Proliferation Of Mesenchymal Stromal Cells Research Articles

Impact of growth factor content on proliferation of mesenchymal stromal cells derived from adipose tissue.

Autologous adipose tissue (AT) transfer has gained widespread acceptance and is used for a broad variety of regenerative clinical indications. It is assumed that the successful outcome of AT transfer essentially depends on the amount of autocrine-generated growth factors (GF). It is supposed that several GF enhance and improve the anatomic and functional integration of the transplanted AT grafts at the site of implantation. In the present study we have investigated for the first time the correlation between the concentration of GF of freshly isolated AT and the proliferation and migration capacity of mesenchymal stroma cells (MSCs) derived from the respective AT sample. We here show that the proliferation and migration capacity of MSCs strongly depends on the GF content of the AT the cells were isolated from but in an inversely proportional manner. The lower the GF content of an AT sample was, the higher was the proliferation and migration capacity of the respective MSC population contained in the AT and vice versa. Furthermore, we found that supplementation with recombinant GFs only in the case of AT samples with low but not with higher growth factor contents led to a significant enhancement of proliferation and migration of the AT-resident MSCs. As we further show, this inefficiency of GFs to enhance MSC proliferation and migration in AT samples with high GF contents indicates a GF-mediated negative feedback mechanism leading to an impaired GF signaling in MSC obtained from those AT samples. Our results might explain why the successful use of AT grafting is frequently limited by low and unpredictable survival rates, and we suggest to use the knowledge of GF content of harvested AT as a predictive clinical parameter for risk assessment of the therapeutic outcome of autologous AT transfer.

Effects of amotosalen treatment on human platelet lysate bioactivity: A proof-of-concept study.

Clinical application of mesenchymal stromal cells (MSCs) usually requires an in vitro expansion step to reach clinically relevant numbers. In vitro cell expansion necessitates supplementation of basal mammalian cell culture medium with growth factors. To avoid using supplements containing animal substances, human platelet lysates (hPL) produced from expired and pathogen inactivated platelet concentrates can be used in place of fetal bovine serum. However, globally, most transfusion units are currently not pathogen inactivated. As blood banks are the sole source of platelet concentrates for hPL production, it is important to ensure product safety and standardized production methods. In this proof-of-concept study we assessed the feasibility of producing hPL from expired platelet concentrates with pathogen inactivation applied after platelet lysis by evaluating the retention of growth factors, cytokines, and the ability to support MSC proliferation and tri-lineage differentiation. Bone marrow-derived MSCs (BM-MSCs) were expanded and differentiated using hPL derived from pathogen inactivated platelet lysates (hPL-PIPL), with pathogen inactivation by amotosalen/ultraviolet A treatment applied after lysis of expired platelets. Results were compared to those using hPL produced from conventional expired pathogen inactivated platelet concentrates (hPL-PIPC), with pathogen inactivation applied after blood donation. hPL-PIPL treatment had lower concentrations of soluble growth factors and cytokines than hPL-PIPC treatment. When used as supplementation in cell culture, BM-MSCs proliferated at a reduced rate, but more consistently, in hPL-PIPL than in hPL-PIPC. The ability to support tri-lineage differentiation was comparable between lysates. These results suggest that functional hPL can be produced from expired and untreated platelet lysates by applying pathogen inactivation after platelet lysis. When carried out post-expiration, pathogen inactivation may provide a valuable solution for further standardizing global hPL production methods, increasing the pool of starting material, and meeting future demand for animal-free supplements in human cell culturing.

GARP is a key molecule for mesenchymal stromal cell responses to TGF-β and fundamental to control mitochondrial ROS levels.

Multipotent mesenchymal stromal cells (MSCs) have emerged as a promising cell therapy in regenerative medicine and for autoimmune/inflammatory diseases. However, a main hurdle for MSCs‐based therapies is the loss of their proliferative potential in vitro. Here we report that glycoprotein A repetitions predominant (GARP) is required for the proliferation and survival of adipose‐derived MSCs (ASCs) via its regulation of transforming growth factor‐β (TGF‐β) activation. Silencing of GARP in human ASCs increased their activation of TGF‐β which augmented the levels of mitochondrial reactive oxygen species (mtROS), resulting in DNA damage, a block in proliferation and apoptosis. Inhibition of TGF‐β signaling reduced the levels of mtROS and DNA damage and restored the ability of GARP−/lowASCs to proliferate. In contrast, overexpression of GARP in ASCs increased their proliferative capacity and rendered them more resistant to etoposide‐induced DNA damage and apoptosis, in a TGF‐β‐dependent manner. In summary, our data show that the presence or absence of GARP on ASCs gives rise to distinct TGF‐β responses with diametrically opposing effects on ASC proliferation and survival.

