Proliferative activity of mesenchymal stem cells from different parts of the placenta

The ability of mesenchymal stem cells (MSCs) isolated from the decidua, chorionic tissue and amniotic membrane of the placenta to the self-renewal and the proliferation was investigated. Our results revealed that the number of colony forming unit-fibroblast (CFU-F) and the growth rate of MSCs were higher in the decidua and chorionic tissue compared to the amniotic membrane. Decidua MSCs and chorionic MSCs possessed a similar powerful proliferative potential and increased in 1010- fold in cultures for 3 months, that is 103 times more then the amniotic MSCs. The cumulative population doubling (PD) of placenta-derived MSCs was significantly higher at all passages then PD of bone marrow derived MSCs. The decidua and chorionic tissue of the placenta are ideal MSCs sources for cell based therapy.

Dissecting the Hematopoietic Niche to Improve Transplant: Hedgehog and Wnt Pathways Activation in Fetal Mesenchymal Cells (MSC) Are Associated with Better Support to Hematopoietic Progenitors and Higher Proliferative Capacity Compared to Adult MSC

Abstract 4809 Background. Mesenchymal Stem Cells (MSC) are among major players of the hematopoietic niche. In addition, they have shown to be useful in clinical studies both as immune-modulators as well as in regenerative medicine. However, a major obstacle for proving their usefulness is to obtain a clinically effective cell dose, since often less than 10̂6 /kg of adult MSC have been used. Thus, their potential efficacy can be either disregarded or not formally proved. In addition, forcing in vitro the proliferative potential of adult BM derived MSC (BM-MSC) implies the risk of generating chromosomal abnormalities. The aim of the study is to investigate the potential use of fetal derived MSC (FT-MSC) which could provide means to overcome the above limits. In addition, we attempted to identify some molecular mechanisms regulating the proliferative potential of FT-MSC and their supportive capacity of hematopoietic progenitors. Methods: Specimens from 5 fetal samples were collected after mechanically induced abortion (median gestational age 11 weeks). FT-MSC were obtained from kidneys and femurs and adult MSC from normal BM. After 15 days of culture adherent cells were sub-cultured until 80% confluence. Immunophenotype and karyotype were evaluated. The telomere length was evaluated using the Telomere PNA system. FT-MSC were injected into NOD/SCID mice to rule out any tumorigenic potential. The immunosuppressive activity of FT-MSC and adult BM-MSC was evaluated by standard assays. To quantify the supportive capacity of FT-MSC we weekly measured the colony output using normal BM MNC layered over confluent FT-MSC compared to stromal cell line MS-5 and to BM-MSC. Protein expression of the key regulators of Hedgehog and Wnt pathways were investigated in FT-MSC and BM-MSC by immunofluorescence using monoclonal antibodies against Indian Hedghog (IHh), Sonic Hedgehog (SHh), Desert Hedgehog (DHh) and b-catenin. Fluorescent signal was detected by confocal microscope. Results. MSC successfully grew from 100% of fetal samples. FT-MSC maintained the typical MSC markers (CD73, CD105, CD44, CD106, CD166, HLA class I, negative for HLA class II) and possessed the same capacity to inhibit T cell proliferation as BM-MSC. FT-MSC compared to adult BM-MSC showed a higher proliferative potential: they reached the fifteenth generation and had a median cumulative population doubling of 26 (range 4–47) while BM-MSC grew until the sixth passage showing a median cumulative population doubling of 4 (range 2–5) (p<0.05 at the fourth, fifth and sixth passage). This might be ascribed to both Hedghog and Wnt pathways which were highly activated in FT-MSC compared to BM-MSC. The mean value of SHh protein expression was 11,5 RU (relative units) compared to 5,5 RU in BM-MSC and b-catenin was 17 RU in FT-MSC vs 3.7 RU in adult. Differently from BM-MSC, FT-MSC maintained a stable karyotype without any sign of tumorigenic potential in the mouse model and stable telomere length during culture. By contrast, 5 out of 32 adult MSC developed genetic abnormalities during in vitro expansion. Importantly, FT-MSC display a better support of hematopoiesis compared to BM-MSC or MS-5 cell line, in fact, during five weeks of culture normal bone marrow samples produced an overall colony number higher when FT-MSC were used as feeder layer compared to BM-MSC or MS-5 (p= 0.07 for both). IHh was significantly higher in FT-MSC compared to BM-MSC (121 RU vs 9 RU). This is in keeping with what already reported by Kobune et al. (Blood 2006) regarding the role of IHh in supporting hematopoiesis and provides the molecular basis for the high supportive capacity of FT-MSC. In conclusion, FT-MSC seem to be candidate for being used to support hematopoiesis in transplant settings. FT-MSC overcome the limitation imposed by adult MSC providing a remarkable yield in culture and they show a reassuring safety profile. FT-MSC show a peculiar profile of Hedgehog and Wnt activation possibly responsible for their biological properties described in this study. Disclosures: Saglio: Novartis Pharmaceutical: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Speakers Bureau; Pfizer: Consultancy.

