Characterization of a pluripotent stem cell-derived matrix with powerful osteoregenerative capabilities.

Approximately 10% of fractures will not heal without intervention. Current treatments can be marginally effective, costly, and some have adverse effects. A safe and manufacturable mimic of anabolic bone is the primary goal of bone engineering, but achieving this is challenging. Mesenchymal stem cells (MSCs), are excellent candidates for engineering bone, but lack reproducibility due to donor source and culture methodology. The need for a bioactive attachment substrate also hinders progress. Herein, we describe a highly osteogenic MSC line generated from induced pluripotent stem cells that generates high yields of an osteogenic cell-matrix (ihOCM) in vitro. In mice, the intrinsic osteogenic activity of ihOCM surpasses bone morphogenic protein 2 (BMP2) driving healing of calvarial defects in 4 weeks by a mechanism mediated in part by collagen VI and XII. We propose that ihOCM may represent an effective replacement for autograft and BMP products used commonly in bone tissue engineering.

Quantitative Efficacy and Fate of Mesenchymal Stromal Cells Targeted to Cardiac Sites by Radiofrequency Catheter Ablation.

Engraftment and functional integration of stem cells or stem cell-derived cells within cardiac tissue is an important prerequisite for cell replacement therapy aiming at the treatment of heart disease. Recently, a novel intravenous approach for application of mesenchymal stromal cells (MSCs) to cardiac sites has been established using radiofrequency catheter ablation (RFCA)-guided targeting, bypassing the need for open chest surgery or direct myocardial cell injection. However, little is known about the quantitative efficacy and longevity of this strategy. We performed selective power-controlled RFCA with eight ablation pulses (30 W, 60 s each) to induce heat-mediated lesions at the right atrial appendices (RAAs) of pigs. Different concentrations of human bone marrow-derived MSCs (105 to 1.6 × 106 cells/kg bodyweight) labeled with superparamagnetic iron oxide (SPIO) particles were infused intravenously in nine pigs one d after RFCA treatment and hearts were explanted 8 d later to quantify the number of engrafted cells. Prussian blue staining revealed high numbers of SPIO-labeled cells in areas surrounding the RFCA-induced lesions. Cell numbers were evaluated by quantitative real-time polymerase chain reaction using specific primers for human MSCs (hMSCs), which indicated that up to 106 hMSCs, corresponding to ∼3.9% of the systemically applied human cells, engrafted within the RAAs of RFCA-treated pigs. Of note, infused hMSCs were observed in nontargeted organs, as well, but appeared at very low concentrations. To assess long-term deposition of MSCs, RAAs of three pigs were analyzed after 6 months, which revealed few persisting hMSCs at targeted sites. RFCA-mediated targeting of MSCs provides a novel minimal invasive strategy for cardiac stem cell engraftment. Qualitative and quantitative results of our large animal experiments indicate an efficient guidance of MSCs to selected cardiac regions, although only few cells remained at targeted sites 6 mo after cell transplantation.

Three-dimensional in vitro modeling of malignant bone disease recapitulates experimentally accessible mechanisms of osteoinhibition.

Malignant bone disease (MBD) occurs when tumors establish in bone, causing catastrophic tissue damage as a result of accelerated bone destruction and inhibition of repair. The resultant so-called osteolytic lesions (OL) take the form of tumor-filled cavities in bone that cause pain, fractures, and associated morbidity. Furthermore, the OL microenvironment can support survival of tumor cells and resistance to chemotherapy. Therefore, a deeper understanding of OL formation and MBD progression is imperative for the development of future therapeutic strategies. Herein, we describe a novel in vitro platform to study bone-tumor interactions based on three-dimensional co-culture of osteogenically enhanced human mesenchymal stem cells (OEhMSCs) in a rotating wall vessel bioreactor (RWV) while attached to micro-carrier beads coated with extracellular matrix (ECM) composed of factors found in anabolic bone tissue. Osteoinhibition was recapitulated in this model by co-culturing the OEhMSCs with a bone-tumor cell line (MOSJ-Dkk1) that secretes the canonical Wnt (cWnt) inhibitor Dkk-1, a tumor-borne osteoinhibitory factor widely associated with several forms of MBD, or intact tumor fragments from Dkk-1 positive patient-derived xenografts (PDX). Using the model, we observed that depending on the conditions of growth, tumor cells can biochemically inhibit osteogenesis by disrupting cWnt activity in OEhMSCs, while simultaneously co-engrafting with OEhMSCs, displacing them from the niche, perturbing their activity, and promoting cell death. In the absence of detectable co-engraftment with OEhMSCs, Dkk-1 positive PDX fragments had the capacity to enhance OEhMSC proliferation while inhibiting their osteogenic differentiation. The model described has the capacity to provide new and quantifiable insights into the multiple pathological mechanisms of MBD that are not readily measured using monolayer culture or animal models.