Paracrine Proangiogenic Function of Human Bone Marrow-Derived Mesenchymal Stem Cells Is Not Affected by Chronic Kidney Disease.

Background Cell-based therapies are being developed to meet the need for curative therapy in chronic kidney disease (CKD). Bone marrow- (BM-) derived mesenchymal stromal cells (MSCs) enhance tissue repair and induce neoangiogenesis through paracrine action of secreted proteins and extracellular vesicles (EVs). Administration of allogeneic BM MSCs is less desirable in a patient population likely to require a kidney transplant, but potency of autologous MSCs should be confirmed, given previous indications that CKD-induced dysfunction is present. While the immunomodulatory capacity of CKD BM MSCs has been established, it is unknown whether CKD affects wound healing and angiogenic potential of MSC-derived CM and EVs. Methods MSCs were cultured from BM obtained from kidney transplant recipients (N = 15) or kidney donors (N = 17). Passage 3 BM MSCs and BM MSC-conditioned medium (CM) were used for experiments. EVs were isolated from CM by differential ultracentrifugation. BM MSC differentiation capacity, proliferation, and senescence-associated β-galactosidase activity was assessed. In vitro promigratory and proangiogenic capacity of BM MSC-derived CM and EVs was assessed using an in vitro scratch wound assay and Matrigel angiogenesis assay. Results Healthy and CKD BM MSCs exhibited similar differentiation capacity, proliferation, and senescence-associated β-galactosidase activity. Scratch wound migration was not significantly different between healthy and CKD MSCs (P = 0.18). Healthy and CKD BM MSC-derived CM induced similar tubule formation (P = 0.21). There was also no difference in paracrine regenerative function of EVs (scratch wound: P = 0.6; tubulogenesis: P = 0.46). Conclusions Our results indicate that MSCs have an intrinsic capacity to produce proangiogenic paracrine factors, including EVs, which is not affected by donor health status regarding CKD. This suggests that autologous MSC-based therapy is a viable option in CKD.

Iron Overload Impairs Bone Marrow Mesenchymal Stromal Cells from MDS Patients and Promotes Leukemia Transformation

Bone marrow microenvironment plays an important role in the development of myelodysplastic syndrome (MDS). Iron overload (IO) may be related to the prognosis of the MDS patients. The effects of IO damaging mesenchymal stromal cells (MSCs) from MDS patients and promoting leukemia transformation were investigated. In our study, the IO group showed the decreased quantity and weakened abilities on proliferation and differentiation of MSCs. The gene expressions of VEGFA, CXCL12 and TGF-β1 in MSCs regulating hematopoiesis were significantly downregulated in IO group. The levels of reactive oxygen species (ROS) and apoptosis in IO group were significantly higher than those in non-iron overload (NIO) group, with highly expressed caspase3 and β-catenin. Meanwhile, the damages of MSCs caused by IO could be partially reversed by antioxidant or iron chelator. Due to the higher gene mutation frequency in IO group, the effects of IO promoting leukemia transformation were investigated. The IO MDS/AML group had a lower quantity of MSCs compared with IO MDS group, with significantly decreased expression of p-AKT. However, there was no significant differences in apoptosis, gene expressions of VEGF, CXCL12 supporting hematopoiesis, and β-catenin involving leukemia transformation in MSCs between them. In conclusion, IO damages MSCs of MDS patients by inducing ROS related apoptosis and activating caspase3 signals, which could be partially reversed by antioxidant or iron chelator. The increased genetic mutations and activated β-catenin signals in MSCs of MDS patients caused by IO may involve in promoting leukemia transformation. Disclosures No relevant conflicts of interest to declare.

Effect of Bioactive Peptide Complex Isolated from Bovine Serum on Proliferation and Migration of Mesenchymal Stromal Cells In Vitro and Reparation of Bone Defects In Vivo.

