Inhibition of collagen deposition in the extracellular matrix prevents the establishment of a stroma supportive of hematopoiesis in long-term murine bone marrow cultures.

Abstract

Long-term production of murine hematopoietic cells in vitro is dependent on establishment of a complex microenvironment consisting of a variety of stromal cells and an extensive extracellular matrix which includes collagen, fibronectin, laminin, proteoglycans, and other undefined components adherent to the culture dishes. Cis4-hydroxyproline (CHP), a relatively specific inhibitor of collagen secretion, was used to examine the role of extracellular collagen deposition in supporting hematopoiesis in long-term C57BI/6J mouse bone marrow cell cultures. Throughout the 10-wk culture period, all culture dishes contained either 0, 10, 25, or 50 sg/ml of CHP. All medium and nonadherent cells were removed at weekly intervals and replaced with fresh medium containing the previous concentrations of CHP. Nonadherent cells were assayed weekly for total cells and pluripotent, erythroid, megakaryocytic, and granulocytic-macrophage progenitor cells. Dishes were killed at selected intervals to assess protein and collagen synthesis in the adherent layer. Adherent cell numbers, as judged by microscopic examination and DNA assays, correlated inversely with CHP concentrations used and paralleled degree of collagen synthesis inhibition. The decreased hemopoietic progenitor cell production correlated closely with percent inhibition of collagen synthesis and stromal cellularity. The CHP concentrations tested were not directly toxic to hemopoietic progenitor cells. These studies demonstrate that collagen deposition in the extracellular matrix of murine bone marrow cell cultures is essential to the establishment of a functional stromal microenvironment that is supportive of long-term hematopoiesis.

Extracellular matrix promotes the growth and differentiation of murine hematopoietic cells in vitro.

“…(10). Furthermore, the addition of cis-hydroxyproline (which specifically inhibits collagen synthesis) resulted in a decrease of stem cell number in these cultures (8). Keating et al (25) found that proteoglycans are also a major component of the adherent layer of long-term cultures.…”

“…Recent studies suggest that the ECM also plays a role in the in vitro proliferation and differentiation of hematopoietic cells. Inhibitors of matrix formation are known to decrease stem cell production (8), while compounds stimulating matrix production increase stem cell proliferation (9). Finally, the stromal cell population is known to produce a variety of ECM proteins including fibronectin, laminin, and type IV collagen (10).…”

In vitro requirement for periostin in B lymphopoiesis

“…Previous in vitro studies have shown that the ECM protein collagen type I is required for hematopoiesis, 14,15 and, similarly, fibronectin has been shown both in vitro and in vivo to promote proliferation of HSCs. 16 Whether the ECM protein periostin is also important for hematopoiesis, specifically B lymphopoiesis, has not been investigated.…”

The role of novel and known extracellular matrix and adhesion molecules in the homeostatic and regenerative bone marrow microenvironment

“…143 Direct adhesion of HSPCs to fibronectin attenuates HSPC proliferation and differentiation, which is mediated by integrins. 144 In addition, the abundant ECM protein tenascin-C supports the regeneration of haematopoietic cells after myeloablation.…”