Marrow stromal cells as a source of progenitor cells for nonhematopoietic tissues in transgenic mice with a phenotype of osteogenesis imperfecta

Abstract

Marrow stromal cells from wild-type mice were infused into transgenic mice that had a phenotype of fragile bones resembling osteogenesis imperfecta because they expressed a human minigene for type I collagen. In mice that were irradiated with potentially lethal levels (700 cGy) or sublethal levels (350 cGy), DNA from the donor marrow stromal cells was detected consistently in marrow, bone, cartilage, and lung either 1 or 2.5 mo after the infusions. The DNA also was detected but less frequently in the spleen, brain, and skin. There was a small but statistically significant increase in both collagen content and mineral content of bone 1 mo after the infusion. Similar results were obtained with infusion of relatively large amounts of wild-type whole marrow cells into the transgenic mice. In experiments in which male marrow stromal cells were infused into a female osteogenesis imperfecta-transgenic mouse, fluorescense in situ hybridization assays for the Y chromosome indicated that, after 2.5 mo, donor male cells accounted for 4-19% of the fibroblasts or fibroblast-like cells obtained in primary cultures of the lung, calvaria, cartilage, long bone, tail, and skin. In a parallel experiment in which whole marrow cells from a male mouse were infused into a female immunodeficient rag-2 mouse, donor male cells accounted for 4-6% of the fibroblasts or fibroblast-like cells in primary cultures. The results support previous suggestions that marrow stromal cells or related cells in marrow serve as a source for continual renewal of cells in a number of nonhematopoietic tissues.

Figure 1

ALP activity in mouse MSCs. Cells were cultured, and ALP activity per μg protein was assayed as described in text. Values are means ± SD (n = 3).

Figure 2

PCR assay for ratio of DNA from the endogenous murine Col1a1 gene (mCol1a1) to the mCol1a1 gene plus the human mini COL1A1 gene (hCol1a1). (Upper Left) Standard curve prepared with mixtures of DNA from tails of wild-type and OI-transgenic mice. Values are means ± 2 SD of three separate assays in duplicate (n = 6). (Lower Left) Assay of tail DNA from an OI-transgenic mouse. The PCR products were separated by using 7% PAGE containing 7 M urea, and the image on a phosphor storage plate was scanned. The human mini-COL1A1 is present in a high copy number (≈100). (Upper Right) Assay of marrow DNA from an OI-transgenic mouse that was marrow-ablated with 700 cGy/min and infused with 12 × 10⁶ wild-type MSCs 1 mo earlier. (Lower Right) Assay of bone DNA from the same OI-transgenic mouse as shown in Upper Right.

Figure 3

FISH assay for Y chromosome and immunofluorescence for type I collagen in primary cultures of cells. Wild-type MSCs (11 × 10⁶) from an isogenic male mouse together with WMCs (2 × 10⁶) from an OI-transgenic mouse were infused into a 3-week-old female OI-transgenic mouse that was marrow ablated (Experiment 1 in Table 4). After 2.5 mo primary cultures were prepared. (Upper Left) Primary cultures from skin assayed by FISH. Cell at Upper Left contains a Y chromosome. (Upper Right) Primary culture of tail from same mouse. Cell in Upper Left contains the Y chromosome. (Lower Left) Primary culture of tail from a control male mouse. (Lower Right) Cells from same primary culture of skin as in Upper Left stained with antibodies to type I collagen.