We studied the effect of a bioactive peptide complex isolated from bovine serum on the proliferative potential and migration rate of mesenchymal stromal cells in vitro, as well as on the healing of modeled bone defects in rats. This bioregulatory preparation stimulated proliferation of mesenchymal stromal cells from deciduous tooth pulp in vitro, but did not affect the rate of their migration in two-dimensional cultures. In vivo experiments showed that application of this preparation in combination with hydroxyapatite and chitosan gel accelerated bone tissue regeneration, thus ensuring restoration of morphologically normal bone matrix. Thus, cattle blood serum is an available source for the production of bioregulatory preparations for medical purposes.

Enhancement of Cellular Adhesion and Proliferation in Human Mesenchymal Stromal Cells by the Direct Addition of Recombinant Collagen I Peptide to the Culture Medium.

Mesenchymal stromal cells (MSCs) have considerable potential for a wide range of clinical applications and regenerative medicine and cell therapy. As a consequence, there is considerable interest in developing robust culture methods for producing large number of MSCs for use in repair of injured tissues or treatment of diseases. In general, tissue culture plates or flasks that have been precoated with substrates derived from animal tissues are used in the production of MSCs. However, these substrates can potentially cause serious problems due to contamination of the MSCs with animal-derived components. In this study, we evaluated the use of a type I collagen-based recombinant peptide (RCP) for MSC culture in an attempt to avoid the problems associated with animal cell-derived substances. This RCP is xeno free, has an increased RGD (Arg–Gly–Asp) sequence, and has high molecular weight uniformity. The effect of RCP on promotion of cellular adhesion and proliferation of MSCs was investigated in cultures in which RCP was included in the culture medium. The effects of RCP on promotion of cellular adhesion and proliferation of MSCs were investigated by comparing cultures in which the additive was present in the culture medium and those where the culture plates were coated with RCP. In addition, changes in gene expression profiles during cell culture were monitored by real time-polymerase chain reaction. Our analyses showed that RCP enhanced cellular adhesion and proliferation in cultures in which the additive was included in the culture medium. Our findings indicate that adding RCP to the culture medium could save time and cost in MSC culture. Our gene expression analysis indicated that RCP enhanced expression of genes encoding proteins associated with the extracellular matrix and cell adhesion.

Optically Transparent Anionic Nanofibrillar Cellulose Is Cytocompatible with Human Adipose Tissue-Derived Stem Cells and Allows Simple Imaging in 3D.

The anti-inflammatory and immunomodulatory properties of human mesenchymal stromal cells (MSCs) are a focus within regenerative medicine. However, 2D cultivation of MSCs for extended periods results in abnormal cell polarity, chromosomal changes, reduction in viability, and altered differentiation potential. As an alternative, various 3D hydrogels have been developed which mimic the endogenous niche of MSCs. Nevertheless, imaging cells embedded within 3D hydrogels often suffers from low signal-to-noise ratios which can be at least partly attributed to the high light absorbance and light scattering of the hydrogels in the visible light spectrum. In this study, human adipose tissue-derived MSCs (ADSCs) are cultivated within an anionic nanofibrillar cellulose (aNFC) hydrogel. It is demonstrated that aNFC forms nanofibres arranged as a porous network with low light absorbance in the visible spectrum. Moreover, it is shown that aNFC is cytocompatible, allowing for MSC proliferation, maintaining cell viability and multilineage differentiation potential. Finally, aNFC is compatible with scanning electron microscopy (SEM) and light microscopy including the application of conventional dyes, fluorescent probes, indirect immunocytochemistry, and calcium imaging. Overall, the results indicate that aNFC represents a promising 3D material for the expansion of MSCs whilst allowing detailed examination of cell morphology and cellular behaviour.

MRI Tracking of SPIO- and Fth1-Labeled Bone Marrow Mesenchymal Stromal Cell Transplantation for Treatment of Stroke.

