Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells

Abstract

Marrow stromal cells are adult stem cells from bone marrow that can differentiate into multiple nonhematopoietic cell lineages. Previous reports demonstrated that single-cell-derived colonies of marrow stromal cells contained two morphologically distinct cell types: spindle-shaped cells and large flat cells. Here we found that early colonies also contain a third kind of cell: very small round cells that rapidly self-renew. Samples enriched for the small cells had a greater potential for multipotential differentiation than samples enriched for the large cells. Also, the small cells expressed a series of surface epitopes and other proteins that potentially can be used to distinguish the small cells from the large cells. The results suggested it will be important to distinguish the major subpopulations of marrow stromal cells in defining their biology and their potential for cell and gene therapy.

Figure 1

Phase-contrast photomicrograph of two different colonies of human MSCs 6 days after the cells were plated at three cells/cm². Arrows indicate one large mMSC and three doublets of recently replicated RS cells. (×200.)

Figure 2

(A–C) Phase-contrast photomicrographs of a dividing RS cell taken at intervals of 20 min. (D) Electron micrographs of two RS cells. (E) Electron micrograph of an mMSC. As indicated, the mMSCs were vacuolated and frequently binucleate. (Bar = 5 μm.)

Figure 3

Time course for the expansion of cells and alkaline phosphatase activity in cultures of MSCs. MSCs were plated at an initial density of about 3 cells/cm² in 75-cm² plates.

Figure 4

Assays for differentiation. (A) Cell preparations enriched for RS cells incubated in osteogenic medium for 21 days (21). The mineral in the cultures was detected by staining with Alizarin red. (B) Cell fraction enriched for mMSCs incubated in osteogenic medium for 21 days. (C) Cell fraction enriched for RS cells incubated in adipogenic medium for 21 days (21). Fat droplets in the cells were stained with Oil Red O. (D) Cell fraction enriched for mMSCs incubated in adipogenic medium for 21 days. (E) Cell fraction for RS cells incubated under chondrogenic conditions for 21 days (21). Proteoglycans in paraffin sections of the pellet were stained with Safranin O. (F) Cell fraction enriched for mMSCS incubated under chondrogenic conditions for 21 days. Paraffin sections of the pellet were stained with Safranin O.