Bone marrow-derived mesenchymal stromal cells improve vascular regeneration and reduce leukocyte-endothelium activation in critical ischemic murine skin in a dose-dependent manner

BACKGROUND AIMS Stem cells participate in vascular regeneration following critical ischemia. However, their angiogenic and remodeling properties, as well as their role in ischemia-related endothelial leukocyte activation, need to be further elucidated. Herein, we investigated the effect of bone marrow-derived mesenchymal stromal cells (BM-MSCs) in a critically ischemic murine skin flap model. METHODS Groups received either 1 × 10(5), 5 × 10(5), or 1 × 10(6) BM-MSCs or cell-free conditioned medium (CM). Controls received sodium chloride. Intravital fluorescence microscopy was performed for morphological and quantitative assessment of micro-hemodynamic parameters over 12 days. RESULTS Tortuosity and diameter of conduit-arterioles were pronounced in the MSC groups (P < 0.01), whereas vasodilation was shifted to the end arteriolar level in the CM group (P < 0.01). These effects were accompanied by angiopoietin-2 expression. Functional capillary density and red blood cell velocity were enhanced in all treatment groups (P < 0.01). Although a significant reduction of rolling and sticking leukocytes was observed in the MSC groups with a reduction of diameter in postcapillary venules (P < 0.01), animals receiving CM exhibited a leukocyte-endothelium interaction similar to controls. This correlated with leukocyte common antigen expression in tissue sections (P < 0.01) and p38 mitogen-activated protein kinase expression from tissue samples. Cytokine analysis from BM-MSC culture medium revealed a 50% reduction of pro-inflammatory cytokines (interleukin [IL]-1β, IL-6, IL-12, tumor necrosis factor-α, interferon-γ) and chemokines (keratinocyte chemoattractant, granulocyte colony-stimulating factor) under hypoxic conditions. DISCUSSION We demonstrated positive effects of BM-MSCs on vascular regeneration and modulation of endothelial leukocyte adhesion in critical ischemic skin. The improvements after MSC application were dose-dependent and superior to the use of CM alone.

Bovine coccygeal intervertebral discs contain multipotent Tie2+ cells which can differentiate into osteogenic and adipogenic lineages

Question: The intervertebral disc (IVD) has a limited regenerative potential and low back pain represents a leading cause of disability [1]. IVD repair strategies require an appropriate cell source that is able to regenerate the damaged tissue such as progenitor stem cells. Recently, progenitor cells that are positive for the angiopoietin re- ceptor (Tie2) in the nucleus pulposus were identified [2]. Here we isolated primary cells from bovine IVD and sorted bovine nucleus pulposus progenitor cells (NPPC) for the marker Tie2. Furthermorewe tested whether Tie2 expressing cells can differentiate into os- teogenic and adipogenic lineages in vitro. Methods: NP cells were obtained from 1 year old bovine tails by sequential digestion with pronase for 1 h and collagenase over- night. Sorted Tie2- and Tie2+ cells were cultured in osteogenic and adipogenic medium for 3 weeks. The formed cell layers from both subpopulations were stained for calcium deposition and fat droplets. Colony forming units were prepared for both cell sus- pensions in methylcellulose-based medium and formed colonies ([10 cells) were analyzed macroscopically after 8 days. Results: After 3 weeks of culture, sorted Tie2+ cells were able to differentiate into osteocytes and adipocytes as characterized by cal- cium deposition and fat droplet formation. By contrast, Tie2- cells generated a weak staining for calcium and no fat droplets were ob- tained (Fig. 1). Sorted Tie2- and Tie2+ subpopulations of cells both formed colonies, however with different morphologies. The colonies formed from Tie2+ cells were spheroid in shape whereas those from Tie2- cells were spread and fibroblastic. Conclusion: Our data showed that Tie2+ cells of the nucleus pul- posus cells are progenitor-like cells that are able to differentiate into osteogenic and adipogenic lineages. Sorting of NPPC for Tie2 may represent a promising strategy with the potential to be used in the clinics for treatment of intervertebral disc damage. References 1. Freemont AJ (2009) The cellular pathobiology of the degenerate intervertebral disc and discogenic back pain. Rheumatology (Oxford) 48:5–10 2. Sakai D, Nakamura Y, Nakai T et al (2012) Exhaustion of nucleus pulposus progenitor cells with ageing and degeneration of the intervertebral disc. Nat Commun 3:1264 Acknowledgments: This project was funded by two projects of the Swiss National Science Foundation grant number #IZK0Z3_154384 and #310030_153411.