We aimed to identify a suitable method for long-term monitoring of the migration and proliferation of mesenchymal stromal cells in stroke models of rats using ferritin transgene expression by magnetic resonance imaging (MRI). Bone marrow mesenchymal stromal cells (BMSCs) were transduced with a lentivirus containing a shuttle plasmid (pCDH-CMV-MCS-EF1-copGFP) carrying the ferritin heavy chain 1 (Fth1) gene. Ferritin expression in stromal cells was evaluated with western blotting and immunofluorescent staining. The iron uptake of Fth1-BMSCs was measured with Prussian blue staining. Following surgical introduction of middle cerebral artery occlusion, Fth1-BMSCs and superparamagnetic iron oxide- (SPIO-) labeled BMSCs were injected through the internal jugular vein. The imaging and signal intensities were monitored by diffusion-weighted imaging (DWI), T2-weighted imaging (T2WI), and susceptibility-weighted imaging (SWI) in vitro and in vivo. Pathology was performed for comparison. We observed that the MRI signal intensity of SPIO-BMSCs gradually reduced over time. Fth1-BMSCs showed the same signal intensity between 10 and 60 days. SWI showed hypointense lesions in the SPIO-BMSC (traceable for 30 d) and Fth1-BMSC groups. T2WI was not sensitive enough to trace Fth1-BMSCs. After transplantation, Prussian blue-stained cells were observed around the infarction area and in the infarction center in both transplantation models. Fth1-BMSCs transplanted for treating focal cerebral infarction were safe, reliable, and traceable by MRI. Fth1 labeling was more stable and suitable than SPIO labeling for long-term tracking. SWI was more sensitive than T2W1 and suitable as the optimal MRI-tracking sequence.

3170 – ABERRANT BONE MARROW MICROENVIRONMENT IN THERAPY RELATED MYELOID NEOPLASM

Introduction Therapy related myeloid neoplasm (t-MN) is a lethal second hematological malignancy following chemotherapy (CT) and radiotherapy (RT) for primary cancers. It accounts for 15-20% of myelodysplastic syndromes (MDS) and acute myeloid leukemia. Prognosis of t-MN is poor with no effective therapies. Although CT/RT can damage the bone marrow microenvironment, very limited studies assessed it in t-MN. Aim To compare bone marrow microenvironment milieu and mesenchymal stromal cells (MSC) from primary MDS (pMDS), t-MN and double cancers (DC, primary cancer and MDS but without a history of CT/RT for primary cancer). Methods Bone Marrow plasma cytokines/chemokines (CCK) and bone marrow MSC proliferation, senescence, morphology and differentiation potential of pMDS, t-MN and DC was compared. Results Proliferation and clonogenic potential of MSC from t-MN were significantly lower than pMDS and DC (Fig.1A). MSC from t-MN could not be maintained in culture and had higher senescence rates, assessed by β-galactosidase positive cells, compared to pMDS and DC (p Conclusions Our data demonstrate that MSC from t-MN patients have significantly altered morphology, a lower proliferation rate, and higher senescence rate compared to pMDS and DC. Osteogenic differentiation of t-MN MSC was higher at the cost of reduced adipogenic differentiation. This data suggests that RT/CT leads to long term damages to bone marrow microenvironment which may play a role in t-MN pathogenesis.

C1q/tumor necrosis factor-related protein-3-engineered mesenchymal stromal cells attenuate cardiac impairment in mice with myocardial infarction

Mesenchymal stromal cells (MSCs) transplantation offers an attractive alternative in myocardial infarctive therapy. However, poor cell engraftment and survival limit their restorative capacity. C1q/tumor necrosis factor-related protein-3 (CTRP3) inhibits reverse remodeling after myocardial infarction (MI) and was found to be secreted by MSCs in our preliminary experiments. We examined whether the overexpression of CTRP3 improved the survival of transplanted MSCs and augmented their efficacy on MI and whether silencing CTRP3 attenuated these effects. For gain-of-function analysis, MSCs overexpressing CTRP3 (LvC3-MSCs), control virus-transfected MSCs (LvNull-MSCs), MSCs alone, or phosphate-buffered saline (PBS) were injected into the peripheral areas of the infarction immediately after coronary artery ligation. For loss-of-function analysis, mice subjected to MI were randomized into groups and administered CTRP3-knockdown MSCs (LvshC3-MSCs), Lvshctrl-MSCs, MSCs, or PBS. Survival rates, cardiac function, and myocardial remodeling in mice were evaluated after 4 weeks. Injection of MSCs or LvNull-MSCs improved the left ventricular ejection fraction, inhibited cardiac fibrosis, and regulated cellular profiles of the infarction border zone 4 weeks after MI compared with those in the PBS group. Furthermore, overexpression of hCTRP3 promoted the efficacy of MSCs in the treatment of MI. However, knocking down CTRP3 impaired that. Coculture experiments confirmed that hCTRP3-enriched conditioned medium (CM) promoted MSCs migration and protected against H2O2-induced cell damage. Conversely, CM from C3−/− MSCs (CTRP3 knock out) significantly reduced the migration and antioxidative effects of MSCs. CTRP3 protein alone promoted MSCs proliferation and migration by upregulating matrix metalloproteinase 9 (MMP9) and protecting against oxidation by increasing superoxide dismutase 2 (SOD2) and metallothionein 1/2 (MT1/2) expression; and these effects were blocked by pretreatment with the extracellular signal-regulated kinase (ERK1/2) inhibitor U0126. Overexpression of CTRP3 significantly improved the MSCs-based efficacy on MI by increasing cell survival and retention via a mechanism involving ERK1/2-MMP9 and ERK1/2-SOD2/MT1/2 signaling.

