Bone Marrow Mesenchymal Stem Cells for Improving Hematopoietic Function: An In Vitro and In Vivo Model. Part 2: Effect on Bone Marrow Microenvironment

The aim of the present study was to determine how mesenchymal stem cells (MSC) could improve bone marrow (BM) stroma function after damage, both in vitro and in vivo. Human MSC from 20 healthy donors were isolated and expanded. Mobilized selected CD34+ progenitor cells were obtained from 20 HSCT donors. For in vitro study, long-term bone marrow cultures (LTBMC) were performed using a etoposide damaged stromal model to test MSC effect in stromal confluence, capability of MSC to lodge in stromal layer as well as some molecules (SDF1, osteopontin,) involved in hematopoietic niche maintenance were analyzed. For the in vivo model, 64 NOD/SCID recipients were transplanted with CD34+ cells administered either by intravenous (IV) or intrabone (IB) route, with or without BM derived MSC. MSC lodgement within the BM niche was assessed by FISH analysis and the expression of SDF1 and osteopontin by immunohistochemistry. In vivo study showed that when the stromal damage was severe, TP-MSC could lodge in the etoposide-treated BM stroma, as shown by FISH analysis. Osteopontin and SDF1 were differently expressed in damaged stroma and their expression restored after TP-MSC addition. Human in vivo MSC lodgement was observed within BM niche by FISH, but MSC only were detected and not in the contralateral femurs. Human MSC were located around blood vessels in the subendoestal region of femurs and expressed SDF1 and osteopontin. In summary, our data show that MSC can restore BM stromal function and also engraft when a higher stromal damage was done. Interestingly, MSC were detected locally where they were administered but not in the contralateral femur

Dasatinib as a Bone-Modifying Agent: Anabolic and Anti-Resorptive Effects

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al.[Background]: Bone loss, in malignant or non-malignant diseases, is caused by increased osteoclast resorption and/or reduced osteoblast bone formation, and is commonly associated with skeletal complications. Thus, there is a need to identify new agents capable of influencing bone remodeling. We aimed to further pre-clinically evaluate the effects of dasatinib (BMS-354825), a multitargeted tyrosine kinase inhibitor, on osteoblast and osteoclast differentiation and function. [Methods]: For studies on osteoblasts, primary human bone marrow mensenchymal stem cells (hMSCs) together with the hMSC-TERT and the MG-63 cell lines were employed. Osteoclasts were generated from peripheral blood mononuclear cells (PBMC) of healthy volunteers. Skeletally-immature CD1 mice were used in the in vivo model. [Results]: Dasatinib inhibited the platelet derived growth factor receptor-β (PDGFR-β), c-Src and c-Kit phosphorylation in hMSC-TERT and MG-63 cell lines, which was associated with decreased cell proliferation and activation of canonical Wnt signaling. Treatment of MSCs from healthy donors, but also from multiple myeloma patients with low doses of dasatinib (2-5 nM), promoted its osteogenic differentiation and matrix mineralization. The bone anabolic effect of dasatinib was also observed in vivo by targeting endogenous osteoprogenitors, as assessed by elevated serum levels of bone formation markers, and increased trabecular microarchitecture and number of osteoblast-like cells. By in vitro exposure of hemopoietic progenitors to a similar range of dasatinib concentrations (1-2 nM), novel biological sequelae relative to inhibition of osteoclast formation and resorptive function were identified, including F-actin ring disruption, reduced levels of c-Fos and of nuclear factor of activated T cells 1 (NFATc1) in the nucleus, together with lowered cathepsin K, αVβ3 integrin and CCR1 expression. [Conclusions]: Low dasatinib concentrations show convergent bone anabolic and reduced bone resorption effects, which suggests its potential use for the treatment of bone diseases such as osteoporosis, osteolytic bone metastasis and myeloma bone disease. © 2012 Garcia-Gomez et al.This work was supported by grants from the Spanish Ministry of Science and Innovation – ISCIII (PI081825); Mutua Madrileña Medical Research Foundation (AP27262008); Centro en Red of Regenerative Medicine and Cellular Therapy from Castilla y León, Consejería de Sanidad JCyL – ISCIII; the Cooperative Research Thematic Network in Cancer (RTICC; RD06/0020/0006 and RD03/0020/0041); and Spanish FIS (PS09/01897). AG-G and CS are supported by the Centro en Red of Regenerative Medicine and Cellular Therapy from Castilla y León Project.Peer Reviewe

