Clonal analysis and hierarchy of human bone marrow mesenchymal stem and progenitor cells

Abstract

Objective: This study was performed to assess adult human bone marrow mesenchymal stem/progenitor cells at a single-cell level and to determine a hierarchy based on proliferative potential.

Materials and methods: Adult bone marrow mesenchymal cells expressing the enhanced green fluorescent protein (EGFP) were sorted as single cells into 24-well plates, each well confirmed with single EGFP-positive cells by fluorescence microscopy, and counted every 3 days. Colonies derived from single cells were expanded then sorted and evaluated using established differentiation protocols for adipogenic, chondrogenic, and osteogenic lineages. Cells were further analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) (peroxisome proliferator-activated receptor[PPAR]-gamma2, LEP, LPL, LUM, COMP, BIG, RUNX2, IBSP, BGLAP) and immunocytochemistry (PPAR-gamma1/2, collagen II, bone sialoprotein II) specific for trilineage differentiation.

Results: Bone marrow mesenchymal cells were found to contain high proliferative potential (HPP) mesenchymal colony-forming cells (MCFC) (7%), low proliferative potential (LPP) MCFC (29%), mesenchymal cell clusters (MCC, 26%), and mature mesenchymal cells (MMC, 38%). All LPP-MCFC, MCC, and MMC colonies reached senescence at the end of the evaluation period. However, HPP-MCFC continued to grow, showed differentiation toward all three lineages, and demonstrated the capacity to give rise to secondary HPP-MCFC upon replating at a clonal level.

Conclusion: These findings suggest that there is a low frequency of bone marrow-derived HPP-MCFC that can both self-renew at a single-cell level and differentiate toward multiple lineages of mesenchymal origin.

Figure 1. Plating Protocol

The overall plan for cell plating is shown.

Figure 2. Fluorescence images of mesenchymal cell growth

Images of EGFP expressing cells obtained on day 1 (A, E, I, M) post-sorting, day 3 (B, F, J, N), day 6 (C, G, K, O), and day 9 (D, H, L, P). Four distinct cell populations derived from single mesenchymal cells included high proliferative potential-mesenchymal colony forming cells (HPP-MCFC; A–D), low proliferative potential-mesenchymal colony forming cells (LPP-MCFC; E–H), mesenchymal cell clusters (MCC; I–L), and mature mesenchymal cells (MMC; M–P) which were observed over time in culture. Images shown in each row were taken from the same well over time.

Figure 3. Growth of single mesenchymal cells

Single fluorescent mesenchymal cells expressing EGFP (green, insert) were sorted into 24-well plates with irradiated mesenchymal cells as feeders (EGFP-negative). The insert demonstrates the presence of two EGFP-expressing cells (day 3) within the irradiated feeder cells. EGFP-positive cells were counted every 3 days for 12 days. Wells containing ≥2 cells were excluded from the study. Single HPP-MCFC showed a significantly higher growth rate compared to LPP-MCFC, MCC, or MMC (p<0.05). All LPP-MCFC, MCC, and MMC reached senescence, whereas HPP-MCFC continued to proliferate. *p<0.05.

Figure 4. Differentiation of mesenchymal cell clonal colonies

Colonies of fluorescent HPP-MCFC were differentiated toward adipogenic (A, D), osteogenic (B, E), and chondrogenic (C, F) lineages. Adipogenic-differentiated cells were stained with Oil Red-O (red channel only, A, 10X objective) and co-expressed PPAR γ1/γ2 (red, D, 40X objective) with the EGFP-HPP-MCFC. Osteogenic-differentiated cells were stained with Von Kossa (red channel only, B, 10X objective) and co-expressed BSP (red, E, 20X objective) with the EGFP-HPP-MCFC. Sections of chondrogenic-differentiated pellets were stained with Safranin-O (red channel only, C, 10X objective) and co-expressed Collagen II (red, F, 20X objective) with the EGFP-HPP-MCFC. No staining (other than the EGFP of the HPP-MCFC progeny) was observed in secondary antibody controls (G–I). Blue = DAPI staining of nuclei.

Figure 5. PCR analysis of differentiated HPP-MCFC

Genes specific for adipogenic (A), chondrogenic (B), and osteogenic (C) lineages were analyzed after sorting EGFP-positive differentiated HPP-MCFC from feeder mesenchymal cells (fdMC, EGFP-negative). Significantly greater levels of adipogenic (A) and osteogenic (C) gene expression were observed in HPP-MCFC compared to fdMC (p<0.05). This effect was not as profound for chondrogenic (B) differentiation when compared to the other two lineages. *p<0.05.

Figure 6. Growth of single mesenchymal cells after sorting

HPP-MCFC from the initial sorting of single cells were pooled, sorted, and monitored for 12 days. The percentage of HPP-MCFC (>9 population doublings) and MMC (≤3 population doublings) appeared to increase from the first sort to the second sort, while LPP-MCFC (>6–9 population doublings) and MCC (>3–6 population doublings) were observed to decrease.

Figure 7. Cumulative growth of HPP-MCFC

Single HPP-MCFC proliferated to 3.8×10⁶ ± 6.6×10⁵ cells over 24 days.