Maik Stiehler

Culture media for the differentiation of mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) can be isolated from various tissues such as bone marrow aspirates, fat or umbilical cord blood. These cells have the ability to proliferate in vitro and differentiate into a series of mesoderm-type lineages, including osteoblasts, chondrocytes, adipocytes, myocytes and vascular cells. Due to this ability, MSCs provide an appealing source of progenitor cells which may be used in the field of tissue regeneration for both research and clinical purposes. The key factors for successful MSC proliferation and differentiation in vitro are the culture conditions. Hence, we here summarize the culture media and their compositions currently available for the differentiation of MSCs towards osteogenic, chondrogenic, adipogenic, endothelial and vascular smooth muscle phenotypes. However, optimal combination of growth factors, cytokines and serum supplements and their concentration within the media is essential for the in vitro culture and differentiation of MSCs and thereby for their application in advanced tissue engineering.

Prevalence of Low Back Pain in Adolescent Athletes – an Epidemiological Investigation

Low back pain (LBP) is a common symptom in the populations of western countries, and adolescent athletes seem to be prone to LBP. The main objective of this study was to analyze the point (LBP within the last 48 h), 1-year (LBP within the last 12 months) and lifetime (LBP within the entire life) prevalence rates of LBP in adolescent athletes participating in various sports. We also assessed the characteristics of LBP and its association with potential risk factors. To this end, 272 competitive adolescent athletes involved in 31 different sports (158 males, 113 females, 15.4 ± 2.0 years, body mass index [BMI] 20.3 ± 2.4 kg/m(2)) were enrolled in a 10-month prospective clinical trial that included a questionnaire and physical examination. We found a point prevalence of 14%, a 1-year prevalence of 57%, and a lifetime prevalence of 66% for LBP. The mean age of first appearance of LBP was 13.1 ± 2.0 years. The lifetime prevalence was significantly higher in volleyball than in biathletes (74.3 vs. 45.7%, p = 0.015). Our findings confirm that LBP is a common symptom in adolescent athletes; LBP prevalence correlates with sports participation and individual competitive level. Adolescent athletes with LBP should receive a thorough diagnostic work-up and adapt training and technique correspondingly when indicated.

Mesenchymal stromal cells from patients with myelodyplastic syndrome display distinct functional alterations that are modulated by lenalidomide

The contribution of the bone marrow microenvironment in myelodysplastic syndrome is controversial. We therefore analyzed the functional properties of primary mesenchymal stromal cells from patients with myelodysplastic syndrome in the presence or absence of lenalidomide. Compared to healthy controls, clonality and growth were reduced across all disease stages. Furthermore, differentiation defects and particular expression of adhesion and cell surface molecules (e.g. CD166, CD29, CD146) were detected. Interestingly, the levels of stromal derived factor 1-alpha in patients’ cells culture supernatants were almost 2-fold lower (P<0.01) than those in controls and this was paralleled by a reduced induction of migration of CD34+ hematopoietic cells. Co-cultures of mesenchymal stromal cells from patients with CD34+ cells from healthy donors resulted in reduced numbers of cobblestone area-forming cells and fewer colony-forming units. Exposure of stromal cells from patients and controls to lenalidomide led to a further reduction of stromal derived factor 1-alpha secretion and cobblestone area formation, respectively. Moreover, lenalidomide pretreatment of mesenchymal stromal cells from patients with low but not high-risk myelodysplastic syndrome was able to rescue impaired erythroid and myeloid colony formation of early hematopoietic progenitors. In conclusion, our analyses support the notion that the stromal microenvironment is involved in the pathophysiology of myelodysplastic syndrome thus representing a potential target for therapeutic interventions.

Salvianolic acid B protects human endothelial progenitor cells against oxidative stress-mediated dysfunction by modulating Akt/mTOR/4EBP1, p38 MAPK/ATF2, and ERK1/2 signaling pathways.

