Bone marrow mesenchymal stem cell niches and regenerative medicine

Mesenchymal progenitors are a powerful tool in regenerative medicine, but suffer from a rapid loss of differentiation potential during in vitro expansion. The recent discovery that well-characterized stem cells, like HSC, maintain their stemness during self-renewal through the interaction with specialized microenvironments, called stem cell niches, prompted us to investigate the existence of a niche compartment for also mesenchymal progenitors.
In Chapter 4 of this thesis we described the establishment of a niche/progenitor system in vitro for bone marrow mesenchymal stem cells (MSC). We asked whether the non-adherent fraction of human bone marrow cultures contained early progenitors which can constitute a reservoir for the mesenchymal compartment and whether the adherent cells, instead, could provide a niche function for the maintenance and regulation of these progenitors.
Replating the non-adherent fraction in a new dish at the first medium change, we found that a population of bone marrow non-adherent mesenchymal progenitors (BM-NAMP) was present and their number was 20.43.6% of the initial CFU-f. However, further investigation showed that, when serially replated in new dishes, BM-NAMP were able to steadily increase in number, self-renewing as non-adherent progenitors while generating at the same time adherent colonies. The diameter size evaluation showed that BM-NAMP could produce colonies with 2-fold larger diameter, indicating a significantly higher proliferation capacity. However, the colonies produced in the following replating steps were progressively smaller, indicating a gradual loss of BM-NAMP proliferation potential. Together with increased proliferation, first-replated BM-NAMP progeny cells displayed a higher differentiation potential compared to standard CFU-f both in vitro and in vivo. Taken together, these data indicate together that BM-NAMP show features of earlier progenitor features and suggest a biological difference between BM-NAMP and the initially adhrering CFU-f.
Serial replating experiments performed with serum alone showed that BM-NAMP critically required FGF-2 for their initial selection and maintenance in culture. Interestingly, blocking receptor experiments showed that the maintenance of BM-NAMP in culture was mediated through FGFR2c signaling, which has been shown to be involved in vivo in the balance between proliferation and differentiation of skeletal progenitors.
We also hypothesized that BM-NAMP were in close interaction with the adherent cells, and that these provide a niche function for them. BM-NAMP were not able to survive when replated either on agarose-coated dishes or on human fibroblasts. This suggests that BM-NAMP required specific signals from the adherent progeny and that this fraction constitutes a unique environment for BM-NAMP survival and self-renewal. In fact, when kept in contact with initial CFU-f progeny for 14 days instead of being serially replated, BM-NAMP were able to produce 3-fold more colonies. Furthermore, the colony diameter analysis showed that, unlike the serial replating which caused a gradual loss of BM-NAMP proliferative activity, the continuous culture in the primary plate could preserve BM-NAMP proliferation potential. Furthermore, if kept in the original plate, BM-NAMP could generate a progeny that also displayed a higher differentiation capacity. Taken together, these results suggest together that CFU-f progeny provides a niche function for BM-NAMP.
In Chapter 5 we sought at investigating the presence of a class of non-adherent progenitors in human adipose tissue stromal vascular fraction (SVF), which constitute an abundant source of mesenchymal progenitors, to determine whether the NAMP compartment was specific to bone marrow or they could constitute a reservoir also in other tissues.
NAMP were present in adipose tissue SVF cultures (AT-NAMP) with a similar frequency as observed in the bone marrow and the replating of the non-adherent fraction in the same dish revealed that they were stably non-adherent. The main difference compared to BM-NAMP was the inability of AT-NAMP to self-renew as non-adherent progenitors upon serial replating, since only few colonies were present in the last replating step. However, these colonies had a significantly increased diameter. This suggests that, when serially replated, AT-NAMP do not undergo proliferation but rather a selection for the very rare progenitors with the highest proliferation ability. Similarly to BM-NAMP, when kept in contact with the initially adhering CFU-f, AT-NAMP could proliferate without loss of their proliferation capacity. This suggests that, as for bone marrow cells, adherent CFU-f provide a niche function for the non-adherent progenitors, regulating the maintenance of their early-progenitor properties.
In conclusion, these data show that, although displaying important tissue-specific biological differences, NAMP are present in the mesenchymal progenitor compartment of different tissues and they represent a reservoir of earlier progenitors compared to standard CFU-f.