How stem cell composition in bone marrow aspirate relates to clinical outcomes when used for cervical spine fusion.

Anterior cervical discectomy and fusion (ACDF) is performed to relieve pain caused by degenerative disk disease and nerve obstruction. As an alternative to bone graft, autologous concentrated bone marrow aspirate (CBMA) is used to achieve vertebral fusion with a satisfactory success rate. This has been attributed in part to bone marrow-resident mesenchymal stromal cells (MSCs) with the capacity to differentiate into osteoblasts and generate bone tissue. To date, there has been no study comparing cellular yields, MSC frequencies and their osteogenic potential with ACDF outcome. Patients (n = 24) received ACDF with CBMA and allograft bone matrix. Colony forming unit fibroblast (CFU-F) and CFU-osteoblasts (CFU-O) assays were performed on CBMA samples to enumerate MSCs (CFU-F) and osteogenic MSCs (CFU-O). CFUs were normalized to CBMA volume to define yield and also to mononuclear cells (MNC) to define frequency. After 1-year, fusion rates were good (86.7%) with pain and disability improved. There was a negative relationship between MNC and CFU-F measurements with age of patient and CFU-Os negatively correlated with age in females but not males. Tobacco use did not affect CBMA but was associated with poorer clinical outcome. Surprisingly, we found that while high-grade fusion was not associated with CFU-O, it correlated strongly (p<0.0067) with CBMA containing the lowest frequencies of CFU-F (3.0x10(-6)-5.83x10(-5) CFU-F/MNC). MNC levels alone were not responsible for the results. These observations suggest that osteogenesis by human bone marrow is controlled by homeostatic ratio of MSCs to other cellular bone marrow components rather than absolute level of osteogenic MSCs, and that a lower ratio of MSCs to other cellular components in marrow tends to predict effective osteogenesis during ACDF. The results presented herein challenge the current dogma surrounding the proposed mechanism of MSCs in bone healing.

Rapid Osteogenic Enhancement of Stem Cells in Human Bone Marrow Using a Glycogen-Synthease-Kinase-3-Beta Inhibitor Improves Osteogenic Efficacy In Vitro and In Vivo.

Non-union defects of bone are a major problem in orthopedics, especially for patients with a low healing capacity. Fixation devices and osteoconductive materials are used to provide a stable environment for osteogenesis and an osteogenic component such as autologous human bone marrow (hBM) is then used, but robust bone formation is contingent on the healing capacity of the patients. A safe and rapid procedure for improvement of the osteoanabolic properties of hBM is, therefore, sought after in the field of orthopedics, especially if it can be performed within the temporal limitations of the surgical procedure, with minimal manipulation, and at point-of-care. One way to achieve this goal is to stimulate canonical Wingless (cWnt) signaling in bone marrow-resident human mesenchymal stem cells (hMSCs), the presumptive precursors of osteoblasts in bone marrow. Herein, we report that the effects of cWnt stimulation can be achieved by transient (1-2 hours) exposure of osteoprogenitors to the GSK3ß-inhibitor (2'Z,3'E)-6-bromoindirubin-3'-oxime (BIO) at a concentration of 800 nM. Very-rapid-exposure-to-BIO (VRE-BIO) on either hMSCs or whole hBM resulted in the long-term establishment of an osteogenic phenotype associated with accelerated alkaline phosphatase activity and enhanced transcription of the master regulator of osteogenesis, Runx2. When VRE-BIO treated hBM was tested in a rat spinal fusion model, VRE-BIO caused the formation of a denser, stiffer, fusion mass as compared with vehicle treated hBM. Collectively, these data indicate that the VRE-BIO procedure may represent a rapid, safe, and point-of-care strategy for the osteogenic enhancement of autologous hBM for use in clinical orthopedic procedures. Stem Cells Translational Medicine 2018;7:342-353.