The vascular endothelium is specifically sensitive to oxidative stress, and this is one of the mechanisms that causes widespread endothelial dysfunction in most cardiovascular diseases and disorders. Protection against reactive oxygen species (ROS)-mediated oxidative damage via antioxidant mechanisms is essential for tissue maintenance and shows therapeutic potential for patients suffering from cardiovascular and metabolic disorders. Salvianolic acid B (SalB), a natural bioactive component known from Traditional Chinese Medicine, has been reported to exert cellular protection in various types of cells. However, the underlying mechanisms involved are not fully understood. Here, we showed that SalB significantly promoted the migratory and tube formation abilities of human bone marrow derived-endothelial progenitor cells (BM-EPCs) in vitro, and substantially abrogated hydrogen peroxide (H2O2)-induced cell damage. SalB down-regulated Nox4 and eNOS, as well as nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase expression upon H2O2 induction that in turn prevents oxidative-induced endothelial dysfunction. Moreover, SalB suppressed the Bax/Bcl-xL ratio and caspase-3 activation after H2O2 induction. Furthermore, our results provide mechanistic evidence that activation of the mTOR/p70S6K/4EBP1 pathways is required for both SalB-mediated angiogenic and protective effects against oxidative stress-induced cell injury in BM-EPCs. Suppression of MKK3/6-p38 MAPK-ATF2 and ERK1/2 signaling pathways by SalB significantly protected BM-EPCs against cell injury caused by oxidative stress via reduction of intracellular ROS levels and apoptosis. Taken together, by providing a mechanistic insight into the modulation of redox states in BM-EPCs by SalB, we suggest that SalB has a strong potential of being a new proangiogenic and cytoprotective therapeutic agent with applications in the field of endothelial injury-mediated vascular diseases.

Icariin Promotes Angiogenic Differentiation and Prevents Oxidative Stress‐Induced Autophagy in Endothelial Progenitor Cells

Reduced tissue levels of endothelial progenitor cells (EPCs) and functional impairment of endothelium are frequently observed in patients with diabetes and cardiovascular disease. The vascular endothelium is specifically sensitive to oxidative stress, and this is one of the mechanisms that causes widespread endothelial dysfunction in most cardiovascular diseases and disorders. Hence attention has increasingly been paid to enhance mobilization and differentiation of EPCs for therapeutic purposes. The aim of this study was to investigate whether Icariin, a natural bioactive component known from traditional Chinese Medicine, can induce angiogenic differentiation and inhibit oxidative stress‐induced cell dysfunction in bone marrow‐derived EPCs (BM‐EPCs), and, if so, through what mechanisms. We observed that treatment of BM‐EPCs with Icariin significantly promoted cell migration and capillary tube formation, substantially abrogated hydrogen peroxide (H2O2)‐induced apoptotic and autophagic programmed cell death that was linked to the reduced intracellular reactive oxygen species levels and restored mitochondrial membrane potential. Icariin downregulated endothelial nitric oxide synthase 3, as well as nicotinamide‐adenine dinucleotide phosphate‐oxidase expression upon H2O2 induction. These antiapoptotic and antiautophagic effects of Icariin are possibly mediated by restoring the loss of mammalian target of rapamycin /p70S6K/4EBP1 phosphorylation as well as attenuation of ATF2 and ERK1/2 protein levels after H2O2 treatment. In summary, favorable modulation of the angiogenesis and redox states in BM‐EPCs make Icariin a promising proangiogenic agent both enhancing vasculogenesis and protecting against endothelial dysfunction. Stem Cells 2015;33:1863–1877

Salidroside exerts angiogenic and cytoprotective effects on human bone marrow‐derived endothelial progenitor cells via Akt/mTOR/p70S6K and MAPK signalling pathways

With the increase of age, increased susceptibility to apoptosis and senescence may contribute to proliferative and functional impairment of endothelial progenitor cells (EPCs). The aim of this study was to investigate whether salidroside (SAL) can induce angiogenic differentiation and inhibit oxidative stress‐induced apoptosis in bone marrow‐derived EPCs (BM‐EPCs), and if so, through what mechanism.

Recommendations and considerations for the use of biologics in orthopedic surgery.

Reconstruction of extensive bone defects remains technically challenging and has considerable medical and financial impact on our society. Surgical procedures often require a bone/substitute graft to enhance and accelerate bone repair. Bone autografts are associated with morbidity related to bone harvesting and are limited in quantity. Alternatively, bone allografts expose the patient to the risk of transmission of infectious disease. Synthetic bone graft substitutes, such as calcium sulfates, hydroxyapatite, tricalcium phosphate, and combinations, circumvent some of the disadvantages of auto- and allografts, but have limited indications. Biomedical research has made possible the stimulation of the bodys own healing mechanisms, either by delivering exogenous growth factors locally, or by stimulating their local production by gene transfer. Among all known factors having osteoinductive properties, only two bone morphogenetic proteins (for specific indications) and demineralized bone matrix have been approved for clinical use. In addition, ongoing research is exploring the efficacy of cell therapy and tissue engineering. The present report examines the composition, biological properties, indications, clinical experience and regulations of several of the biotherapeutics employed for bone reconstruction.

