In Vivo Parathyroid Hormone Treatment Expands All Multipotent Primitive Hematopoietic Cell Subsets.

Abstract 1449

Poster Board I-472

Osteoblastic cells have been identified as a component of the hematopoietic stem cell (HSC) niche. This identification provides a novel strategy for in vivo expansion of HSCs by stimulating their regulatory microenvironment. This therapeutic approach could potentially expand the clinical use of HSCs in conditions where their number limits use. Activation of osteoblastic cells by Parathyroid Hormone (PTH) increases bone, osteoblastic and osteoclastic cell number and expands the HSC pool. Since HSCs do not express the PTH receptor, and genetic activation of PTH receptors in osteoblastic cells is sufficient to expand HSCs, the HSC increase must be initiated by PTH-dependent activation of osteoblastic cells. The HSC-enriched lineage-, sca-1+, and c-kit+ (LSK) compartment is heterogeneous and contains at least three subpopulations of cells with multilineage potential, but with progressively decreased quiescence and more limited self-renewal. In the initial evaluation of PTH effects on the HSC niche, the effects of osteoblastic activation on HSC subsets were not characterized in detail. The effect of PTH and osteoblastic activation on individual HSC subsets could suggest additional therapeutic uses if PTH increases not only Long Term-HSC (LT-HSC) but also the more proliferative Short-Term HSCs/ Multi Potent Progenitors (ST-HSCs/MPPs). In this study, our goal was to identify changes in the proportion of LT-HSC vs ST-HSC and MPPs after PTH treatment by utilizing flow cytometric analysis and competitive repopulation assays (primary, secondary and tertiary reconstitution). We developed an accelerated PTH treatment regimen in mice (40μg/kg body weight ip three times daily for 10 days), which minimizes the effects of normal aging on bone and hematopoiesis. We first analyzed the effect of this PTH regimen on bone. PTH-treated mice had increased trabecular bone volume (% BV/TV VEH 25±2 vs PTH 43±4 p<0.001) compared to their vehicle-treated controls when analyzed by micro-CT. Histologic analysis demonstrated a dramatic increase in trabecular bone as well as osteoblastic cells lining the trabecular surfaces and TRAP+ osteoclasts in the PTH-treated animals. PTH-treated mice also had increased primitive hematopoietic cells defined through flow cytometry as LSK (VEH 0.19±0.02 vs PTH 0.27±0.01 p<0.005). LSKs were further subdivided to phenotypically identify HSC subsets using the SLAM receptors. Surprisingly the frequency of subpopulations was increased in PTH treatment increased not only LT-HSCs (VEH 0.025±0.002 vs PTH 0.040±0.002 p<0.0001), but also the less quiescent ST-HSCs/MPPs (VEH 0.041±0.001 vs PTH 0.065±0.004 p<0.0001). HSC subsets can also be identified within the LSK pool based on expression of Flt3 and Thy1.1. This phenotypic analysis of bone marrow cells from mice treated with the intensive PTH regimen demonstrated a significant increase in all LSK subsets including MPPs (VEH 0.043±0.003 vs PTH 0.061±0.004 p<0.005), identified as LSK Flt3⁺ Thy1.1^low cells, LSK Flt3⁻ Thy1.1^int (VEH 0.056±0.005 vs PTH 0.077±0.004 p<0.01), enriched for ST-HSCs, and the LT-HSC enriched LSK Flt3⁻ Thy1.1^int subset (VEH 0.042±0.004 vs PTH 0.054±0.002 p<0.05). Taken together, these data suggest that systemic PTH treatment not only expands the most quiescent HSCs, but also increases ST-HSC/MPPs. Since PTH treatment increased all phenotypic HSC subsets, transplantation of hematopoietic bone marrow cells from PTH treated mice would be expected to have superior engraftment both in the short and long term. Accordingly, a significant increase in both early and late hematopoietic reconstitution was observed in primary transplant recipients of bone marrow from PTH-treated animals, as well as secondary and tertiary transplant recipients. These results also suggest that PTH treatment equally expands all HSC subsets. The novel finding that the less quiescent, multipotent ST-HSC and MPPs are also expanded by PTH may be of great therapeutic relevance, as it supports the use of osteoblastic stimulation by PTH not only to increase long term repopulation, but also to critically improve accelerated hematopoietic recovery after myeloablation. Thus, manipulation of the HSC niche may result in beneficial effects beyond their ability to increase the most quiescent HSCs. Further studies will elucidate the specific mechanisms causing this novel global increase of multipotent primitive hematopoietic cells.