Current developments in the use of stem cell for therapeutic neovascularisation: is the future therapy "cell-free"?

The plasticity and self-regenerative properties of stem cells have opened new avenues in regenerative medicine. Greater understanding of the biology of stem cells is followed by growing expectations of a rapid translation into alternative therapeutic options. Recent preclinical studies and clinical trials employing stem and progenitor cells from different sources have shown encouraging results. However, their underlying mechanisms are still poorly understood, the potential adverse effects and the discrepancy in efficacy remain to be further investigated. Their essential role in vessel regeneration has made endothelial progenitor cells (EPC) a suitable candidate for therapeutic applications aiming at tissue revascularisation. Recent evidence suggests that EPC contribute to neovascularisation not only by direct participation in tissue homeostasis but mainly via paracrine mechanisms. In future, novel therapeutic strategies could be based on EPC paracrine factors or synthetic factors, and replace cell transplantation.

Functional Characterization and Comparison of Intercellular Communication in Stem Cell-Derived Cardiomyocytes

One novel treatment strategy for the diseased heart focuses on the use of pluripotent stem cell-derived cardiomyocytes (SC-CMs) to overcome the heart's innate deficiency for self-repair. However, targeted application of SC-CMs requires in-depth characterization of their true cardiogenic potential in terms of excitability and intercellular coupling at cellular level and in multicellular preparations. In this study, we elucidated the electrical characteristics of single SC-CMs and intercellular coupling quality of cell pairs, and concomitantly compared them with well-characterized murine native neonatal and immortalized HL-1 cardiomyocytes. Firstly, we investigated the electrical properties and Ca2+ signaling mechanisms specific to cardiac contraction in single SC-CMs. Despite heterogeneity of the new cardiac cell population, their electrophysiological activity and Ca2+ handling were similar to native cells. Secondly, we investigated the capability of paired SC-CMs to form an adequate subunit of a functional syncytium and analyzed gap junctions and signal transmission by dye transfer in cell pairs. We discovered significantly diminished coupling in SC-CMs compared with native cells, which could not be enhanced by a coculture approach combining SC-CMs and primary CMs. Moreover, quantitative and structural analysis of gap junctions presented significantly reduced connexin expression levels compared with native CMs. Strong dependence of intercellular coupling on gap junction density was further confirmed by computational simulations. These novel findings demonstrate that despite the cardiogenic electrophysiological profile, SC-CMs present significant limitations in intercellular communication. Inadequate coupling may severely impair functional integration and signal transmission, which needs to be carefully considered for the prospective use of SC-CMs in cardiac repair.

In vitro analysis of osteogenic inhibition in non-union spinal fusion caused by nucleus pulposus cells.

Discectomy and spinal fusion is the gold standard for spinal surgery to relieve pain. However, fusion can be hindered for yet unknown reasons that lead to non-fusions with pseudo-arthrosis. Clinical observations indicate that presence of residual intervertebral disc (IVD) tissue might hinder the ossification. We hypothesize that BMP-antagonists are constantly secreted by IVD cells and potentially prevent the ossification process. Furthermore, L51P, the engineered BMP2 variant, stimulates osseo-induction of bone marrow-derived mesenchymal stem cells (MSC) by antagonizing BMP-inhibitors. Human MSCs, primary nucleus pulposus (NPC) and annulus pulposus cells (AFC) were isolated and expanded in monolayer cultures up to passage 3. IVD cells were seeded in 1.2% alginate beads (4Mio/mL) and separated by culture inserts from MSCs. MSCs were kept in 1:control medium, 2:osteogenic medium±alginate beads, 3:osteogenic medium+NPC (±L51P) and 4:osteogenic medium+AFC (±L51P) for 21 days. Relative gene expression of bone-related genes, alkaline phosphatase assay and histological staining were performed. Osteogenesis of MSCs was hindered as shown by reduced alizarin red staining in the presence of NPC. No such inhibition was observed if co-cultured with alginate only or in the presence of AFC. The results were confirmed on the RNA and protein level. Addition of L51Pto the co- cultures, however, induced mineralization of MSCs in presence of NPC. We demonstrated that NPC secrete BMP-antagonists that prevent osteogenesis of MSCs and L51P can antagonize BMP-antagonists and induce bone formation.