An allograft generated from adult stem cells and their secreted products efficiently fuses vertebrae in immunocompromised athymic rats and inhibits local immune responses.

BACKGROUND CONTEXT: Spine pain and the disability associated with it are epidemic in the United States. According to the National Center for Health Statistics, more than 650,000 spinal fusion surgeries are performed annually in the United States, and yet there is a failure rate of 15%-40% when standard methods employing current commercial bone substitutes are used. Autologous bone graft is the gold standard in terms of fusion success, but the morbidity associated with the procedure and the limitations in the availability of sufficient material have limited its use in the majority of cases. A freely available and immunologically compatible bone mimetic with the properties of live tissue is likely to substantially improve the outcome of spine fusion procedures without the disadvantages of autologous bone graft. PURPOSE: This study aimed to compare a live human bone tissue analog with autologous bone grafting in an immunocompromised rat model of posterolateral fusion. DESIGN/SETTING: This is an in vitro and in vivo preclinical study of a novel human stem cell-derived construct for efficacy in posterolateral lumbar spine fusion. METHODS: Osteogenically enhanced human mesenchymal stem cells (OEhMSCs) were generated by exposure to conditions that activate the early stages of osteogenesis. Immunologic characteristics of OEhMSCs were evaluated in vitro. The secreted extracellular matrix from OEhMSCs was deposited on a clinical-grade gelatin sponge, resulting in bioconditioned gelatin sponge (BGS). Bioconditioned gelatin sponge was used alone, with live OEhMSCs (BGS+OEhMSCs), or with whole human bone marrow (BGS+hBM). Efficacy for spine fusion was determined by an institutionally approved animal model using 53 nude rats. RESULTS: Bioconditioned gelatin sponge with live OEhMSCs did not cause cytotoxicity when incubated with immunologically mismatched lymphocytes, and OEhMSCs inhibited lymphocyte expansion in mixed lymphocyte assays. Bioconditioned gelatin sponge with live OEhMSC and BGS+hBM constructs induced profound bone growth at fusion sites in vivo, with a comparable rate of fusion with syngeneic bone graft (negative [0 of 10], BGS alone [0 of 10], bone graft [7 of 10], BGS+OEhMSC [10 of 15], and BGS+hBM [8 of 8]). CONCLUSIONS: Collectively, these studies demonstrate that BGS+OEhMSC constructs possess low immunogenicity and drive vertebral fusion with efficiency matching syngeneic bone graft in rodents. We also demonstrate that BGS serves as a promising scaffold for spine fusion when combined with hBM.

Disseminated refractory pyoderma gangraenosum during an ulcerative colitis flare. Treatment with infliximab.

BACKGROUND: Pyoderma gangraenosum is an immune-mediated, inflammatory, neutrophilic dermatosis of unknown etiology, which represents one of the extraintestinal manifestations of inflammatory bowel disease. It is a rare disease that occurs in less than 1% of patients with inflammatory bowel disease and with the same ratio in patients with Crohn's disease and ulcerative colitis. MAIN OBSERVATIONS: A 36-year-old woman was diagnosed with ulcerative colitis 6 years before admission to our dermatology department with an acute disseminated pyoderma gangraenosum with mucosal involvement, during a flare of ulcerative colitis. Disease progression was interrupted by intravenous administration of the tumor necrosis factor-α inhibitor infliximab at 5 mg/kg at weeks 0, 2, and 6 (1st cycle) and every 8 weeks thereafter. Improvement of intestinal, skin and oral manifestations was evident already after the 1st cycle of treatment and has been maintained since (at least 16 months). CONCLUSIONS: This case report is one of very few on disseminated pyoderma gangraenosum with oral involvement complicating ulcerative colitis, where infliximab was shown to have a rapid efficacy on skin, mucosal and bowel symptoms.