Use of adult mesenchymal stromal cells in tissue repair: impact of physical exercise.

Physical exercise (PE) has unquestionable beneficial effects on health, which likely extend into several organ-to-cell physiological processes. At the cell scale, endogenous mesenchymal stromal cells (MSCs) contribute to tissue repair, although their repair capacities may be insufficient in paucicellular or severely damaged tissues. For this reason, MSC transplantation holds great promise for tissue repair. With the goals of understanding if PE has beneficial effects on MSC biology and if PE potentiates their role in tissue repair, we reviewed literature reports regarding the effects of PE on MSC properties (specifically, proliferation, differentiation, and homing) and of a combination of PE and MSC transplantation on tissue repair (specifically neural, cartilage, and muscular tissues). Contradictory results have been reported; interpretation is complicated because various and different species, cell sources, and experimental protocols, specifically exercise programs, have been used. On the basis of these data, the effects of exercise on MSC proliferation and differentiation depend on exercise characteristics (type, intensity, duration, etc.) and on the characteristics of the tissue from which the MSCs were collected. For the in vitro studies, the level of strain (and other details of the mechanical stimulus), the time elapsed between the end of exposure to strain and MSC collection, the age of the donors, as well as the passage number at which the MSCs are evaluated also play a role. The combination of PE and MSC engraftment improves neural, cartilage, and muscular tissue recovery, but it is not clear whether the effects of MSCs and exercise are additive or synergistic.

Pooling of Patient-Derived Mesenchymal Stromal Cells Reduces Inter-Individual Confounder-Associated Variation without Negative Impact on Cell Viability, Proliferation and Osteogenic Differentiation.

Patient-derived mesenchymal stromal cells (MSCs) play a key role in bone tissue engineering. Various donor-specific factors were identified causing significant variability in the biological properties of MSCs impairing quality of data and inter-study comparability. These limitations might be overcome by pooling cells of different donors. However, the effects of pooling on osteogenic differentiation, proliferation and vitality remain unknown and have, therefore, been evaluated in this study. MSCs of 10 donors were cultivated and differentiated into osteogenic lineage individually and in a pooled setting, containing MSCs of each donor in equal parts. Proliferation was evaluated in expansion (assessment of generation time) and differentiation (quantification of dsDNA content) conditions. Vitality was visualized by a fluorescence-microscopy-based live/dead assay. Osteogenic differentiation was assessed by quantification of alkaline phosphatase (ALP) activity and extracellular calcium deposition. Compared to the individual setting, generation time of pooled MSCs was shorter and proliferation was increased during differentiation with significantly lower variances. Calcium deposition was comparable, while variances were significantly higher in the individual setting. ALP activity showed high variance in both groups, but increased comparably during the incubation period. In conclusion, MSC pooling helps to compensate donor-dependent variability and does not negatively influence MSC vitality, proliferation and osteogenic differentiation.

Asprosin improves the survival of mesenchymal stromal cells in myocardial infarction by inhibiting apoptosis via the activated ERK1/2-SOD2 pathway

