Vitamin D metabolism in human bone marrow stromal (mesenchymal stem) cells

Abstract

There are many human extra-renal tissues and cells that biosynthesize 1α,25-dihydroxyvitamin D (1α,25(OH)(2)D) by the action of CYP27B1/1α-hydroxylase. Human marrow stromal cells (hMSCs), also known as mesenchymal stem cells, were isolated from marrow discarded from well-characterized, consented subjects during common orthopedic procedures. Human MSCs can give rise to osteoblasts, chondrocytes, adipocytes, and other lineages. Their in vitro differentiation to osteoblasts is stimulated by 1α,25(OH)(2)D, and recent evidence indicates that they have the capacity to metabolize vitamin D in a regulated manner. Human MSCs express the vitamin D receptor, 25-hydroxylases, 1α-hydroxylase, and 24-hydroxylase; stimulation of in vitro osteoblastogenesis by 25(OH)D depends on the activity of CYP27B1/1α-hydroxylase. The finding that hMSCs are a both a producer and target of 1α,25(OH)(2)D suggests a potential autocrine/paracrine role of vitamin D metabolism in osteoblast differentiation. Expression and enzyme activity of CYP27B1/1α-hydroxylase are upregulated by substrate 25(OH)D and Parathyroid Hormone (PTH) and are downregulated by 1α,25(OH)(2)D. With subject age, there are decreases in basal osteoblast potential and in stimulation of osteoblastogenesis by 1α,25(OH)(2)D, 25(OH)D, and PTH. In vitro treatment with a combination of 25(OH)D and PTH rejuvenated osteoblastogenesis with hMSCs from elders; this was attributable to increases in CYP27B1/1α-hydroxylase and in receptor for each hormone by the reciprocal factor. Other clinical variables beside age, i.e. low serum 25(OH)D or low estimated glomerular filtration rate, are correlated with reduced osteoblastogenesis. These studies suggest that osteoblastogenesis may not be optimal unless there is sufficient serum 25(OH)D substrate for hMSCs to synthesize and respond to local 1α,25(OH)(2)D.

Figure 1. Ficoll isolation method for hMSCs

Discarded human bone and marrow tissues are minced in PBS with EDTA to release cells. After passage through a sieve to remove bone particles, cells are collected by centrifugation and resuspension in PBS. Ficoll-Histopaque 1077 (FH-1077, a medium with density of 1.077 g/mL) is carefully added beneath the aqueous cell suspension. Centrifugation separates marrow cells into three major fractions. The fat cells float at the top, and the mature, differentiated erythrocytes and leukocytes sediment at the bottom. Low-density undifferentiated cells accumulate at the interface between the aqueous and FH-1077 layers. This fraction contains progenitors for both hematopoietic and mesenchymal cells. They are collected, washed, and seeded into tissue culture dishes. After 2–3 days incubation, the non-adherent hematopoietic lineage cells, which includes osteoclast progenitors, are removed, allowing the adherent stromal cells to expand. The enriched adherent cells are called hMSCs, which are positive for CD90, CD105, CD166, and STRO-1, and negative for CD34 and CD45.

Figure 2. Vitamin D metabolism, regulation, and action in hMSCs

Vitamin D₃ (cholecalciferol) is hydroxylated at carbon-25 by CYP27A1 to 25(OH)D₃ (calcidiol), which downregulates CYP27A1 and upregulates CYP27B1 and, at higher concentrations, upregulates CYP24A1. In a dose-dependent manner, 25(OH)D₃ is hydroxylated at carbon-1α by CYP27B1 to 1α,25(OH)₂D₃ (calcitriol). Higher concentrations of 1α,25(OH)₂D₃ downregulate CYP27B1 and upregulate CYP24A1. Both 25(OH)D₃ and 1α,25(OH)₂D₃ upregulate IGF-I which mediates their stimulation of osteoblast differentiation in hMSCs. These findings indicate an autocrine/paracrine role for vitamin D metabolism in human osteoblastogenesis in hMSCs.

Figure 3. Summary of the interactions among PTH, IGF-I, and 1,25(OH)₂D₃ on CYP27B1/1α-hydroxylase expression and bone formation in hMSCs

PTH upregulates IGF-I [12] which stimulates bone formation in hMSCs [8]. PTH upregulates CYP27B1 expression and stimulates 1α,25(OH)₂D₃ production [12], and at higher concentration, 1α,25(OH)₂D₃ downregulates CYP27B1 [8]. In addition, IGF-I upregulates CYP27B1 expression and stimulates 1α,25(OH)₂D₃ biosynthesis [8]. 1α,25(OH)₂D₃ induces IGF-I and bone formation in hMSCs [8].