Investigation of bone inhibition in non-union spinal fusion caused by intervertebral disc cells in vitro

Introduction: Discectomy and spinal fusion is the gold standard for spinal surgery to relieve pain. However, fusion can be hindered for yet unknown reasons that lead to non-fusions with pseudo-arthrose. It is hence appealing to develop biomaterials that can enhance bone formation. Clinical observations indicate that presence of residual intervertebral disc (IVD) tissue might hinder the ossification. We hypothesize that BMP-antagonists are constantly secreted by IVD cells and potentially prevent the ossification process. Furthermore, L51P, the engineered BMP2 variant, stimulates osteoinduction of bone marrow-derived mesenchymal stem cells (MSC) by antagonizing BMP-inhibitors. Methods: Human MSCs, primary nucleus pulposus (NPC) and annulus pulposus cells (AFC) were isolated and expanded in monolayer cultures up to passage 3. IVD cells were seeded in 1.2% alginate beads (4Mio/mL) and separated by culture inserts from MSCs in a co-culture set-up. MSCs were kept in 1:control medium, 2:osteogenic medium+alginate control, 3:osteogenic medium+NPC (±L51P) and 4:osteogenic medium+AFC (±L51P) for 21 days. Relative gene expression of bone-related genes, Alkaline Phosphatase (ALP) assay and histological staining were performed. Results: Osteogenesis of MSCs was hindered as shown by reduced alizarin red staining in the presence of NPC. No such inhibition was observed if co-cultured with alginate only or in the presence of AFC. The results were confirmed on the RNA and protein level. Addition of L51P to the co-cultures induced mineralization of MSCs, however a reduced ALP was observed. Conclusion: We demonstrated that NPC secrete BMP-antagonists that prevent osteogenesis of MSCs and L51P can antagonize BMP-antagonists and induce bone formation.

Osteogenic differentiation of bone marrow stromal cells is hindered by the presence of intervertebral disc cells.

BACKGROUND Clinical observations indicate that the presence of nucleus pulposus (NP) tissue during spinal fusion hinders the rate of disc ossification. While the underlying mechanism remains unknown, this observation could be due to incomplete removal of NP cells (NPCs) that secrete factors preventing disc calcification, such as bone morphogenetic protein (BMP) antagonists including noggin and members of the DAN (differential screening selected gene aberrative in neuroblastoma) family. METHODS Monolayer human bone marrow-derived mesenchymal stem cells (MSCs) were cocultured withNPCs and annulus fibrosus cells (AFCs) embedded in alginate for 21 days. At the end of coculture, MSCs were stained for mineral deposition by alizarin red, and relative expression of bone-related genes [Runt-related transcription factor 2, (RUNX2), Osteopontin (OPN), and Alkaline phosphatase (ALP)] and ALP activity were analyzed. Relative expression of three BMP antagonists, chordin (CHRD), gremlin (GREM1), and noggin (NOG), was determined in primary human NPCs and AFCs. These cells were also stained for Gremlin and Noggin by immunocytochemistry. RESULTS Alizarin red staining showed that MSC osteogenesis in monolayer cultures was inhibited by coculture with NPCs or AFCs. ALP activity and RT-PCR analyses confirmed these results and demonstrated inhibition of osteogenesis of MSC in the presence of disc cells. NOG was significantly up-regulated in MSCs after coculture. Relative gene expression of intervertebral disc (IVD) cells showed higher expression of GREM1 in NPCs than in AFCs. CONCLUSIONS We show that primary IVD cells inhibit osteogenesis of MSCs. BMP inhibitors NOG, GREM1 and CHRD were expressed in IVD cells. GREM1 appears to be differentially expressed in NPCs and AFCs. Our results have implications for the design and development of treatments for non-union in spinal fusion.