Sequential intravenous allogeneic mesenchymal stromal cells as a potential treatment for thromboangiitis obliterans (Buerger’s disease)

Abstract Thromboangiitis obliterans (TAO), also known as Buerger’s Disease, is an occlusive vasculitis linked with high morbidity and amputation risk. To date, TAO is deemed incurable due to the lack of a definitive treatment. The immune system and inflammation are proposed to play a central role in TAO pathogenesis. Due to their immunomodulatory effects, mesenchymal stromal cells (MSCs) are the subject of intense research for the treatment of a wide range of immune-mediated diseases. Thus far, local intramuscular injections of autologous or allogeneic MSCs have shown promising results in TAO. However, sequential intravenous allogeneic MSC administration has not yet been explored, which we hypothesized could exert a systemic anti-inflammatory effect in the vasculature and modulate the immune response. Here, we report the first case of a TAO patient at amputation risk treated with four sequential intravenous infusions of bone marrow-derived allogeneic MSCs from a healthy donor. Following administration, there was significant regression of foot skin ulcers and improvements in rest pain, Walking Impairment Questionnaire scores, and quality of life. Sixteen months after the infusion, the patient had not required any further amputations. This report highlights the potential of sequential allogeneic MSC infusions as an effective treatment for TAO, warranting further studies to compare this approach with the more conventionally used intramuscular MSC administration and other cell-based therapies

Characterization of human MSC.

<p><b>A</b>) Images from MSC expansion. <b>B</b>) Mean fluorescence intensity of stained MSC (blue) and control non-stained MSC (green). <b>E</b>) <i>In vitro</i> differentiation of MSC to osteoblasts (a, d), adipocytes (b, e) and chondrocytes (c, f) under control conditions (a, b, c) or with differentiation medium (d, e, f).</p

<i>In vitro</i> TP-MSC-lodging capacity.

<p>Results expressed as percentages of TP-MSC counted after FISH staining of sex chromosomes (n = 10).</p

Human <i>in vivo</i> MSC engraftment detected by FISH.

<p>Results expressed as median number of human MSC detected by slide (range).</p><p>MSC: Mesenchymal stem cells; IV: intravenous injection; IB: intrabone injection.</p

Example of human MSC detection by FISH.

<p>In this case cells were expanded from murine BM 6 weeks after transplantation human cells from sex-mismatched donors, female HSC and male MSC (green for X chromosome and red for Y chromosome).</p

SDF-1α and osteopontin <i>in vitro</i> expression by MSC.

<p><b>A</b>) SDF-1 expression in control stromas: positive cluster (on the left) surrounded by negative cells (right) showing a polygonal shape (<b>B</b>). <b>C</b>) In etoposide-treated stromas SDF-1 expression was weaker and cells had different shapes (<b>D</b>). <b>E</b>) In treated stromas cultured with TP-MSC supernatant, there was an increase in the number of positive cells, which were polygon-shaped (<b>F</b>). Regarding osteopontin, in control stromas it was expressed in polygonal cells (<b>G, H</b>). In etoposide-treated stromas, osteopontin expression was stronger than in controls and was located in unusually shaped cells in standard culture medium (<b>I, J</b>) and it was also high expressed in treated-stromas cultured with TP-MSC supernatant (<b>K, L</b>). <b>M</b>) As confirmed by western blot, osteopontin expression was lower in untreated stromas than in etoposide-treated stromas (1 = Control; 2 = Etoposide-treated; 3 = Etoposide-treated+TP-MSC supernatant). Finally, the expression of both molecules osteopontin (<b>N</b>) and SDF-1 (<b>O</b>) was confirmed by PCR.</p