Reversible commitment to differentiation by human multipotent stromal cells in single-cell-derived colonies

Abstract

Objective: Human multipotent stromal cells readily form single-cell-derived colonies when plated at clonal densities. However, the colonies are heterogeneous because cells from a colony form new colonies that vary in size and differentiation potential when replated at clonal densities. The experiments here tested the hypothesis that cells in the inner regions of colonies are partially differentiated, but the differentiation is reversible.

Materials and methods: Cells were separately isolated from the dense inner (IN) regions and less-dense outer regions (OUT) of single-cell-derived colonies. Cells were then compared by assays of their transcriptomes and proteins, and for clonogenicity and differentiation.

Results: IN cells expressed fewer cell-cycle genes and higher levels of genes for extracellular matrix than the OUT cells. When transferred to differentiation medium, differentiation of the colonies occurred primarily in the IN regions. However, the IN cells were indistinguishable from OUT cells when replated at clonal densities and assayed for rates of propagation and clonogenicity. Also, colonies formed by IN cells were similar to colonies formed by OUT cells because they had distinct IN and OUT regions. Cultures of IN and OUT cells remained indistinguishable through multiple passages (30 to 75 population doublings), and both cells formed colonies that were looser and less dense as they were expanded.

Conclusions: The results demonstrated that cells in the IN region of single-cell-derived colonies are partially differentiated, but the differentiation can be reversed by replating the cells at clonal densities.

Figure 1. Expansion of a single-cell derived colony

Recovered passage 1 human MSCs (donor 5064) were plated at 0.5 cells per cm² and incubated for 12 days without medium change. Phase photomicrographs were taken approximately every 24 hours for 12 days. Location of single cell after 2 hour incubation was marked by right angle cuts on bottom of dish. Scale bar: 500 μm. Arrows: single cells after 2 hour and 1 day incubation.

Figure 2. Isolation and microarray assays of IN and OUT samples

MSCs (donor 5064) were incubated on laser microdissection slides at 2 cells per cm² for 12 days without medium change. Cells from IN and OUT were isolated with LMPC. The colonies in the figure were stained with crystal violet for illustrative purposes only. (A) Intact colony. (B) Colony after IN was captured. (C) Colony after IN and OUT were captured. (D) Heat map of microarray data from IN and OUT samples using 199 differentially expressed genes. Enriched GeneOntology terms with p values < 0.001 are shown for both clusters. On the heat map, red indicates upregulation and blue downregulation based on gene-wise standardized values. Three OUT samples (2OUT, 4OUT, 5OUT) and three IN samples (1IN, 2IN, 5IN) were used in the microarray assays. Scale bar in A, B, and C: 500 μm. Abbreviations: AURKB, aurora kinase B; CDC, cell division cycle; COL21A1, collagen type 21 alpha 1; ESPL, extra spindle pole bodies homolog; FGF; fibroblast growth factor; GPC, glypican; GTSE, G-2 and S-phase expressed; H2AFX, H2A histone family member X; IN, inner region of a colony; LMPC, laser capture microdissection and pressure catapulting; LAMA, laminin alpha; LUM, lumican; MKI67, antigen identified by monoclonal antibody Ki-67; MYB, v-myb myeloblastosis viral oncogene homolog; OUT, outer region of a colony; POLE, polymerase epsilon; SGOL, shugoshin-like; SPAG, sperm associated antigen; TACC, transforming acidic coiled-coil containing protein; THBS, thrombospondin.

Figure 3. Assays of mRNA from IN and OUT regions by microarrays and real-time RT-PCR