Stem cell mobilization chemotherapy with gemcitabine is effective and safe in myeloma patients with bortezomib-induced neurotoxicity.

Vinorelbine chemotherapy with granulocyte-colony stimulating factor (G-CSF) stimulation is a widely applied non-myelosuppressive mobilization regimen in Switzerland for myeloma patients, but its neurotoxic potential limits its use in patients with bortezomib-induced polyneuropathy. In this single-center study, we alternatively evaluated safety and effectiveness of gemcitabine chemotherapy with G-CSF for mobilization of autologous stem cells. Between March 2012 and February 2013, all bortezomib-pretreated myeloma patients planned to undergo first-line high-dose melphalan chemotherapy received a single dose of 1250 mg/m(2) gemcitabine, with G-CSF started on day 4. The 24 patients in this study had received a median of four cycles of bortezomib-dexamethason-based induction. Bortezomib-related polyneuropathy was identified in 21 patients (88%) by clinical evaluation and a standardized questionnaire. Administration of gemcitabine mobilization did not induce new or aggravate pre-existing neuropathy. Stem cell mobilization was successful in all 24 patients, with a single day of apheresis being sufficient in 19 patients (78%). The median yield was 9.51 × 10(6) CD34+ cells/kg. Stem collection could be accomplished at day 8 in 67%. Our data suggest that single-dose gemcitabine together with G-CSF is an effective mobilization regimen in myeloma patients and a safe alternative non-myelosuppressive mobilization chemotherapy for myeloma patients with bortezomib-induced polyneuropathy.

Neurotoxicity of stem cell mobilization chemotherapy with vinorelbine in myeloma patients after bortezomib treatment.

Vinorelbine chemotherapy with G-CSF stimulation is the standard mobilization regimen in Switzerland for multiple myeloma patients. However, with the increasing use of bortezomib during induction treatment, adding the neurotoxic compound vinorelbine for mobilization may aggravate bortezomib-induced polyneuropathy. In this retrospective single-center study, we aimed to explore vinorelbine mediated neuropathy in 106 consecutive bortezomib pretreated myeloma patients. We confirmed that vinorelbine with G-CSF represents a reliable and effective regimen for mobilization of autologous stem cells. However, the single administration of 35 mg/m(2) vinorelbine added significant neurotoxicity. We found that 24 patients (24%) reported vinorelbine mediated neurotoxicity: Aggravation of bortezomib-induced neuropathy was observed in 17 patients (17%), and vinorelbine mobilization induced first occurrence of polyneuropathy in additional 7 patients (7%). We observed that development of polyneuropathy was not associated with differing survival rates. Finally, affected patients reported polyneuropathy associated disease burden as "very high" in 13% and "high" in 50%. Our data indicate that a single administration of vinorelbine to mobilize autologous stem cells is associated with significant additional polyneuropathy in bortezomib pretreated myeloma patients. The efficacy of vinorelbine mobilization should be balanced against its neurotoxic potential.

Three-dimensional culture and characterization of mononuclear cells from human bone marrow

BACKGROUND AIMS The diverse phenotypic changes and clinical and economic disadvantages associated with the monolayer expansion of bone marrow-derived mesenchymal stromal cells (MSCs) have focused attention on the development of one-step intraoperative cells therapies and homing strategies. The mononuclear cell fraction of bone marrow, inclusive of discrete stem cell populations, is not well characterized, and we currently lack suitable cell culture systems in which to culture and investigate the behavior of these cells. METHODS Human bone marrow-derived mononuclear cells were cultured within fibrin for 2 weeks with or without fibroblast growth factor-2 supplementation. DNA content and cell viability of enzymatically retrieved cells were determined at days 7 and 14. Cell surface marker profiling and cell cycle analysis were performed by means of multi-color flow cytometry and a 5-ethynyl-2'-deoxyuridine incorporation assay, respectively. RESULTS Total mononuclear cell fractions, isolated from whole human bone marrow, was successfully cultured in fibrin gels for up to 14 days under static conditions. Discrete niche cell populations including MSCs, pericytes and hematopoietic stem cells were maintained in relative quiescence for 7 days in proportions similar to that in freshly isolated cells. Colony-forming unit efficiency of enzymatically retrieved MSCs was significantly higher at day 14 compared to day 0; and in accordance with previously published works, it was fibroblast growth factor-2-dependant. CONCLUSIONS Fibrin gels provide a simple, novel system in which to culture and study the complete fraction of bone marrow-derived mononuclear cells and may support the development of improved bone marrow cell-based therapies.