Bone regeneration with osteogenically enhanced mesenchymal stem cells and their extracellular matrix proteins.

Although bone has remarkable regenerative capacity, about 10% of long bone fractures and 25% to 40% of vertebral fusion procedures fail to heal. In such instances, a scaffold is employed to bridge the lesion and accommodate osteoprogenitors. Although synthetic bone scaffolds mimic some of the characteristics of bone matrix, their effectiveness can vary because of biological incompatibility. Herein, we demonstrate that a composite prepared with osteogenically enhanced mesenchymal stem cells (OEhMSCs) and their extracellular matrix (ECM) has an unprecedented capacity for the repair of critical-sized defects of murine femora. Furthermore, OEhMSCs do not cause lymphocyte activation, and ECM/OEhMSC composites retain their in vivo efficacy after cryopreservation. Finally, we show that attachment to the ECM by OEhMSCs stimulates the production of osteogenic and angiogenic factors. These data demonstrate that composites of OEhMSCs and their ECM could be utilized in the place of autologous bone graft for complex orthopedic reconstructions.

Identification of biomarkers of human skin ageing in both genders. Wnt signalling - a label of skin ageing?

The goal of our work has been to investigate the mechanisms of gender-independent human skin ageing and examine the hypothesis of skin being an adequate model of global ageing. For this purpose, whole genome gene profiling was employed in sun-protected skin obtained from European Caucasian young and elderly females (mean age 26.7±4 years [n1 = 7] and 70.75±3.3 years [n2 = 4], respectively) and males (mean age 25.8±5.2 years [n3 = 6] and 76±3.8 years [n4 = 7], respectively) using the Illumina array platform. Confirmation of gene regulation was performed by real-time RT-PCR and immunohistochemistry. 523 genes were significantly regulated in female skin and 401 genes in male skin for the chosen criteria. Of these, 183 genes exhibited increased and 340 decreased expression in females whereas 210 genes showed increased and 191 decreased expression in males with age. In total, 39 genes were common in the target lists of significant regulated genes in males and females. 35 of these genes showed increased (16) or decreased (19) expression independent of gender. Only 4 overlapping genes (OR52N2, F6FR1OP2, TUBAL3 and STK40) showed differential regulation with age. Interestingly, Wnt signalling pathway showed to be significantly downregulated in aged skin with decreased gene and protein expression for males and females, accordingly. In addition, several genes involved in central nervous system (CNS) ageing (f.i. APP, TAU) showed to be expressed in human skin and were significanlty regulated with age. In conclusion, our study provides biomarkers of endogenous human skin ageing in both genders and highlight the role of Wnt signalling in this process. Furthermore, our data give evidence that skin could be used as a good alternative to understand ageing of different tissues such as CNS.

Inhibition of TGF-ß/Smad signaling by BAMBI blocks differentiation of human mesenchymal stem cells to carcinoma-associated fibroblasts and abolishes their protumor effects.

