting in the nuclear exclusion and reduction of p21(Waf1). The in vivo importance of FGFR1 signaling for the control of proliferation in mesenchymal progenitor populations is underscored by defects in ventral mesoderm formation during development upon inhibition of its signaling. Collectively, these studies demonstrate that FGFR1 signaling mediates the continuation of MSC growth and establishes a receptor target for enhancing the expansion of mesenchymal progenitors while maintaining their multilineage potential.

First In Europe International Cooperation For Clinical Use Of Wharton Jelly-Derived Mesenchymal Stem Cells

IntroductionMesenchymal stem cells (MSC) are now in the limelight of stem cell researchers. The growing number of preclinical studies gives feedback for using MSC in the various fields of medicine. Their immunomodulatory function gives them a scientific rationale to be used in Graft-versus-Host Disease (GvHD) treatment. The MSC can be isolated from bone marrow (BM), adipose tissue, cord blood (CB) or umbilical cord (UC). However, BM harvest puts a donor at risk of procedure complication. In the contrary, Wharton Jelly (WJ) - derived MSC can be collected safely, easily and they are reach in MSC, which makes them more preferable source than CB.We would like to present the first in Europe international cooperation that led to the clinical application of WJ-derived MSC in patients with GvHD. In the present publication we describe the results of collection, transport, culture, investigation, cryopreservation and the first examples of clinical usage of MSC derived from more than 500 UC collected by our group of stem cells banks (www.famicord.eu). MethodsWJ-derived MSC were obtained from third party unrelated donors after natural deliveries as well as caesarian sections. They were collected to the sterile vessel containing 0,9% natrium chloratum and 1% antibiotic and transported in the temperature of 18-24°C. After an isolation by mechanical dissection of cord's blood vessels, they underwent culture in the 37°C in the atmosphere of 5% CO2 in the air with human MSC growth medium as well as supplement containing fetal bovine serum and antibiotic. They were enumerated and their viability was evaluated. Then the cells were cryopreserved in the presence of DMSO and placed in the vapour phase of liquid nitrogen in <150°C. The repeated cell counting, viability test, flow cytometric immunophenotyping, and functional in vitro differentiation assays were performed from the thawed reference samples. ResultsLow contamination level (less than 2%) of the UC tissue collected after both natural deliveries and caesarian section was reported. We have not noticed any differences in growth, cell number and morphology in the primary cultures of tissue fragments from placental, central and baby side of the cord. The first adherent cells with fibroblast-like morphology were well-distinguishable within a week after the initiation of the cell culture. The immunophenotype remained stable (CD45-/CD34-/CD19-/CD14-/HLA-DR-/CD73+/CD90+/CD105+) during the whole period of culture (with extreme limit of 15 passages). MSC were capable of differentiation into adipogenic, chondrogenic and osteogenic cells. The WJ-derived MSC have been applied to the ten patients with steroid-refractory GvHD always after approval of bioethical committee. Three patients were diagnosed with chronic form and 7 with acute one. Five children had multiple infusions, up to 4 doses with 1-2 week intervals. No adverse effects were described during infusions apart from low grade fever in 1 adult patient. ConclusionThe results described above demonstrate a repeatable method to obtain an adequate number of cells for the clinical use. The international cooperation between Polish, Hungarian, Romanian and Spanish stem cell banks, enabled us to use WJ-derived MSC in the setting of GvHD. No serious adverse effects were described. Third party donor WJ-derived MSCs are safe and effective treatment of GvHD, however further studies are needed. Disclosures:No relevant conflicts of interest to declare.