Total RNA was isolated from IN and OUT cells obtained with LMPC (donor 5064) or live cell isolation method (donors 7012 and 240). Genes shown have significant changes in their expression based on real-time RT-PCR. (A) Genes upregulated in the OUT sample (donor 5064). (B) Genes upregulated in the IN sample (donor 5064). (C) Genes up-regulated in the OUT region of all three donors (5064, 7012, and 240). (D) Genes up-regulated in the IN region of all three donors. Values are fold changes. Asterisk: changes not significant in the microarray assays (< 2-fold). Error bars: 95 % confidence intervals; n=3. Abbreviations: AGC, aggrecan; ANGPT, angiopoietin; AURKB, aurora kinase B; CCND, cyclin D; CTNNB, catenin beta; CXCL12, CXC chemokine ligand 12; DCN, decorin; DKK, dickkopf homolog; E2F7, E2F transcription factor 7; FGF, fibroblast growth factor; FZD, frizzled homolog; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GAS, growth arrest-specific; GDF, growth differentiation factor; GLB, galactosidase beta; GPC, glypican; GSN, gelsolin; HMMR, hyaluronan-mediated motility receptor; IL, interleukin; IN, inner region of a colony; ITGA, integrin alpha; LIF, leukemia inhibitory factor; LMPC, laser capture microdissection and pressure catapulting; LPXN, leupaxin; LUM, lumican; MCM, minichromosome maintenance deficient; MKI67, antigen identified by monoclonal antibody Ki-67; MMP, matrix metalloproteinase; NOTCH, notch homolog; OUT, outer region of a colony; PODXL, podocalyxin-like; RT-PCR, reverse transcriptase polymerase chain reaction; RARB, retinoic acid receptor beta; RUNX, runt-related transcription factor; SOCS, suppressor of cytokine signaling; SOX, sex determining region Y-box; STAT, signal transducer and activator of transcription; SYNPO, synaptopodin; SYTL, synaptotagmin-like; THBS, thrombospondin; VCAM, vascular cell adhesion molecule; VIL, villin; WNT, wingless-type MMTV integration site; WWTR, ww domain containing transcription regulator.

Figure 4. Immunocytochemistry of MKI67, PODXL, and VCAM1 in colonies

Three regions of MSC colonies from donor 5064 are shown. Nuclear counterstain: DAPI. Insets: enlarged regions of the same slides. Magnification: 40X, 100X (insets). Abbreviations: MKI67, antigen identified by monoclonal antibody Ki-67; PODXL, podocalyxin-like; VCAM, vascular cell adhesion molecule.

Figure 5. Differentiation of colonies

Representative colonies (donor 7012) after adipogenic or osteogenic differentiation. Colonies were fixed with formalin and stained with either Oil-Red-O (adipogenesis) or Alizarin Red S (osteogenesis). Under both conditions, differentiation initiated in the IN regions of the colonies. Scale bar: 1 mm.

Figure 6. Expansion and clonogenicity of isolated IN and OUT cells

MSCs (donors 5064, 7012, and 240) were plated at 2 cells per cm² and incubated for 10 to 12 days. Cells (passage 2) were isolated either from the OUT or the IN and re-plated at 100 cells per cm² for growth curve assay and at 2 cells per cm² for CFU-F assay. (A) Growth curve assay for donor 5064. Inset: Fold changes per day. (B) CFU-F assays of cells from IN and OUT of all three donors. Subcloning was continued for up to five cycles of isolation and re-plating (passage 7). Error bars: standard deviations; n=3. Abbreviations: CFU-F, colony forming unit fibroblast; IN, inner region of a colony; OUT, outer region of a colony; P, passage.

Figure 7. Changes in colony morphology upon subcloning

MSC colonies were stained with crystal violet, measured, and classified either as type I or type II. Type I colonies were at least 4 mm in size with a dense IN. Type II colonies were either less than 4 mm in size with dense IN or larger but loose. (A) Representative large and small type I colonies (upper panel) and type II colonies (lower panel) from one culture dish of donor 240 MSCs (passage 3). See Supplemental Figure 3 for densitometry of stained type I and type II colonies. Distribution of type I and II colonies during subcloning for donor: (B) 5064, (C) 240, and (D) 7012. Error bars: standard deviations; n=3. Scale bar: 1 mm. Abbreviations: CFU-F, colony forming unit fibroblast; IN, inner region of a colony; OUT, outer region of a colony; P, passage.

Figure 8. Colony size distribution upon subcloning

MSC colonies were divided into four groups based on their size (2.0-3.5 mm, 4.0-5.5 mm, 6.0-7.5 mm, and 8.0-9.5 mm). Size distribution of colonies derived from donor: (A) 5064, (B) 240, and (C) 7012. Error bars: standard deviations; n=3. Abbreviations: IN, inner region of a colony; OUT, outer region of a colony; P, passage.