Bone marrow mesenchymal stem cells (BM-MSCs) have multiple therapeutic potentials for regenerative, anti-inflammatory, and immunomodulatory purposes and also show promise as vehicles for gene therapy of various metastatic cancers based on their tumor-tropic capacity. However, BM-MSCs are also a source of carcinoma-associated fibroblasts (CAFs) and may promote growth and metastasis of cancer. Transforming growth factor ß (TGF-ß) signaling is required to induce CAF differentiation of mouse BM-MSCs in vivo and can induce expression of some CAF markers in human BM-MSCs in vitro. To determine whether inhibiting TGF-ß signaling in human BM-MSCs can block their differentiation to CAFs induced by tumor microenvironments and the consequent protumor effects, we transduced human BM-MSCs with a lentiviral vector encoding bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI), a decoy TGF-ß receptor. BAMBI transduction significantly inhibited TGF-ß/Smad signaling and expression of CAF markers in human BM-MSCs treated with TGF-ß1 or tumor-conditioned medium or cocultured with cancer cells, but did not alter the stem cell properties and the tumor-tropic property of MSCs. In addition, BAMBI transduction disrupted the cytokine network mediating the interaction between MSCs and breast cancer cells. Consequently, BAMBI transduction abolished protumor effects of BM-MSCs in vitro and in an orthotopic breast cancer xenograft model, and instead significantly inhibited growth and metastasis of coinoculated cancer. These results indicated that TGF-ß signaling is essential for differentiation of human BM-MSCs to CAFs in tumor microenvironments and the consequent protumor effects, and inhibiting TGF-ß/Smad pathway may improve the safety of MSC-based therapies in cancer patients.

Human mesenchymal stem cell-derived matrices for enhanced osteoregeneration.

The methodology for the repair of critical-sized or non-union bone lesions has unpredictable efficacy due in part to our incomplete knowledge of bone repair and the biocompatibility of bone substitutes. Although human mesenchymal stem cells (hMSCs) differentiate into osteoblasts, which promote bone growth, their ability to repair bone in vivo has been variable. We hypothesized that given the multistage process of osteogenesis, hMSC-mediated repair might be maximal at a specific time point of healing. Using a mouse model of calvarial healing, we demonstrate that the osteo-repair capacity of hMSCs can be substantially augmented by treatment with an inhibitor of peroxisome proliferator-activated receptor γ, but efficacy is confined to the rapid osteogenic phase. Upon entry into the bone-remodeling phase, hMSC retention signals are lost, resulting in truncation of healing. To solve this limitation, we prepared a scaffold consisting of hMSC-derived extracellular matrix (ECM) containing the necessary biomolecules for extended site-specific hMSC retention. When inhibitor-treated hMSCs were coadministered with ECM, they remained at the injury, well into the remodeling phase of healing, which resulted in reproducible and complete repair of critical-sized bone defects in mice in 3 weeks. These data suggest that hMSC-derived ECM and inhibitor-treated hMSCs could be used at optimal times to substantially and reproducibly improve bone repair.

Potential of modulating Wnt signaling pathway toward the development of bone anabolic agent.

Normal bone homeostasis is the result of a cross-talk between the anabolic axis (osteoblast differentiation) and catabolic axis (osteoclast remodeling). A disruption of this tightly regulated relationship leads to imbalanced bone turnover which ultimately results in diseases of the skeleton. Given that the majority of disease states are characterized by an inadequate renewal of osteoblasts, and the canonical wingless (Wnt) pathway is critical for their differentiation from progenitors, this represents an intriguing target for bone therapy. This mini-review focuses on the different options available for pharmaceutical enhancement of osteogenic differentiation through targeting the various proteins involved in Wnt signaling.

Assays of osteogenic differentiation by cultured human mesenchymal stem cells.

One of the most noteworthy characteristics of mesenchymal stem cells (MSCs) is their ability to differentiate into osteoblasts in vitro and in vivo. In vitro, this is easily achieved by culturing in the appropriate induction medium. It is because of the reliability and ease of this process that osteogenic differentiation has become a popular assay for the demonstration of MSC plasticity. Although the conditions required for inducing osteogenic differentiation by MSCs typically do not vary particularly between investigators, many methods are employed to measure the extent of differentiation. These methods include, but are not limited to, reverse transcriptase PCR (RT-PCR) for detection of osteogenic transcripts, enzyme linked immunosorbent assay (ELISA) for secreted protein markers, colorimetric assays for osteogenic enzymes, and direct staining of matrix components. This chapter reviews the protocols most commonly utilized for the evaluation of osteogenic differentiation for cultured MSCs.

