Archives

  • 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • 2024-04
  • 2024-05

    • Previous in vitro cell culture

    2018-11-08

    Previous in-vitro cell culture studies in microgravity have shown decreased osteoblast differentiation (Nabavi et al., 2011; Landis et al., 2000; Carmeliet et al., 1997), reduced osteoblast number (Hughes-Fulford and Lewis, 1996), and reduced osteoblast cell integrity (Nabavi et al., 2011). Furthermore, increased mRNA expression of alkaline phosphatase in animals exposed to microgravity, a glycoprotein indicating bone formation activities of osteoblasts, has been found in both spaceflight and hindlimb unloading (HU) studies (Bikle et al., 1994). Conversely osteoclasts showed increased maturation and resorptive activity in microgravity (Tamma et al., 2009) and in HU mice (Vico et al., 1987). Furthermore, investigators found that differentiating MSCs from HU animals proliferate more slowly than those isolated from 1g controls, and hypothesized that this was either due to decreases in the absolute number and recruitment of osteoprecursors, or to decreased proliferation of committed osteoprogenitor UNC 0642 (Kostenuik et al., 1997). Although we also found increased ALP protein expression in osteoblast cultures at D7, we find no increase in ALP mRNA expression in Affymetrix analysis immediately after microgravity exposure (data not shown) indicating that increased osteoblast formation only occurs upon reloading. Our ex-vivo cell differentiation assays therefore indicate that bone marrow exposed to mechanical unloading in microgravity, and then reloaded at 1g, has greater differentiation potential to form both osteoblasts and osteoclasts. Since both assays performed rely on the induction of early stage progenitors by growth factors, these results suggest that the bone marrow cell pool from microgravity mice may either contain more early-stage differentiation-arrested progenitors, or that the progenitor cells present are more readily induced to differentiate following reloading and growth factor treatment. To help resolve this question we investigated expression patterns of over 120 genes in the bone marrow related to stem cell maintenance and self-renewal, and hematopoietic and mesenchymal lineage differentiation. Our gene expression results indicate a broad down-regulation of mRNAs related to hematopoietic and mesenchymal lineage differentiation but no alterations in multipotent stem cell markers for early stage hematopoietic and mesenchymal stem cells. Specifically, we found no alterations in SOX2 and NOTCH1, which are important for both hematopoietic and mesenchymal stem cell proliferation and self-renewal capabilities (Go et al., 2008; Weber and Calvi, 2010). We did, however, find down-regulation of markers involved in proliferation, survival and maintenance of HSCs, as well as decreases in HSC cell surface markers, such as PTPRC and CD90 (Kiel et al., 2005). Down-regulation of KIT, MYB, PTEN, and BMI1, which are involved in the regulation of hematopoiesis and the cell cycle, may indicate a down-regulation in hematopoietic differentiation (Kodama et al., 1994; Sandberg et al., 2005; Park et al., 2003; Zhang et al., 2006). PTEN, a molecule also involved in regulation of the cell cycle by preventing rapid growth of cells, was also found to be down-regulated. Furthermore, MSC markers were found to be down-regulated, including KDR, MCAM, ICAM1, and NT5E (Bianco et al., 2008; Bernardo et al., 2007; Gargett et al., 2009; Majumdar et al., 2003; Jiang et al., 2002). However, these genes also have many other roles in the bone marrow compartment, including as endothelial growth factors, adhesion molecules, and differentiation molecules. Because of the relatively low abundance of MSCs and HSCs in the pool of cells found within the bone marrow compartment, the down-regulation of these genes we observed may not be specific to a down-regulation in stem cell function. Microgravity samples also showed up-regulation of PDGFRβ, a molecule that is required for MSC migration through binding of α5β1-integrin to extracellular matrix, FAK expression, and activation of Pi3K in tissue sites where differentiation into progenitors is required (Veevers-Lowe et al., 2011). Up-regulation of PDGFRβ may therefore indicate the initiation of MSC migration and differentiation in microgravity. LIF, an important molecule for repressing osteoclast and osteoblast activation and maintenance of stem cells was also down-regulated (Escary et al., 1993). Our results therefore indicate that genes associated with maintenance of self-renewal in early stem cells (NOTCH1 and SOX2) are unaltered in microgravity, offering no evidence that stem cell maintenance is altered. However, many genes associated both with somatic bone marrow stem cells and differentiation of the hematopoietic and mesenchymal lineage, show a broad pattern of significant down-regulation, suggesting decreased differentiation ability rather than an alteration in stem cell proliferation (Fig. 4).