Human lung-derived mesenchymal stem cell-conditioned medium exerts in vitro antitumor effects in malignant pleural mesothelioma cell lines.

BACKGROUND The soluble factors secreted by mesenchymal stem cells are thought to either support or inhibit tumor growth. Herein, we investigated whether the human lung-derived mesenchymal stem cell-conditioned medium (hlMSC-CM) exerts antitumor activity in malignant pleural mesothelioma cell lines H28, H2052 and Meso4. METHODS hlMSC-CM was collected from the human lung-derived mesenchymal stem cells. Inhibition of tumor cell growth was based on the reduction of cell viability and inhibition of cell proliferation using the XTT and BrdU assays, respectively. Elimination of tumor spheroids was assessed by the anchorage-independent sphere formation assay. The cytokine profile of hlMSC-CM was determined by a chemiluminescence-based cytokine array. RESULTS Our data showed that hlMSC-CM contains a broad range of soluble factors which include: cytokines, chemokines, hormones, growth and angiogenic factors, matrix metalloproteinases, metalloproteinase inhibitors and cell-cell mediator proteins. The 48- and 72-hour hlMSC-CM treatments of H28, H2052 and Meso4 cell lines elicited significant decreases in cell viability and inhibited cell proliferation. The 72-hour hlMSC-CM incubation of H28 cells completely eliminated the drug-resistant sphere-forming cells, which is more potent than twice the half maximal inhibitory concentration of cisplatin. CONCLUSIONS Our findings indicate that the cell-free hlMSC-CM confers in vitro antitumor activities via soluble factors in the tested mesothelioma cells and, hence, may serve as a therapeutic tool to augment the current treatment strategies in malignant pleural mesothelioma.

Human graft-derived mesenchymal stromal cells potently suppress alloreactive T-cell responses

After organ transplantation, recipient T cells contribute to graft rejection. Mesenchymal stromal cells from the bone marrow (BM-MSCs) are known to suppress allogeneic T-cell responses, suggesting a possible clinical application of MSCs in organ transplantation. Human liver grafts harbor resident populations of MSCs (L-MSCs). We aimed to determine the immunosuppressive effects of these graft-derived MSCs on allogeneic T-cell responses and to compare these with the effects of BM-MSCs. BM-MSCs were harvested from aspirates and L-MSCs from liver graft perfusates. We cultured them for 21 days and compared their suppressive effects with the effects of BM-MSCs on allogeneic T-cell responses. Proliferation, cytotoxic degranulation, and interferon-gamma production of alloreactive T cells were more potently suppressed by L-MSCs than BM-MSCs. Suppression was mediated by both cell-cell contact and secreted factors. In addition, L-MSCs showed ex vivo a higher expression of PD-L1 than BM-MSCs, which was associated with inhibition of T-cell proliferation and cytotoxic degranulation in vitro. Blocking PD-L1 partly abrogated the inhibition of cytotoxic degranulation by L-MSCs. In addition, blocking indoleamine 2,3-dioxygenase partly abrogated the inhibitive effects of L-MSCs, but not BM-MSCs, on T-cell proliferation. In conclusion, liver graft-derived MSC suppression of allogeneic T-cell responses is stronger than BM-MSCs, which may be related to in situ priming and mobilization from the graft. These graft-derived MSCs may therefore be relevant in transplantation by promoting allohyporesponsiveness.