Pharmaceutical inhibition of glycogen synthetase kinase-3ß reduces multiple myeloma-induced bone disease in a novel murine plasmacytoma xenograft model.

Multiple myeloma (MM) is a malignancy of plasma cells that accumulate in the bone marrow. MM is incurable with approximately 100 000 patients currently in the United States and 20 000 new cases diagnosed yearly. The malignancy causes displacement of hematopoiesis and formation of osteolytic bone lesions also known as myeloma bone disease (MBD). At diagnosis, 79% of patients suffer from MBD associated with severe pain and increased mortality. Wnt inhibitors secreted by MM cells inhibit osteogenesis and promote osteoclastogenesis, therefore rapid targeting of Wnt inhibitors is necessary to prevent potentially irreversible effects on the stroma, which could lead to incurable MBD. Inhibition of glycogen synthetase kinase-3ß (GSK3ß) causes accelerated Wnt signaling and enhanced osteogenesis in mesenchymal stem/progenitor cells, irrespective of the extracellular concentration of Wnt inhibitors. Our primary goal of this study was to evaluate a GSK3ß inhibitor (6-bromoindirubin-3'-oxime BIO) for amelioration of bone destruction in a murine model of MBD. When measured using histomorphometry, peritumoral BIO administration improved bone quality at the bone-tumor interface and, surprisingly, increased histologically apparent tumor necrosis. Furthermore, in vitro assays demonstrated a proapoptotic effect on numerous MM cell lines. These preliminary data suggest that pharmaceutical GSK3ß inhibition may improve bone quality in myeloma and other malignant bone diseases.

Pharmaceutical modulation of canonical Wnt signaling in multipotent stromal cells for improved osteoinductive therapy.

Human mesenchymal stem cells (hMSCs) from bone marrow are regarded as putative osteoblast progenitors in vivo and differentiate into osteoblasts in vitro. Positive signaling by the canonical wingless (Wnt) pathway is critical for the differentiation of MSCs into osteoblasts. In contrast, activation of the peroxisome proliferator-activated receptor-gamma (PPARgamma)-mediated pathway results in adipogenesis. We therefore compared the effect of glycogen-synthetase-kinase-3beta (GSK3beta) inhibitors and PPARgamma inhibitors on osteogenesis by hMSCs. Both compounds altered the intracellular distribution of beta-catenin and GSK3beta in a manner consistent with activation of Wnt signaling. With osteogenic supplements, the GSK3beta inhibitor 6-bromo-indirubin-3'-oxime (BIO) and the PPARgamma inhibitor GW9662 (GW) enhanced early osteogenic markers, alkaline phosphatase (ALP), and osteoprotegerin (OPG) by hMSCs and transcriptome analysis demonstrated up-regulation of genes encoding bone-related structural proteins. At higher doses of the inhibitors, ALP levels were attenuated, but dexamethasone-induced biomineralization was accelerated. When hMSCs were pretreated with BIO or GW and implanted into experimentally induced nonself healing calvarial defects, GW treatment substantially increased the capacity of the cells to repair the bone lesion, whereas BIO treatment had no significant effect. Further investigation indicated that unlike GW, BIO induced cell cycle inhibition in vitro. Furthermore, we found that GW treatment significantly reduced expression of chemokines that may exacerbate neutrophil- and macrophage-mediated cell rejection. These data suggest that use of PPARgamma inhibitors during the preparation of hMSCs may enhance the capacity of the cells for osteogenic cytotherapy, whereas adenine analogs such as BIO can adversely affect the viability of hMSC preparations in vitro and in vivo.

Human neonatal thymus-derived mesenchymal stromal cells: characterization, differentiation, and immunomodulatory properties.

