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

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.

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.

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.

Saglio:Novartis Pharmaceutical: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Speakers Bureau; Pfizer: Consultancy.