Spatial Distribution of Stem Cell-Like Keratinocytes in Dissected Compound Hair Follicles of the Dog

Hair cycle disturbances are common in dogs and comparable to some alopecic disorders in humans. A normal hair cycle is maintained by follicular stem cells which are predominately found in an area known as the bulge. Due to similar morphological characteristics of the bulge area in humans and dogs, the shared particularity of compound hair follicles as well as similarities in follicular biomarker expression, the dog is a promising model to study human hair cycle and stem cell disorders. To gain insight into the spatial distribution of follicular keratinocytes with stem cell potential in canine compound follicles, we microdissected hair follicles in anagen and telogen from skin samples of freshly euthanized dogs. The keratinocytes isolated from different locations were investigated for their colony forming efficiency, growth and differentiation potential as well as clonal growth. Our results indicate that i) compound and single hair follicles exhibit a comparable spatial distribution pattern with respect to cells with high growth potential and stem cell-like characteristics, ii) the lower isthmus (comprising the bulge) harbors most cells with high growth potential in both, the anagen and the telogen hair cycle stage, iii) unlike in other species, colonies with highest growth potential are rather small with an irregular perimeter and iv) the keratinocytes derived from the bulbar region exhibit characteristics of actively dividing transit amplifying cells. Our results now provide the basis to conduct comparative studies of normal dogs and those with hair cycle disorders with the possibility to extend relevant findings to human patients.

Differentiation of Human Embryonic Stem Cell (hESC) Derived Pyramidal Neurons

The mammalian cerebral neocortex is a complex six-layered structure containing multiple types of neurons. Pyramidal neurons of the neocortex are formed during development in an inside-out manner, by which deep layer (DL) neurons are generated first, and upper layer (UL) neurons are generated last. Neurons within the six-layered neocortex express unique markers for their position, showing whether they are subplate, deep layer, upper layer, or Cajal-Retzius neurons. The sequential generation of cortical layers, which exists in vivo, has been partially recapitulated in vitro by differentiation of mouse embryonic stem cells (Gaspard et al., 2008) and human embryonic stem cells (hESC) (Eiraku et al., 2008). The timeline of generation of cortical neurons from hESC is still not well defined, and could be very important in the future of cell therapy. In this study we will define timeline for UL and DL neurons for our experimental paradigm as well as test the effects of fibroblast growth factors (FGF) 2 and 8 on this neuronal differentiation. Recent papers suggest that FGFs are critical for forebrain patterning (Storm et al., 2003). Neuronal differentiation after treatment with either FGF2 or FGF8 from hESCs will be examined and the proportion of specific neuronal markers will be analyzed using immunocytochemistry. Our results show that the generated pyramidal neurons will express DL and UL laminar markers in vitro as they do in vivo and that the presence of FGF8 in induction media creates a proliferative effect, while FGF2 induces hESC to differentiate at a higher rate.

Analyzing the function of myogenin in adult satellite cells through the construction of a myogenin conditional allele

Although mechanisms regulating the formation of embryonic skeletal muscle are well characterized, less is known about muscle formation in postnatal life. This disparity is unfortunate because the largest increases in skeletal muscle mass occur after birth. Adult muscle stem cells (satellite cells) appear to recapitulate the events that occur in embryonic myoblasts. In particular, the myogenic basic helix-loop-helix factors, which have crucial functions in embryonic muscle development, are assumed to have similar roles in postnatal muscle formation. Here, I test this assumption by determining the role of the myogenic regulator myogenin in postnatal life. Myogenin-null mice die at birth, necessitating the generation of floxed alleles of myogenin and the use of cre-recombinase lines to delete myogenin. Removing myogenin before embryonic muscle development resulted in myofiber deficiencies identical to those observed in myogenin-null mice. However, mice in which myogenin was deleted following embryonic muscle development had normal skeletal muscle, except for modest alterations in MRF4 and MyoD expression. Notably, myogenin-deleted mice were 30% smaller than controls, suggesting that myogenin's absence disrupted general body growth. These results suggest that skeletal muscle growth in postnatal life is controlled by mechanisms distinct from those occurring in embryonic muscle development. ^