Mesenchymal stromal cells represent an attractive cell population for cell transplantation and tissue engineering purposes. The aim of this study was to search for neonatal thymus-derived mesenchymal stromal cells (nTMSC) and further characterize the differentiation and immunomodulatory properties thereof. The thymus glands of 13 infants undergoing congenital cardiac surgery were removed. After in vitro isolation and expansion, we identified adherent stromal cells with substantial proliferation potential. As characterized by FACS, the pattern of surface antigen expression of nTMSC resembled bone marrow stromal cells. Full mesenchymal differentiation potential is maintained during proliferation as confirmed by cultures for osteogenic, chondrogenic, and adipogenic lineages. After 5-azacytidine enrichment, morphological characteristics of cardiomyocytes were achieved. For immunologic investigations, the influence of nTMSC on the proliferative behavior of peripheral blood mononuclear cells was studied as a measure of the immune response. The nTMSC did not stimulate an allogeneic reaction in this coculture. Further, the expression of immunologically relevant markers was measured. Alike MSC from other origins, nTMSC did not express MHC-II. In contrast to mature MSC, some nTMSC even lack the expression of MHC-I. Our results confirm that the neonatal thymus contains mesenchymal stromal cells (nTMSC) with full mesenchymal differentiation potential and immunomodulatory properties.

Primitive and committed human hematopoietic progenitor cells interact with primary murine neural cells and are induced to undergo self-renewing cell divisions.

OBJECTIVE: Studies in animal models have indicated that hematopoietic progenitor cells (HPC) migrate and home to the central nervous system and might acquire neural features under specific circumstances. The interaction between HPC and the neural environment and the functional effect on hematopoiesis have not yet been defined. METHODS: CD34(+)133(+) cells from mobilized peripheral blood were cocultured with primary murine neurons or astrocytes. Chemotaxis and adhesive interactions were studied by applying beta(1)- and beta(2)-integrin function-blocking anibodies. The impact of neural feeder layers on integrin expression of HPC and the presence of appropriate adhesion ligands on neural cells were determined by immunostaining and flow cytometry. The hematopoietic long-term fate was monitored by time-lapse microscopy of individual cell-division history followed by long-term culture-initiating cell (LTC-IC) and colony-forming cell (CFC) assays. Neural differentiation was assessed by immunostaining against specific neuronal and glial antigens. RESULTS: The 23.0% +/- 4.9% of HPC showed stromal cell-derived factor-1-induced migration toward neural cells, and 20.2% +/- 1.6% displayed firm beta(1)-integrin-mediated adhesion to astrocytes. The latter expressed appropriate adhesion ligands, stabilized beta(1)-integrin expression, and increased beta(2)-integrin expression of HPC. Neural differentiation of HPC could not be identified but astrocytes were able to induce limited self-renewing cell divisions of HPC and thus maintain 25.8% +/- 3.4% of the initial LTC-IC and 80.7% +/- 1.9% of the initial CFC. CONCLUSION: Human HPC are able to interact with neural cells and interaction maintains, albeit to a limited extent, the self-renewal capability of HPC.

Atrial-radiofrequency catheter ablation mediated targeting of mesenchymal stromal cells.

Sinus node dysfunction and high-degree heart block are the major causes for electronic pacemaker implantation. Recently, genetically modified mesenchymal stromal cells (MSCs, also known as "mesenchymal stem cells") were demonstrated to generate pacemaker function in vivo. However, experimental approaches typically use open thoracotomy for direct cell injection into the myocardium. Future clinical implementation, however, essentially requires development of more gentle methods to precisely and efficiently apply specified stem cells at specific cardiac locations. In a "proof of concept" study, we performed selective power-controlled radiofrequency catheter ablation (RFCA) with eight ablation pulses (30 W, 60 seconds each) to induce heat-mediated lesions at the auricles of the cardiac right atrium of four healthy foxhounds. The next day, allogeneic MSCs (4.3 x 10(5) cells per kilogram of body weight) labeled with superparamagnetic iron oxide particles (SPIOs) were infused intravenously. Hearts were explanted 8 days later. High numbers of SPIO-labeled cells were identified in areas surrounding the RFCA-induced lesions by Prussian blue staining. Antibody staining revealed SPIO-labeled cells being positive for the typical MSC surface antigen CD44. In contrast, low levels of calprotectin, an antigen found on monocytes and macrophages, indicated negligible infiltration of monocytes in MSC-positive areas. Thus, RFCA allows targeting of MSCs to the cardiac right atrium, adjacent to the sinoatrial node, providing an opportunity to rescue or generate pacemaker function without open thoracotomy and direct injection of MSCs. This method presents a new strategy for cardiac stem cell application leading to an efficient guidance of MSCs into the myocardium. Disclosure of potential conflicts of interest is found at the end of this article.