AimsSeveral adipokines have been proven to improve the therapeutic efficacy of mesenchymal stromal cells (MSCs) when used to treat ischemic heart disease. Asprosin (ASP) is a newly-discovered adipokine. ASP might also predict the severity of coronary pathology. We investigated the role of ASP on MSCs and the effects of ASP-pretreated MSCs on myocardial infarction (MI). Main methodsMSCs were labelled with a lentivirus carrying green fluorescent protein (GFP). For in vivo study, after pretreatment with vehicle or ASP, MSCs were injected into infarcted hearts. Cardiac function and fibrosis were then evaluated 4 weeks after the induction of MI and survival of MSCs evaluated after 1 week. MSCs proliferation and migration were investigated after ASP treatment in vitro. MSCs apoptosis induced by hydrogen peroxide (H2O2) was assessed using flow cytometry. Key findingsCompared to vehicle-pretreated MSCs, ASP-pretreated MSCs significantly improved the left ventricular ejection fraction (LVEF), and inhibited myocardial fibrosis 4 weeks after MI. ASP pretreatment may have promoted homing of transplanted MSCs. In vitro results showed that ASP had no significant effect on MSC proliferation and migration, but protected these cells from H2O2-induced apoptosis. Among 21 molecules associated with antioxidation and cell death, the antioxidant enzyme SOD2 was significantly upregulated by ASP. Furthermore, ASP treatment inhibited H2O2-induced ROS generation and apoptosis via the activated ERK1/2-SOD2 pathway. SignificanceThis is the first evidence that ASP can regulate MSCs function and enhance MSCs therapy for ischemic heart disease. Furthermore, we demonstrate that ASP protects MSCs from oxidative stress-induced apoptosis via the ERK1/2-SOD2 pathway.

Improving wave-induced motion bioreactor performance for human mesenchymal stromal cell expansion

Abstract Mesenchymal Stromal Cells (MSCs) are considered a viable option for the treatment of several diseases in different medical areas. MSCs are present in small proportions in tissues, thus making an in vitro expansion necessary in accordance with the cGMP. For this purpose, the disposable Wave-Induced Motion Bioreactor (WIMB) is a promising alternative for MSC expansion in a safe and low shear environment. However, wave motion leads to the formation of aggregate deposits (microcarriers and cells) on the Cellbag plastic, impairing the MSC proliferation. In this study, a modification in the Cellbag design was proposed and tested in order to overcome this limitation. Human Umbilical Cord Matrix derived Mesenchymal Stromal Cells (hUCM-MSCs) were expanded in WIMB using Cultispher-S microcarriers and α-MEM medium. Two approaches were evaluated for hUCM-MSCs culture in the new Cellbag design: 1) adhesion phase in spinner and expansion in the WIMB (Expansion Factors: 5.8–8.0 fold) and 2) adhesion and expansion phases in the WIMB (Expansion Factors: 11.6–25.6 fold), both with absence of the most damaging particle deposits. These results showed that the use of WIMB with the Cellbag design modification have good potential for hUCM-MSCs expansion, while maintaining cell quality attributes.

Full-time physioxic culture conditions promote MSC proliferation more than hypoxic preconditioning

Background & Aim Cells that are cultured and handled in traditional hyperoxic room air experience a dramatic shift in oxygen levels when injected in vivo. It has been reported that hypoxic pre-conditioning of mesenchymal stromal cells (MSC) in culture can increase cell survival in vivo after administration. Published hypoxic pre-treatments subject room-air expanded cells to lower oxygen atmospheric conditions for lengths of time that range from 15 minutes to 36 hours, followed by another period of high oxygen exposure in room air conditions before administration. Some regimens use cobalt compounds to deplete HIF-1a rather than the cell environment. We previously showed that Human bone marrow MSC yields are improved by enclosing cell-handling processes as well as incubation for a full-time controlled physioxic atmosphere. AIMs To use the Xvivo System (BioSpherix), with fully-enclosed and environmentally-controlled cell incubation and handling chambers, to compare different hypoxic pre-conditioning regimens to full-time physioxic conditions for MSC yields and subjected to subsequent physioxic conditions as if injected venously. Our null hypothesis was that the different culture conditions make no difference to MSC morphology and proliferation rates. Methods, Results & Conclusion MSC were incubated for 1-36 hours at 1% O2 before return to traditional supraphysioxic room air incubator oxygen levels for 24 hours. The cells were then incubated at 5% O2 (venous O2 levels) as if injected in vivo. These conditions were compared with unbroken 5% O2 for both cell handling and culture. Using an immersion oxygen probe, we recorded vessel headspace and pericellular medium oxygen levels during each regimen. For 72 hours in culture to 5% O2, we assayed cells for changes in cell division rates and final cell yield. MSC pericellular O2 levels lagged far behind atmospheric oxygen level changes, so MSC experienced low oxygen conditions for far less time than the pre-conditioning period. Unbroken oxygen conditions were more favorable for keeping MSC in the cell cycle than any pre-conditioning regiment, disproving the null hypothesis. We concluded that hypoxic pre-conditioning is not as favorable for MSC yields as full-time physioxic conditions for cell handling and incubation.

