Sonia Rodriguez-Rodriguez

Impact of interactions of cellular components of the bone marrow microenvironment on hematopoietic stem and progenitor cell function

Hematopoietic stem (HSC) and progenitor (HPC) cell fate is governed by intrinsic and extrinsic parameters. We examined the impact of hematopoietic niche elements on HSC and HPC function by analyzing the combined effect of osteoblasts (OBs) and stromal cells (SCs) on Lineage(-)Sca-1(+)CD117(+) (LSK) cells. CFU expansion and marrow repopulating potential of cultured Lineage(-)Sca-1(+)CD117(+) cells were significantly higher in OB compared with SC cultures, thus corroborating the importance of OBs in the competence of the hematopoietic niche. OB-mediated enhancement of HSC and HPC function was reduced in cocultures of OBs and SCs, suggesting that SCs suppressed the OB-mediated hematopoiesis-enhancing activity. Although the suppressive effect of SC was mediated by adipocytes, probably through up-regulation of neuropilin-1, the OB-mediated enhanced hematopoiesis function was elaborated through Notch signaling. Expression of Notch 2, Jagged 1 and 2, Delta 1 and 4, Hes 1 and 5, and Deltex was increased in OB cultures and suppressed in SC and OB/SC cultures. Phenotypic fractionation of OBs did not segregate the hematopoiesis-enhancing activity but demonstrated that this function is common to OBs from different anatomic sites. These data illustrate that OBs promote in vitro maintenance of hematopoietic functions, including repopulating potential by up-regulating Notch-mediated signaling between HSCs and OBs.

Osteoblast lineage cells expressing high levels of Runx2 enhance hematopoietic progenitor cell proliferation and function

Although osteoblasts (OB) play a key role in the hematopoietic stem cell (HSC) niche, little is known as to which specific OB lineage cells are critical for the enhancement of stem and progenitor cell function. Unlike hematopoietic cells, OB cell surface phenotypic definitions are not well developed. Therefore, to determine which OB lineage cells are most important for hematopoietic progenitor cell (HPC) function, we characterized OB differentiation by gene expression and OB function, and determined whether associations existed between OB and HPC properties. OB were harvested from murine calvariae, used immediately (fresh OB) or cultured for 1, 2, or 3 weeks prior to their co‐culture with Lin−Sca1+c‐kit+ (LSK) cells for 1 week. OB gene expression, alkaline phosphatase activity, calcium deposition, hematopoietic cell number fold increase, CFU fold increase, and fold increase of Lin−Sca1+ cells were determined. As expected, HPC properties were enhanced when LSK cells were cultured with OB compared to being cultured alone. Initial alkaline phosphatase and calcium deposition levels were significantly and inversely associated with an increase in the number of LSK progeny. Final calcium deposition levels and OB culture duration were inversely associated with all HPC parameters, while Runx2 levels were positively associated with all HPC properties. Since calcium deposition is associated with OB maturation and high levels of Runx2 are associated with less mature OB lineage cells, these results suggest that less mature OB better promote HPC proliferation and function than do more mature OB. J. Cell. Biochem. 111: 284–294, 2010.

Impact of maturational status on the ability of osteoblasts to enhance the hematopoietic function of stem and progenitor cells.

Osteoblasts (OBs) exert a prominent regulatory effect on hematopoietic stem cells (HSCs). We evaluated the difference in hematopoietic expansion and function in response to co‐culture with OBs at various stages of development. Murine calvarial OBs were seeded directly (fresh) or cultured for 1, 2, or 3 weeks prior to seeding with 1000 Lin‐Sca1 + cKit+ (LSK) cells for 1 week. Significant increases in the following hematopoietic parameters were detected when comparing co‐cultures of fresh OBs to co‐cultures containing OBs cultured for 1, 2, or 3 weeks: total hematopoietic cell number (up to a 3.4‐fold increase), total colony forming unit (CFU) number in LSK progeny (up to an 18.1‐fold increase), and percentage of Lin‐Sca1+ cells (up to a 31.8‐fold increase). Importantly, these studies were corroborated by in vivo reconstitution studies in which LSK cells maintained in fresh OB co‐cultures supported a significantly higher level of chimerism than cells maintained in co‐cultures containing 3‐week OBs. Characterization of OBs cultured for 1, 2, or 3 weeks with real‐time PCR and functional mineralization assays showed that OB maturation increased with culture duration but was not affected by the presence of LSK cells in culture. Linear regression analyses of multiple parameters measured in these studies show that fresh, most likely more immature OBs better promote hematopoietic expansion and function than cultured, presumably more mature OBs and suggest that the hematopoiesis‐enhancing activity is mediated by cells present in fresh OB cultures de novo.