MSC cultures are heterogeneous
Rat MSC obtained from standard purification methods and maintained in adherent culture consist of a morphologically heterogeneous population. Two major subpopulations can be observed: (a) Cells with an elongated, spindly shape with two processes that extend in opposite directions from the cell body (), and (b) polygonal cells with or without short processes (). In some previous studies (15
), spindle-shaped cells have been classified as “immature,” while polygonal cells have been characterized as “mature.” Colter et al. (15
) used flow cytometry to identify smaller spindle-shaped cells as the “RS1” and “RS2” rapidly-cycling, multipotent phenotypes. Since the correlation between cell shape and stem-cell characteristics has yet to be definitively determined, we describe the two subpopulations simply as “spindle-shaped” and “flat”.
MSC cultures contain morphologically distinct cell populations
Growth patterns and growth kinetics of heterogeneous MSC depend on initial plating density
MSC were plated at different densities, and expanded through passage 5 (20 cells/cm2) or passage 10 (200 and 2000 cells/cm2), with replicate cultures counted each day. MSC plated at 20 cells/cm2 had completed 30.6 population doublings by the end of passage 5 (45 days in culture) with a population doubling time of 35.3 hours. MSC plated at 200 cells/cm2 grew much faster, completing 44.9 population doublings over 10 passages (50 days in culture) with a population doubling time of 26.7 hours. However, a further step up in plating density led to decreased growth. Cells plated at 2000 cells/cm2 displayed a population doubling time of 40.7 hours over 16.5 population doublings (10 passages; 28 days in culture).
From the perspective of rapidly expanding an initial population of adherent MSC, the plating density of 200 cells/cm2 was clearly superior to sparser and denser choices. Cells at this density completed 16 doublings in 65% of the time (19 days) needed for MSC cells plated at higher or lower densities ().
MSC plating density effects growth kinetics across multiple passages
The effect of plating density on MSC growth patterns was also observed during expansion. MSC plated at 20 cells/cm2 grew into few large, very dense colonies. The more numerous colonies formed by cells plated at 200 cells/cm2 were smaller and less dense. MSC plated at 2000 cells/cm2 grew evenly on the plastic ().
Population doubling times vary between heterogeneous and homogeneous cultures
To analyze the differences in growth kinetics between heterogeneous MSC and distinct populations of spindle-shaped or flat MSC, clones were prepared from heterogeneous MSC at passage 2 using the limiting dilution technique. However, distinctions were not absolute. A small percentage of flat cells was observed in populations expanded from a single spindle-shaped MSC. Conversely, small percentages of spindle-shaped cells were observed in populations that had been expanded from a flat MSC founder.
Heterogeneous MSC cultures from passage 5 expansion cultures seeded at 20, 200 and 2000 cell/cm2 were plated and grown for 14 days in culture without passage. At the start of the experiment, cells seeded at 20 cells/cm2 were at PDL 30.6; cells seeded at 200 cells/cm2 were at PDL 19.6, and cells seeded at 2000 cell/cm2 were at PDL 6.9.
Spindle-shaped and flat MSC clones were plated at all three seeding densities and also grown for 14 days without passage. At the start of the experiment, the spindle-shaped clones were at PDL 30.9, the flat clones at PDL 27.2 ().
Growth of MSC at Passage 5 for 14 days.
Heterogeneous MSC cultures exhibited a log phase that lasted 5, 4 and 4 days for MSC plated at 20, 200 and 2000 cells/cm2, respectively. The population doubling times exhibited by these cultures during the log phase were 16.2 hours for those plated at 20 cells/cm2, 15.2 hours for cells plated at 200 cells/cm2 and 16.6 hours for cells plated at 2000 cells/cm2. During the two weeks allotted to this experiment, MSC plated at 20 cells/cm2 completed 10.5 population doublings, cells plated at 200 cells/cm2 underwent 8.3 population doublings, and MSC plated at 2000 cells/cm2 had a total of 6.0 population doublings (, ).
Growth kinetics of heterogeneous cultures and cloned spindle-shaped and flat cultures
Cloned cells grew at slower rates. Spindle-shaped MSC clones plated at 20 cells/cm2 proliferated in the log phase from day 1 through day 7 at an average population doubling time of 20.7 hours, completing 9.5 population doublings over 14 days. Spindle-shaped clones plated at 200 cells/cm2 showed exponential growth from day 1 until day 7 with an average population doubling time of 22.1 hours reaching approximately 8.1 population doublings. Spindle-shaped MSC clones plated at 2000 cells/cm2 demonstrated exponential growth until day 4 and had an average population doubling time of 24.0 hours, completing only 4.8 population doublings over 14 days in culture (, ).