Erythropoietin augments bone formation in a rabbit posterolateral spinal fusion model

We tested the hypothesis that erythropoietin (EPO) enhances bone formation after posterolateral spinal fusion (PLF) in a rabbit model. Thirty‐four adult rabbits underwent posterolateral intertransverse arthrodesis at the L5–L6 level using 2.0 g autograft per side. The animals were randomly divided into two groups receiving subcutaneous daily injections of either EPO or saline for 20 days. Treatment commenced 2 days preoperatively. Hemoglobin was monitored at baseline and 2, 4, and 6 weeks after fusion surgery. After euthanasia 6 weeks postoperatively, manual palpation, radiographic, and histomorphometric examinations were performed. Bone volume of the fusion mass was estimated by CT after 6 weeks. EPO increased bone fusion volume to 3.85 ccm (3.66–4.05) compared with 3.26 ccm (2.97–3.55) in the control group (p < 0.01). EPO treatment improved vascularization of the fusion mass and increased hemoglobin levels (p < 0.01). Fusion rate tended to be higher in the EPO group based on manual palpation, CT, and radiographic examinations. For the first time EPO has shown to augment bone formation after autograft PLF in a rabbit model. Increased vascularization provides a partial explanation for the efficacy of EPO as a bone autograft enhancer.

Breast carcinoma cells modulate the chemoattractive activity of human bone marrow‐derived mesenchymal stromal cells by interfering with CXCL12

We investigated whether breast tumor cells can modulate the function of mesenchymal stromal cells (MSCs) with a special emphasis on their chemoattractive activity towards hematopoietic stem and progenitor cells (HSPCs). Primary MSCs as well as a MSC line (SCP‐1) were cocultured with primary breast cancer cells, MCF‐7, MDA‐MB231 breast carcinoma or MCF‐10A non‐malignant breast epithelial cells or their conditioned medium. In addition, the frequency of circulating clonogenic hematopoietic progenitors was determined in 78 patients with breast cancer and compared with healthy controls. Gene expression analysis of SCP‐1 cells cultured with MCF‐7 medium revealed CXCL12 (SDF‐1) as one of the most significantly downregulated genes. Supernatant from both MCF‐7 and MDA‐MB231 reduced the CXCL12 promoter activity in SCP‐1 cells to 77% and 47%, respectively. Moreover, the CXCL12 mRNA and protein levels were significantly reduced. As functional consequence of lower CXCL12 levels, we detected a decreased trans‐well migration of HSPCs towards MSC/tumor cell cocultures or conditioned medium. The specificity of this effect was confirmed by blocking studies with the CXCR4 antagonist AMD3100. Downregulation of SP1 and increased miR‐23a levels in MSCs after contact with tumor cell medium as well as enhanced TGFβ1 expression were identified as potential molecular regulators of CXCL12 activity in MSCs. Moreover, we observed a significantly higher frequency of circulating colony‐forming hematopoietic progenitors in patients with breast cancer compared with healthy controls. Our in vitro results propose a potential new mechanism by which disseminated tumor cells in the bone marrow may interfere with hematopoiesis by modulating CXCL12 in protected niches.

Cancellous bone allograft seeded with human mesenchymal stromal cells: a potential good manufacturing practice-grade tool for the regeneration of bone defects.

BACKGROUND AIMS Combining autologous bone precursor cells with cancellous bone allograft (CBA) offers an appealing strategy for skeletal regeneration. In this context, multipotent mesenchymal stromal cells (MSC) provide an excellent cell source because they are readily harvested from donors, expanded and differentiated in vitro. The aim of this study was to evaluate the proliferation, morphology, osteogenic differentiation and stem cell-related gene expression during static long-term ex vivo cultivation using human MSC and CBA under good manufacturing practice (GMP)-conforming conditions. METHODS MSC were isolated from healthy donors (n = 5) and cultivated on peracetic acid-sterilized CBA in the presence of 10% human platelet-rich plasma without osteogenic supplements. Total protein content, cell-specific alkaline phosphatase (ALP) activity and osteogenic marker gene expression levels were assessed. Stem cell-related gene expression was compared with MSC monolayer cultivation using microarray analysis. Furthermore, cellular distribution and morphology within the porous CBA were visualized by histology and scanning electron microscopy. RESULTS Effective adhesion, spreading, proliferation and intercellular contact of human MSC within the pores of CBA were observed during the study (< or = 42 days). Cell-specific ALP activity peaked after 3 weeks of cultivation. Gene expression of early, intermediate and late osteogenic marker genes was detectable during long-term cultivation. Microarray-based annotation and biologic interaction network data analysis indicated that expression levels of genes encoding crucial differentiation-regulating proteins and extracellular matrix components involved in the process of osteogenesis were induced in CBA-cultivated MSC. CONCLUSIONS MSC-vitalized CBA offers an attractive GMP-grade bone-filling material. Further research is warranted to evaluate its bone-healing potential in vivo.