Comparison of immunological properties of bone marrow stromal cells and adipose tissue-derived stem cells before and after osteogenic differentiation in vitro.

Mesenchymal stem cells (MSCs) can be isolated from various tissues and represent an attractive cell population for tissue-engineering purposes. MSCs from bone marrow (bone marrow stromal cells [BMSCs]) are negative for immunologically relevant surface markers and inhibit proliferation of allogenic T cells in vitro. Therefore, BMSCs are said to be available for allogenic cell therapy. Although the immunological characteristics of BMSCs have been the subject of various investigations, those of stem cells isolated from adipose tissue (ASCs) have not been adequately described. In addition, the influence of osteogenic differentiation in vitro on the immunological characteristics of BMSCs and ASCs is the subject of this article. Before and after osteogenic induction, the influence of BMSCs and ASCs on the proliferative behavior of resting and activated allogenic peripheral blood mononuclear cells (PBMCs) was studied as a measure of the immune response (mixed lymphocyte culture). At the same points, the expression of immunologically relevant surface markers (e.g., major histocompatibility complex (MHC)-I, MHC-II, CD40, CD40L) was measured, and correlations between the different sets of results were sought. The pattern of surface antigen expression of BMSCs is the same as that of ASCs. Analogous to BMSCs, undifferentiated cells isolated from adipose tissue lack expression of MHC-II; this is not lost in the course of the osteogenic differentiation process. In co-culture with allogenic PBMCs, both cell types fail to lead to any significant stimulation, and they both retain these characteristics during the differentiation process. BMSCs and ASCs suppress proliferation on activated PBMCs before and after osteogenic differentiation. Our results confirm that MSCs are immune modulating cells. These properties are retained even after osteogenic induction in vitro and seem to be similar in BMSCs and ASCs. Our results suggest that allogenic transplantation of BMSCs and ASCs would be possible, for example, in the context of tissue engineering.

Stem cell therapy improves myocardial perfusion and cardiac synchronicity: new application for echocardiography.

OBJECTIVES: Intravenous delivery of mesenchymal stem cell (MSC) is a noninvasive approach for myocardial tissue repair. We aimed to test this strategy in a pig model of myocardial infarction and to examine the usefulness of new echocardiographic applications to monitor cardioprotective effects of stem cell therapy. METHODS: Pigs (n = 8) received autologous or allogeneic MSCs (1 x 10(6)/kg body weight) labeled with fluorescent dye 48 hours after proximal left anterior descending coronary artery occlusion. Infarct size, myocardial function, and perfusion (A x beta) were assessed by myocardial contrast echocardiography and standard histologic methods after 1 month. RESULTS: Morphologic analysis revealed that labeled MSCs migrated in the peri-infarct region resulting in smaller infarct size by myocardial contrast echocardiography (control vs autologous and allogeneic MSC: 38 +/- 10% vs 25 +/- 5% and 28 +/- 6%, P < .01), higher fractional area shortening (23 +/- 3% vs 34.0 +/- 7% and 28 +/- 2%, P < .01), higher cardiac synchrony (167 +/- 36 vs 68 +/- 17 and 85 +/- 26 milliseconds, P < .003), and improved microvascular flow A x beta in the ischemic border zone (0.18 +/- 0.2 vs 0.56 +/- 0.3 and 0.49 +/- 0.2, P < .03). CONCLUSIONS: Systemic delivery of autologous and allogeneic MSCs preserves myocardial viability even in large animals and is, therefore, an attractive approach for tissue repair. Myocardial contrast echocardiography is useful to evaluate microvascular perfusion, which was enhanced by MSCs.