The population doubling time of flat MSC plated at 20 cells/cm2 was 24.8 hours during the log phase and reached the stationary phase at day 9. These MSC completed 9.3 population doublings over 14 days. Flat MSC plated at 200 cells/cm2 exhibited a doubling time of 25.6 hours during the log phase, entered the stationary phase on day 8 and completed approximately 8.9 population doublings by day 14. The log phase of MSC flat clones plated at 2000 cells/cm2 lasted until day 5. During the log phase flat clones had a population doubling time of 20.3 hours and the total number of population doublings was 6.9 (, ).
Time in culture increases percentage of flat cells independent of initial plating density
In order to determine how plating density, time in culture and passage affect MSC morphology, heterogeneous MSC from passages 2, 5 and 10 were plated at 20, 200 and 2000 cells/cm2 and imaged each day until cells were confluent. MSC were classified as either spindle-shaped or flat; data for each phenotype is presented as the percentage of total MSC (). Data are presented from day one after plating, from the day on which cultures were 50% confluent, and from the first day cells were confluent.
Proportion of MSC with flat morphology increases over time in culture regardless of plating density
MSC from passage 2 showed an 80% or better percentage of spindle-shaped MSC on the day after plating at all three seeding densities. The majority of MSC plated at 20 cells/cm2 presented a flat morphology on day 7. MSC plated at 200 cells/cm2 maintained a 60% majority of spindle-shaped cells at day 5, although flat cells had come to dominate the culture (80%) by the time confluence was achieved on day 9. Throughout the culture of the most densely seeded plates (2000 cells/cm2), spindle-shaped cells dominated.
When later-passage MSC were used to found the cultures, a higher proportion of flat cells was generally present. At passage 5, 70% of MSC plated at all three densities were spindle-shaped on day 1. By 50% confluence, that percentage had slipped to between 40% and 50% for the three densities. In all cases, by the final day of imaging less than 10% of MSC displayed spindle-shaped morphology.
Finally, at passage 10, 80% of the MSC plated at 20 cells/cm2 were spindle-shaped on day 1, whereas MSC plated at 200 and 2000 cells/cm2 consisted of approximately 50% spindle-shaped cells. Similar to passage 5 MSC, fewer than 20% of cells were spindle-shaped by the time the cultures attained confluence.
Taken together, these data suggest that over time, regardless of plating density, the proportion of spindle-shaped MSC decreased.
The history of plating density does not affect mesenchymal potential
To determine if the mesenchymal differentiation potential of MSC was altered by plating density or by our cloning procedures, chondrogenic, adipogenic and osteogenic differentiation assays were used to induce differentiation in heterogeneous MSC from passage 5 plated at 20, 200 and 2000 cells/cm2, and in cloned populations. In the chondrogenesis assay, increased pellet size, Type II collagen secretion, and positive Safranin O staining indicates differentiation. Chondrogenesis was evident in MSC plated at all three densities, as shown by positive Safranin O staining (). The pattern of Type II collagen staining corresponded with Safranin O (data not shown). MSC that had consistently been plated at 20 and 200 cells/cm2 produced pellets greater than 2 mm in diameter (), and those plated at 2000 cells/cm2 produced a pellet greater than 1 mm in diameter (). Spindle-shaped MSC produced a similarly-sized pellet and were positive for Safranin O staining (). Only flat MSC produced a pellet without evidence of chondrogenesis (). Thus, high initial plating density does not appear to hinder the ability of MSC to undergo chondrogenic differentiation. However, flat MSC appear to have diminished capacity for chondrogenesis.
Chondrogenic and adipogenic potential is limited in MSC with flat morphology, but is unaffected by plating density
The ability to differentiate to fat was evaluated by analysis of Oil Red O staining after induction with adipogenic medium. MSC maintained in regular growth medium were used as controls; all control wells were negative for Oil Red O staining. Surprisingly, MSC plated at 20 cells/cm2 were also negative for Oil Red O staining (). These assays may have failed because MSC did not reach confluency, which is normally required for adipogenic differentiation. MSC plated at 200 and 2000 cells/cm2 were positive for Oil Red O staining ().
When cloned MSC were assayed for adipogenesis, spindle-shaped MSC clones showed positive Oil Red O staining (). In contrast, flat MSC were negative for adipogenesis (). Thus, the adipogenic potential of flat MSC appears to be limited.
Osteogenesis of MSC was evaluated by scoring calcium deposition after induction with osteogenic medium. Approximately 20 µg of calcium per confluent 35-mm dish is indicative of low levels of mineralization, similar to that which has been observed with fibroblasts. Untreated MSC display about 30 µg calcium. The production of over 40 µg of calcuim is considered a positive result in this in vitro assay. All tested cultures yielded calcium deposition greater than 40 µg in response to treatment with osteogenic medium. MSC from passage 5 plated at 20 cells/cm2 yielded 53 µg, at 200 cells/cm2 46 µg and at 2000 cells/cm2 43 µg calcium. Spindle-shaped and flat MSC both yielded 54 µg calcium.