Simone Pacini

Constitutive Expression of Pluripotency-Associated Genes in Mesodermal Progenitor Cells (MPCs)

Background We recently characterized a progenitor of mesodermal lineage (MPCs) from the human bone marrow of adults or umbilical cord blood. These cells are progenitors able to differentiate toward mesenchymal, endothelial and cardiomyogenic lineages. Here we present an extensive molecular characterization of MPCs, from bone marrow samples, including 39 genes involved in stem cell machinery, differentiation and cell cycle regulation. Methodology/Principal Findings MPCs are cytofluorimetrically characterized and quantitative RT-PCR was performed to evaluate the gene expression profile, comparing it with MSCs and hESCs lines. Immunofluorescence and dot-blot analysis confirm qRT-PCR data. MPCs exhibit an increased expression of OCT4, NANOG, SALL4, FBX15, SPP1 and to a lesser extent c-MYC and KLF4, but lack LIN28 and SOX2. MPCs highly express SOX15. Conclusions/Significance MPCs express many pluripotency-associated genes and show a peculiar Oct-4 molecular circuit. Understanding this unique molecular mechanism could lead to identifying MPCs as feasible, long telomeres, target cells for reprogramming with no up-regulation of the p53 pathway. Furthermore MPCs are easily and inexpensively harvested from human bone marrow.

Identification and purification of mesodermal progenitor cells from human adult bone marrow.

Bone marrow-derived mesodermal stem cells may differentiate toward several lines and are easily cultured in vitro. Some putative progenitors of these cells have been described in both humans and mice. Here, we describe a new mesodermal progenitor population [mesodermal progenitors cells (MPCs)] able to differentiate into mesenchymal cells upon appropriate culture conditions. When cultured in presence of autologous serum, these cells are strongly adherent to plastic, resistant to trypsin detachment, and resting. Mesodermal progenitor cells may be pulsed to proliferate and differentiate by substituting autologous serum for human cord blood serum or fetal calf serum. By these methods cells proliferate and differentiate toward mesenchymal cells and thus may further differentiate into osteoblats, chondrocytes, or adipocytes. Moreover MPCs are capable to differentiate in endothelial cells (ECs) showing characteristics similar to microvessel endothelium cells. Mesodermal progenitors cells have a defined phenotype and carry embryonic markers not present in mesenchymal cells. Moreover MPCs strongly express aldehyde dehydrogenase activity, usually present in hematopoietic precursors but absent in mesenchymal cells. When these progenitors are pulsed to differentiate, they lose these markers and acquire the mesenchymal ones. Interestingly, mesenchymal cells may not be induced to back differentiate into MPCs. Our results demonstrate the adult serum role in maintaining pluripotent mesodermal precursors and allow isolation of these cells. After purification, MPCs may be pulsed to proliferate in a very large scale and then induced to differentiate, thus possibly allowing their use in regenerative medicine.

Deterministic and stochastic approaches in the clinical application of mesenchymal stromal cells (MSCs)

Mesenchymal stromal cells (MSCs) have enormous intrinsic clinical value due to their multi-lineage differentiation capacity, support of hemopoiesis, immunoregulation and growth factors/cytokines secretion. MSCs have thus been the object of extensive research for decades. After completion of many pre-clinical and clinical trials, MSC-based therapy is now facing a challenging phase. Several clinical trials have reported moderate, non-durable benefits, which caused initial enthusiasm to wane, and indicated an urgent need to optimize the efficacy of therapeutic, platform-enhancing MSC-based treatment. Recent investigations suggest the presence of multiple in vivo MSC ancestors in a wide range of tissues, which contribute to the heterogeneity of the starting material for the expansion of MSCs. This variability in the MSC culture-initiating cell population, together with the different types of enrichment/isolation and cultivation protocols applied, are hampering progress in the definition of MSC-based therapies. International regulatory statements require a precise risk/benefit analysis, ensuring the safety and efficacy of treatments. GMP validation allows for quality certification, but the prediction of a clinical outcome after MSC-based therapy is correlated not only to the possible morbidity derived by cell production process, but also to the biology of the MSCs themselves, which is highly sensible to unpredictable fluctuation of isolating and culture conditions. Risk exposure and efficacy of MSC-based therapies should be evaluated by pre-clinical studies, but the batch-to-batch variability of the final medicinal product could significantly limit the predictability of these studies. The future success of MSC-based therapies could lie not only in rational optimization of therapeutic strategies, but also in a stochastic approach during the assessment of benefit and risk factors.

