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

Abstract

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.

Fig. 1

LSK cells were cultured alone or in the presence of freshly prepared OBs for 7 days and the following parameters were measured: (A) total number of hematopoietic cells in culture; (B) total number of CFU produced in culture; (C) percentage of cells expressing Lin-Sca1+ cell surface markers; and (D) plating efficiency of clonogenic cells (number of CFUs/number of cells assayed × 100). Data are presented as the mean ± the standard deviation. Co-culture of LSK cells with OBs significantly increased all hematopoietic parameters analyzed relative to LSK cells cultured alone. *Indicates statistically significant differences (p < .05) compared to LSK cells alone for identical HPC measurements.

Fig. 2

LSK cells were co-cultured with freshly prepared OBs or with OBs cultured in complete medium for 1, 2, or 3 weeks prior to co-culture with LSK cells. After 7 days of co-culture, hematopoietic cells were assayed as follows: (A) total number of hematopoietic cells in culture; (B) total number of CFUs produced in culture; (C) percentage of cells expressing Lin-Sca1+ cell surface markers; and (D) plating efficiency of clonogenic cells (number of CFUs/number of cells assayed × 100). Results are reported as box plots representing the range of data points, with the line representing the average. Error bars represent the standard deviations associated with the mean. Data points more than 2 standard deviations from the mean are identified on the plot but were used in statistical determinations. For all hematopoietic parameters analyzed, results from freshly prepared OB cultures were superior to those from OB cultures previously cultured for 1, 2, or 3 weeks prior to co-culture.

Fig. 3

LSK cells were cultured alone or were co-cultured with freshly prepared OBs or with OBs cultured for 1 week. After 7 days of co-culture, hematopoietic cells were removed by washing and cell cycle status was assessed by staining with propidium iodide. No differences in the percentage of hematopoietic cells in the S/G2 + M phases of cell cycle were observed. In parallel studies, fresh and 1-week OBs were cultured alone and OB cell cycle status determined as above. As would be expected, a higher percentage of fresh OBs were in the active phases of cell cycle than those cultured for 1 week. In data not shown, we also analyzed both hematopoietic and OB cells for the expression of annexin V. In all cases, less than 1% of the cells were annexin V positive, suggesting that apoptosis was not detected.

Fig. 4

Repopulating potential of freshly isolated LSK cells in the presence and absence of fresh OBs and in vitro expanded LSK cells for 5 days in co-cultures of fresh OBs and 3-week OB cultures. Data are from one experiment and 5 mice per group were analyzed. Chimerism in the peripheral blood at 1 and 4 months post-transplantation and in the marrow at 4 months shows significantly better engraftment from LSK cells expanded in fresh OB cultures than with LSK cells expanded in 3-week OB cultures. Data are presented as the mean ± the standard deviation. *Indicates, statistically significant differences (p < .05) compared to fresh LSK (group 1). ^Y Indicates statistically significant differences (p < .05) compared with LSK co-cultured with fresh OBs (group 3).

Fig. 5

Characterization of mRNA expression and calcium deposition in OB cultures (7 days following experiment initiation) from freshly prepared OBs or OBs cultured for 1, 2, or 3 weeks prior to experiment initiation. It should be noted that for all (A–E) gene expression data and (F) calcium deposition data, levels were normalized to levels measured in cultures of fresh OBs. Results are reported as box plots representing the range of data points, with the line representing the average. Error bars represent the standard deviations associated with the mean (all error bars are graphed and in some cases the error bars are within the box plots). There were no data points more than 2 standard deviations from the mean. (A) Runx2 expression was highest in freshly isolated OBs and declined with culture duration. (B) Alkaline phosphatase expression increased with culture duration, peaking at week 2, and this level was maintained in week 3 cultures. As expected, expression of (C) type I collagen, (D) osteopontin, and (E) osteocalcin, as well as (F) calcium deposition, increased as OB culture duration increased.