Human platelet lysate is suitable for the propagation of human bone marrow derived mesenchymal stromal cells

Background & Aim To evaluate the suitability of human platelet lysate (HPL) as a replacement for foetal bovine serum (FBS) for the propagation of human bone marrow derived mesenchymal stromal cells (MSCs). Background FBS is used universally as a cell culture supplement. Currently the use of FBS is still tolerated, however its use in clinical trials is tightly regulated and the US Food and Drug Administration (FDA) is moving towards adopting European guidelines, requiring that xenogeneic materials be eliminated in the preparation of any human therapeutic product containing cultured cells. Therefore, the use of HPL as a replacement for FBS in MSC expansion was evaluated. Methods, Results & Conclusion Methods Two types of HPL were produced by freeze-thawing and pooling either 12 units of expired apheresis platelet concentrates or 10 units of expired pooled platelet concentrates (n=3 batches each). MSCs from two donors were seeded at 5000 cells/cm2 and cultured to approximately 75-80% confluency using DMEM supplemented with either apheresis HPL, pooled HPL or FBS. Similar to FBS, HPL was heat-inactivated for 60 min at 56°C prior to addition to DMEM. The optimum concentration of HPL was determined by titration (2.5%, 5%, 7.5%, or 10%) against MSC culture expansion. Cell proliferation rates and doubling time were determined. Post-thaw immunophenotype and viability (7AAD) of BM-MSCs were determined by flow cytometry. Tri-lineage differentiation capacity was confirmed by microscopy after in vitro stimulation for either 14 days (chondrogenic and adipogenic) or 28 days (osteogenic). Results All 3 batches of HPL demonstrated an optimal culture concentration of 5% for apheresis HPL and 7.5 % for pooled HPL. Both types of HPL induced faster MSC proliferation when compared with matched 10% FBS controls. Apheresis HPL consistently outperformed pooled HPL, presumably as it is richer in growth factors. MSCs expanded under optimum conditions showed >90% expression of CD73, CD90 and CD105, and all were negative for CD45, CD34, CD19, CD11b, and HLA-DR. Mean 7AAD viability was 90% for MSCs grown in apheresis HPL, 88% for pooled HPL, and 84% for FBS. One batch of each type of HPL was used for tri-lineage differentiation capacity analysis. All MSCs were able to differentiate into either chondrogenic, osteogenic, or adipogenic lineages. Conclusion Apheresis and pooled HPL induced higher MSC expansion than FBS. The use of HPL as a substitute for FBS in MSC propagation warrants further investigation.

Green propolis extract promotes in vitro proliferation, differentiation, and migration of bone marrow stromal cells

BackgroundPropolis is a resinous material extracted from bee glue with a complex chemical composition. The unique biological properties of propolis have led to its use in alternative medicine and as a nutritional supplement. Recent research shows that propolis could affect the immune system by decreasing the production of inflammatory cytokines and potentiating an effect on resident stem cells. The exact mechanism, however, is unknown. The goal of this study was to demonstrate whether green propolis extract affects any characteristic properties of mesenchymal stromal cells (MSCs)in vitro. MethodsThe cytocompatibility of propolis extract and the proliferation of bone marrow mesenchymal stromal cells (BMMSCs) in the presence of propolis was evaluated by live/dead cell staining and MTS viability assay over a period of 3 days. Also, we evaluated the effect of propolis extract on trilineage differentiation and migration capacity of undifferentiated and differentiated BMMSCs. ResultsRelative to the control, propolis extract resulted in a significant and linear increase in the proliferation of MSCs and inhibited the osteogenic differentiation of BMMSCs, while there was a potentiation of chondrogenesis and adipogenesis. Finally, in relevance to wound healing, an in vitro scratch assay demonstrated that the migratory potential of differentiated BMMSCs was enhanced in the presence of propolis. ConclusionWe have demonstrated that propolis extract was not toxic to BMMSCs (<400 μg/ml), supported their proliferation, potentiated chondrogenic and adipogenic differentiation processes, and supported cell migrationin vitro. Most interestingly, there was a down-regulation of osteogenesis. These data support the use of propolis extract for enhanced cell proliferation and tissue regeneration; however, it warrants further investigation.

