Improvement of Hematopoietic and Stromal Cells Interaction after PTH Treatment of Long-Term Bone Marrow Cultures from Patients with Aplastic Anemia.

Abstract

Alterations in hematopoietic and stromal cells interactions could be one of the causes of aplastic anemia (AA). Assuming the existence of two types of stromal niches where hematopoietic stem cells (HSC) reside, - osteoblastic niche that regulates survival, self-maintenance abilities, adhesion, homing and quiescence, and vascular niche controlling proliferation and differentiation, - one could suggest an impairment of regulation in both of them in AA patients. As parathyroid hormone (PTH) activates osteoblasts it could potentially improve the survival of HSC in patients with various hematopoietic disorders. The aim of this study was to compare several functional characteristics of stromal cells from healthy donors and AA patients. In order to study stromal microenvironment capable to maintain hematopoiesis, long-term bone marrow cultures were established from 19 patients with AA and 24 healthy donors as a control group. PTH was added for 3 and 6 weeks of cultivation in concentration 10−8, 5x10−8 and 10−7 M once a week while changing half of the media. The proportion of CAFC 7 and CAFC 28–35 from the donor bone marrow survived cocultivation for 2–5 days with irradiated adherent cell layers (ACLs) of AA patients was estimated by limiting dilution assay. To characterize alterations in the expression of several genes in ACLs semi-quantitative analysis of RT-PCR products was performed using PhosphoImager Cyclone, Packard Bell (USA), after Southern blot hybridization with appropriate sequences. The level of b-actin expression was used as a normalization factor. All data from the patients was compared with corresponding gene's expression level in donors ACLs. Among studied signaling molecules involved in niche regulation Ang-1 expression was significantly decreased (2.3-fold) in ACLs of patients after 3 weeks in culture. Further cultivation led to normalization of Ang-1 expression. Another difference in osteoblastic niche regulating genes was revealed in Notch 1 signaling. While in ACLs from donor bone marrow it increased 2-fold during cultivation, it was stable in ACLs from the patients. Expression level of ICAM-1 responsible for adhesion of HSC to osteoblasts in niche also increased (4-fold) during cultivation in ACLs from donors and did not change in AA patients. Expression level of all other studied genes of osteoblastic niche signaling pathways was not altered in ACLs of AA patients. Analysis of genes regulating stromal cells in vascular niche revealed increased level of VEGF (2-fold) accompanied with 2-fold decreased expression of VCAM-1 in ACLs of the patients. Expression level of both genes normalized with further cultivation. The data suggest the functional inhibition of stromal microenvironment in patients with aplastic anemia that could be overcomed by cultivation ex vivo. PTH treatment activating gene expression in ACLs of donors did not affect universally the same genes in patients. Nevertheless treatment with PTH improved survival (2–3-fold) of long-term repopulating HSC (CAFC 28–35) from donors bone marrow explanted on irradiated ACLs of AA patients. The survival rate of short-repopulating HSC (CAFC 7) was not sensitive to PTH treatment. So, in patients with aplastic anemia the impairment of stromal regulation of HSC was observed both in osteoblastic and vascular niches. PTH being the osteoblasts activator could partially improve functioning of stromal microenvironment and hence the HSC survival.