Are MSCs angiogenic cells? New insights on human nestin-positive bone marrow-derived multipotent cells.

Recent investigations have made considerable progress in the understanding of tissue regeneration driven by mesenchymal stromal cells (MSCs). Data indicate the anatomical location of MSC as residing in the “perivascular” space of blood vessels dispersed across the whole body. This histological localization suggests that MSCs contribute to the formation of new blood vessels in vivo. Indeed, MSCs can release angiogenic factors and protease to facilitate blood vessel formation and in vitro are able to promote/support angiogenesis. However, the direct differentiation of MCSs into endothelial cells is still matter of debate. Most of the conflicting data might arise from the presence of multiple subtypes of cells with heterogeneous morpho functional features within the MSC cultures. According to this scenario, we hypothesize that the presence of the recently described Mesodermal Progenitor Cells (MPCs) within the MSCs cultures is responsible for their variable angiogenic potential. Indeed, MPCs are Nestin-positive CD31-positive cells exhibiting angiogenic potential that differentiate in MSC upon proper stimuli. The ISCT criteria do not account for the presence of MPC within MSC culture generating confusion in the interpretation of MSC angiogenic potential. In conclusion, the discovery of MPC gives new insight in defining MSC ancestors in human bone marrow, and indicates the tunica intima as a further, and previously overlooked, possible additional source of MSC.

Selective Culture of Mesodermal Progenitor Cells

We have recently identified mesodermal progenitor cells (MPCs) isolated from adult human bone marrow. These cells show unusual phenotypes, having putative embryonic markers and aldehyde dehydrogenase (ALDH) activity. Interestingly, these resting cells, which have been selected by culturing them in the presence of adult human serum, can easily be induced to differentiate into mature mesenchymal stromal cells (MSCs) after substituting the adult human serum for fetal bovine serum (FBS) or human cord serum. MPC-derived MSCs are, in turn, able to differentiate toward osteoblasts, chondrocytes, and adipocytes. Furthermore, MPCs are able to differentiate into endothelial cells. MPCs have been proven to be strongly adherent to plastic culture bottles and to be trypsin-resistant. In the present article, we show a simple and inexpensive method to isolate highly selected mesodermal progenitors from bone marrow or cord blood. The optimization of standard culture conditions (using commercial human AB sera and appropriate concentrations for cell seeding in plastics) allows a pure population of MPCs to be obtained even after a short culture period. We believe that this simple, repeatable, and standardized method will facilitate studies on MPCs.

Gelatin/PLLA sponge-like scaffolds allow proliferation and osteogenic differentiation of human mesenchymal stromal cells

Tissue engineering has the potential to supply constructs capable of restoring the normal function of native tissue following injury. Poly(L-lactic acid) (PLLA) scaffolds are amongst the most commonly used biodegradable polymers in tissue engineering and previous studies performed on ovine fibroblasts have showed that addition of gelatin creates a favorable hydrophilic microenvironment for the growth of these cells. The attractiveness of using mesenchymal stromal cells (MSCs) in tissue regeneration is that they are able to differentiate into several lines including osteoblasts. In this study, we investigated the ability of gelatin/PLLA sponges to support the adhesion, proliferation, and osteogenic differentiation of human MSCs isolated from the bone marrow of four donors. [Figure: see text].

Mesenchymal cells inhibit expansion but not cytotoxicity exerted by gamma–delta T cells

Background  Multipotent mesenchymal stromal cells (MSCs) exert a relevant immunosuppressive activity by inhibiting T‐ and B‐lymphocytes, natural killer (NK) cells and dendritic cell expansion. Nevertheless, a possible activity on gamma/delta T cells has still not been evaluated.