Characterization of Fibroblast Clones from Periodontal Granulation Tissue in vitro

Connective tissues are known to be composed of heterogeneous fibroblast subpopulations. The significance of this heterogeneity in different physiological and pathological conditions is poorly understood. Granulation tissue is formed in connective tissue during wound healing, chronic inflammation, and certain pathological conditions. In this study, heterogeneity of fibroblasts from granulation tissue was investigated by cell-cloning techniques. Granulation-tissue fibroblasts (GTFs) from both chronically inflamed periodontal lesions and healing wound granulation tissue behaved similarly. GTFs showed a more pronounced decrease in proliferative capacity with increasing cumulative population doubling levels (CPDLs) and 30-40% lower cloning efficiency compared with normal gingival fibroblasts (HGFs). Morphologically, cells in GTF cultures were mainly large, whereas HGFs were mainly small in size. Both cell-line types showed heterogeneity in cell morphology. Clones composed of large stellate-like cells predominated in GTF cultures, whereas clones composed of small spindle-shaped or epithelioid cells predominated in HGF cultures. In both cell-line types, the proportion of clones composed of large cells increased with increasing CPDL. These findings show that the properties of the fibroblasts changed during their in vitro life spans. The finding that normal connective tissue and granulation tissues contain morphologically distinct fibroblast clones in different proportions suggests that local factors could stimulate local fibroblasts to differentiate into GTFs. Alternatively, local factors could select some fibroblast subpopulations to overgrow the others to form granulation tissue.

Endothelial Cell Senescence Inhibits Unidirectional Endothelialization in Vitro

We investigated the effects of cellular senescence on unidirectional endothelialization in vitro, simulating the anastomotic endothelialization of vascular prosthesis. The experiments were carried out with three different cumulative population-doubling levels (CPDLs) of bovine aortic endothelial cells (ECs), which have finite life span. Young ECs with 22 CPDL, middle aged with 46, and senescent with 70 at the time of inoculation were used. The effect of aging on unidirectional endothelialization, as well as cellular morphology and proliferative and migratory potentials of isolated cells, were qualitatively and quantitatively analyzed. The unidirectional endothelialization rate was determined by our newly designed method to prepare the square monolayer sheet with linear margins between cell-adhesion and noncell-adhesion regions. The results showed that endothelial cell senescence retarded not only proliferation and migration but also unidirectional endothelialization. Time-lapsed videomicroscopic study of unidirectional endothelialization process revealed that ECs at several rows back from the leading edge represented much slower rate of migration than did the ECs at the leading edge. These findings suggest that high cellular mobility observed for the ECs at the leading edge may result in localized excessive cell replication. Thus, atherosclerotic vessels containing senescent or injured ECs may have limited capability of anastomotic endothelialization.