Megakaryocyte–Bone Marrow Stromal Cell Aggregates Demonstrate Increased Colony Formation and Alkaline Phosphatase Expressionin Vitro

Abstract

Bone marrow stromal cells (BMSCs) possess certain stem celllike properties and can differentiate to adopt a number of mesenchymal phenotypes. BMSCs are usually investigated in vitro as homogeneous single-cell suspensions; however, these preparations lose much of their osteogenic capacity. Using the fibroblastic colony-forming unit assay, we have compared the proliferation and capacity to express alkaline phosphatase of BMSC-containing aggregates of bone marrow cells with single-cell suspensions of bone marrow cells from the same source. Aggregates were separated from single cells by density gradient centrifugation or cell sieving. The aggregate and single-cell preparations gave rise to similar numbers of colonies; however, the colonies produced by the aggregates were larger and expressed higher levels of alkaline phosphatase. When the aggregates were dissociated, colonies still formed; however, they expressed negligible levels of alkaline phosphatase. Immunomagnetic selection and immunofluorescent staining for CD61, N-methyl-D-aspartate (NMDA) receptor subunit 1, and acetylcholinesterase showed that the majority of the aggregates giving rise to osteoblastic colonies contained megakaryocytes. These data demonstrate that removing BMSCs from their normal environment reduces their osteoblastic capacity and that to achieve their maximal differentiation, BMSCs require direct physical contact with accessory cells such as megakaryocytes. These findings may be of direct relevance to the use of BMSCs for tissue-engineering purposes.

Restoration and reversible expansion of the osteoblastic hematopoietic stem cell niche after marrow radioablation

“…Indeed, there are compelling in vitro data to support the concept that osteoblasts can maintain the growth and differentiation of megakaryocytes 47,48 and that contact between megakaryocytes and osteoblast precursors facilitates proliferation and differentiation of the latter. 49 Although the intent of cytotoxic conditioning regimen is to eradicate host BM cellularity, we noted that many resident megakaryocytes, osteoblasts, and reticular bone lining cells survived and even proliferated up to 48 hours after TBI. Our results implicate SDF-1 as a survival factor, up-regulated after TBI, that could account for the persistence of these mesenchymal elements.…”

Transcriptional regulation of bone marrow thrombopoietin by platelet proteins

“…A large number of reports indicate that megakaryocytes stimulate osteoblastogenesis and inhibit osteoclastogenesis. [34][35][36][37][38][39][40][41] Thus, one might postulate that osteoclasts, while acting to inhibit osteoblast development, might also act indirectly to inhibit megakaryocyte growth by secreting factors that suppress TPO expression and inhibit megakaryocyte development.…”

Megakaryocytes are mechanically responsive and influence osteoblast proliferation and differentiation

“…These mice accumulate immature MKs in the marrow and develop a dramatically high bone mass phenotype with age [25]. Furthermore, in vitro , both osteoblast proliferation and differentiation of mesenchymal progenitors is enhanced by the presence of MKs [25, 26]. This effect requires cell-cell contact between the MKs and the osteoblast lineage cells [25] and evidence suggests this effect may be mediated by gap junctions [27] or integrins [28].…”