Good manufacturing practice-grade fibrin gel is useful as a scaffold for human mesenchymal stromal cells and supports in vitro osteogenic differentiation

BACKGROUND: Recently, there has been an increased interest in using mesenchymal stromal cells (MSCs) in bone tissue engineering coupled with a suitable scaffold of both biological and synthetic origin. The cells and these constructs can be combined in vitro or directly in vivo to enhance tissue repair. MSCs are spindle‐shaped cells capable of self‐renewal and can be induced to differentiate mainly into osteo‐, chondro‐, and adipogenic‐progeny types. Several biomaterials are currently available and, among them, fibrin‐based constructs seem to be suitable for guiding the cells during tissue repair or regeneration due to their biocompatibility and biodegradability.

Mesodermal Progenitor Cells (MPCs) Differentiate into Mesenchymal Stromal Cells (MSCs) by Activation of Wnt5/Calmodulin Signalling Pathway

Background Mesenchymal Stromal Cells (MSCs) remain poorly characterized because of the absence of manifest physical, phenotypic, and functional properties in cultured cell populations. Despite considerable research on MSCs and their clinical application, the biology of these cells is not fully clarified and data on signalling activation during mesenchymal differentiation and proliferation are controversial. The role of Wnt pathways is still debated, partly due to culture heterogeneity and methodological inconsistencies. Recently, we described a new bone marrow cell population isolated from MSC cultures that we named Mesodermal Progenitor Cells (MPCs) for their mesenchymal and endothelial differentiation potential. An optimized culture method allowed the isolation from human adult bone marrow of a highly pure population of MPCs (more than 97%), that showed the distinctive SSEA-4+CD105+CD90neg phenotype and not expressing MSCA-1 antigen. Under these selective culture conditions the percentage of MSCs (SSEA-4negCD105+CD90bright and MSCA-1+), in the primary cultures, resulted lower than 2%. Methodology/Principal Finding We demonstrate that MPCs differentiate to MSCs through an SSEA-4+CD105+CD90bright early intermediate precursor. Differentiation paralleled the activation of Wnt5/Calmodulin signalling by autocrine/paracrine intense secretion of Wnt5a and Wnt5b (p<0.05 vs uncondictioned media), which was later silenced in late MSCs (SSEA-4neg). We found the inhibition of this pathway by calmidazolium chloride specifically blocked mesenchymal induction (ID50 = 0.5 µM, p<0.01), while endothelial differentiation was unaffected. Conclusion The present study describes two different putative progenitors (early and late MSCs) that, together with already described MPCs, could be co-isolated and expanded in different percentages depending on the culture conditions. These results suggest that some modifications to the widely accepted MSC nomenclature are required.

Carboxy‐terminal fragment of osteogenic growth peptide regulates myeloid differentiation through RhoA

The carboxy‐terminal fragment of osteogenic growth peptide, OGP(10–14), is a pentapeptide with bone anabolic effects and hematopoietic activity. The latter activity appears to be largely enhanced by specific growth factors. To study the direct activity of OGP(10–14) on myeloid cells, we tested the pentapeptide proliferating/differentiating effects in HL60 cell line. In this cell line, OGP(10–14) significantly inhibited cell proliferation, and enhanced myeloperoxidase (MPO) activity and nitroblue tetrazolium reducing ability. Moreover, it induced cytoskeleton remodeling and small GTP‐binding protein RhoA activation. RhoA, which is known to be involved in HL60 differentiation, mediated these effects as shown by using its specific inhibitor, C3. Treatment with GM‐CSF had a comparable OGP(10–14) activity on proliferation, MPO expression, and RhoA activation. Further studies on cell proliferation and RhoA activation proved enhanced activity by association of the two factors. These results strongly suggest that OGP(10–14) acts directly on HL60 cells by activating RhoA signaling although other possibilities cannot be ruled out.