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

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

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

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

<p>Human MSC expressing osteopontin and SDF1 could be found close to femur endostium or around blood vessels when they were analyzed in sequential sections (10×) (<b>A–F</b>). In most cases these human cells were positive for both osteopontin and SDF1 (<b>G–I</b>) (100×), but in some cases they were positive only for osteopontin in endostium (<b>J</b>) (100×) or SDF1 around blood vessels (<b>L</b>) (100×). Human hematopoietic cells could be detected forming colonies or groups of human mitochondria positive cells negative for osteopontin and SDF1 (<b>K</b>) (20×).</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