Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow

Abstract

Cultures of plastic-adherent cells from bone marrow have attracted interest because of their ability to support growth of hematopoietic stem cells, their multipotentiality for differentiation, and their possible use for cell and gene therapy. Here we found that the cells grew most rapidly when they were initially plated at low densities (1.5 or 3.0 cells/cm(2)) to generate single-cell derived colonies. The cultures displayed a lag phase of about 5 days, a log phase of rapid growth of about 5 days, and then a stationary phase. FACS analysis demonstrated that stationary cultures contained a major population of large and moderately granular cells and a minor population of small and agranular cells here referred to as recycling stem cells or RS-1 cells. During the lag phase, the RS-1 cells gave rise to a new population of small and densely granular cells (RS-2 cells). During the late log phase, the RS-2 cells decreased in number and regenerated the pool of RS-1 cells found in stationary cultures. In repeated passages in which the cells were plated at low density, they were amplified about 10(9)-fold in 6 wk. The cells retained their ability to generate single-cell derived colonies and therefore apparently retained their multipotentiality for differentiation.

Figures

Figure 1

Size of colonies observed after plating of MSCs at 0.5, 1.5, 3.0, or 6.0 cells/cm2. After incubation for 14 days in 79-cm2 plates, the colonies were stained with 0.5% Crystal violet in methanol for 5–10 min at room temperature. Colonies from cells plated at 6 cells/cm2 are too small to be seen in the photograph.

Figure 2

Effect of plating density on yield of cells, and average doubling times. The cells were plated onto 79-cm2 plates, and yields of cells on days indicated were assayed with a hemocytometer. (A) Yield of cells per 100 cells plated with initial plating densities of 1.5 and 12.0 cells/cm2. (B) Average cell doublings per day when MSCs were plated at 1.5 and 12.0 cells/cm2. Values are 3-day averages from data presented in A.

Figure 3

FACS analysis of cells for size and granularity after different periods of incubation. Preliminary experiments indicated that the precursor–product relationships among the cell populations were more apparent in cultures that grew slowly (not shown). Therefore, the stock sample of MSCs selected for the experiment was previously shown to expand slowly in culture and have an unusually low cfu value of about 12% (see ref. 41). The cells were plated at a suboptimal density of 3 cells/cm2 that was equivalent to 1.0 cell/cm2 or less for samples that expanded more rapidly and had higher cfu values (see Fig. 1). (A) Analysis of cells in stationary culture at day 14. Lines indicate gating used to define RS-1 cells, RS-2 cells, and mMSCs. (B) Analysis on day 5 after initial plating. (C) Analysis on day 7 after initial plating. (D) Analysis on day 10 after initial plating. FSC, forward scattering of light that assays cell size; SSC, side scattering of light that assays cell granularity. Values in arbitrary units.

Figure 4

Time course of number of cells observed after initial plating of cells. Values are taken from the data presented in Fig. 3, adjusted for the total number of cells in the cultures.

Figure 5

Proportionality between percent of RS cells and percent of cfus in samples of MSCs. ▴, samples from cultures that were plated at varying density and incubated for 14 days; *, cultures from the same initial sample of MSCs incubated for 5 days (higher value) or for 14 days (lower value) after plating at 3 cells/cm2. % RS, total number of RS-1 and RS-2 cells in the samples; cfu efficiency, colonies per 100 cells plated.

Figure 6

Population doublings obtained when cells were plated at low density and high density. ▴, cells that were plated at 1.5 cells/cm2, incubated for 10 to 14 days; a large colony was then isolated by ring cloning for each subsequent passage under the same conditions; ■, MSCs from a sample with a comparable value for cfus that was passed by plating at a high density of 5,000 cells/cm2 (see ref. 41).

Figure 7

Scheme for the precursor–product relationships of cells in cultures of MSCs. As discussed in the text, the large mMSCs replicate poorly (2, 41). Therefore, the RS-2 cells that appear during the lag phase must arise from RS-1 cells (Fig. 4). During the early log phase of growth, the RS-2 cell decline in number as the mMSCs appear in large numbers. Therefore, the RS-2 cells are probably precursors of the mMSCs. However, the data do not completely exclude the possibility that RS-2 cells rapidly generate RS-1 cells, and the RS-1 cells then give rise to mMSCs (dashed arrow). Also, the earliest mMSCs probably continue to replicate. During the late log phase, the RS-2 cells decline in number, and the subpopulation of RS-1 expands. Therefore, the RS-2 cells probably recycle into RS-1 cells.