Factors contained in the expansion medium regulate the expression stem cell markers CD146 and SUSD2 on human placenta‐derived mesenchymal stromal cells and modulate their differentiation capacity in vitro

Human mesenchymal stromal cells (MSCs) are investigated in vitro and in vivo manifold to serve as cellular therapy in different clinical situations including the regeneration of muscular tissues eventually. For production of MSCs for experimental purposes expansion media often include fetal bovine serum (FBS) as source of growth factors. In clinical situations xenogenic components such as FBS must be avoided in many countries. Therefore, different xeno‐free MSC expansion media were developed and FBS was replaced e.g. by human serum (hS), platelet lysate (hPL) or hPL plus human plasma (hPLP). However, differences in these medium complements could influence the phenotype and characteristics of MSCs. We therefore compared the expression of cell surface markers, the differentiation capacity and the expression of key cytokines involved in muscular regeneration in human placenta‐derived MSC‐like cells (pMSCs) expanded in different xeno‐free and GMP‐compliant expansion media. We report that addition of 5% hPL to either commercial MSC expansion medium (medium with FBS + 5% hPL) or to GMP‐compliant MSC expansion medium (DMEM + 5% hS + 5% hPL) significantly increased the MSC proliferation (p&lt;0.05), and reduced the expression of CD146 (p&lt;0.05), whereas CD73 and CD90 and other markers remained unchanged, on both transcript and protein levels. Expression of stem cell marker SUSD2 was not significantly regulated by hPL. The osteogenic differentiation capacity was reduced in pMSCs expanded in medium complemented with 5% hP + 5% hPL when compared to pMSCs incubated in medium containing FBS without hPL, but not in DMEM + hS compared to DMEM+ hS + hPL. Significant changes in the expression of neuro‐ or myo‐regenerative cytokines including BDNF, bFGF, GDNF, HGF, IGF‐1, IGF‐2, TGFβ, or VEGF in MSCs was not influenced by the composition of the GMP‐compliant expansion media. However, some interindividual differences between pMSCs from individual donors and between batches of hPL were noted. In contrast, the composition of expansion media did not influence osteogenesis or expression of stem cell markers CD146 and SUSDA on human bone marrow‐derived MSCs. We conclude that the expression of CD146 and the osteogenic differentiation potential of human pMSCs is influenced by the expansion medium. The expression of the regenerative cytokines investigated was not biased by hPL in the expansion medium. Expansion of pMSCs in GMP‐compliant medium enriched by hP + hPL could therefore be an advantage when bone formation by the MSCs applied should be avoided in clinical situations.Support or Funding InformationThis work was supported in part by grants from the DFG, BMBF and by institutional funding.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Roles for HB-EGF in Mesenchymal Stromal Cell Proliferation and Differentiation During Skeletal Growth.

ABSTRACTHB‐EGF, a member of the EGF superfamily, plays important roles in development and tissue regeneration. However, its functions in skeletal stem cells and skeleton development and growth remain poorly understood. Here, we used the Cre/LoxP system to ablate or express HB‐EGF in Dermo1+ mesenchymal stromal cells and their progenies, including chondrocytes and osteoblast lineage cells, and bone marrow stromal cells (BMSCs). Dermo1‐Cre; HB‐EGFf/f mice only showed a modest increase in bone mass, whereas Dermo1‐HB‐EGF mice developed progressive chondrodysplasia, chondroma, osteoarthritis‐like joint defects, and loss of bone mass and density, which were alleviated by treatment with EGFR inhibitor AG1478. The cartilage defects were recapitulated in chondrocyte‐specific HB‐EGF overexpression (Col2‐HB‐EGF) mice with a lesser severity. Dermo1‐HB‐EGF mice showed an increase in proliferation but defects in differentiation of chondrocytes and osteoblasts. HB‐EGF promoted BMSC proliferation via the Akt1 and Erk pathways but inhibited BMSC differentiation via restraining Smad1/5/8 activation. However, Dermo1‐HB‐EGF mice showed normal osteoclastogenesis and bone resorption. These results reveal an important function of autocrine or paracrine HB‐EGF in mesenchymal stromal cell proliferation and differentiation and suggest that EGF signaling needs to be tightly controlled to maintain bone and articular cartilage integrity. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.