Table 1.
Primary and secondary antibody concentrations for; Immunocytochemistry (ICC) and Fluorescence Assisted Cell Sorting (FACS).
Fig 1.
Expansion and morphology of hMSC populations.
A: Growth curve of hMSC populations. Five different hMSC populations were expanded for more than 20 passages, 60 population doublings and 60 days in culture. Population doublings (PD) were calculated using the formula: PD = log [A/(BC)]/log2; where A was the number of collected cells, B was the number of plated cells and C was the attachment efficiency [41]. The five populations examined displayed similar patterns of growth allowing selection of three populations for ongoing experiments. Viability remained greater than 70% for all populations over this time. P+: passage; coloured lines represent each of three different hMSC populations selected for continuing experiments; red: hMSC-19604 (hMSC-1), green: hMSC-20176 (hMSC-2), blue: hMSC-21558 (hMSC-3). Representative phase contrast images of hMSC populations at B: P+5 C: P+7 (D) P+13 chosen to represent each growth phase (Phase A-C). Images shown at 10X magnification. Scale bar represents 70μm.
Table 2.
Total days in culture and population doublings (PD) for individual hMSC populations.
Total number of days in culture and population doublings calculated for each population at each passage representing each phase of growth. Average days in culture and population doublings for the three hMSC populations examined are presented.
Fig 2.
hMSC populations are positive for markers of MSC stemness.
ICC showing positive staining for each of the five markers used to confirm hMSC stemness. Each of three hMSC populations was stained for each marker at each phase of growth. Cells are positive for: A-C: goat anti-mouse-CD44 (1/1000); D-F: goat anti-rabbit-CD29 (1/200); G-I: goat anti-mouse-CD90 (1/500); J-L: goat anti-mouse-CD105 (1/500); and negative for M-O: goat anti-rabbit-CD45 (1/500) at Phase A, Phase B and Phase C. At Phase C populations also become positive for CD45. Secondary antibodies used were anti-mouse-Chromeo546 (yellow; 1/1000) and anti-rabbit-Chromeo488 (green; 1/1000). Cells were mounted in mounting media containing DAPI (blue) to counterstain nuclei. Scale bar represents 70μm.
Table 3.
Mesenchymal markers examined by FACS.
Percentage of the cells positive for each MSC marker examined in MSCS at Phases A-C. Average percentage was calculated between the hMSC populations examined.
Fig 3.
Mesenchymal lineage markers expressed in hMSC by FACS and Western Blotting.
Detection by FACS demonstrates positive expression of three hMSC markers. Populations are more than 95% positive for A-C: CD29 and D-F: CD44. G-I: In addition, hMSC populations are more than 80% positive for the negative marker CD45 at each of the three passages examined during expansion. Dark grey histogram is the marker of interest. Light grey histogram is the secondary antibody only control. Insert C: The MSC marker CD29 (120-130kDa is present in hMSCs during all growth phases when examined by WB. GAPDH was used as a loading control.
Fig 4.
Adipogenic and osteogenic differentiation of MSCs at each passage during expansion.
A: Phase contrast images of differentiated adipogenic and osteogenic cultures showing phenotypic changes as well as lipid droplet accumulation and mineralisation of the cultures. WB analysis demonstrating protein detection of MSC markers CD29, PPARG and COL1A1 in undifferentiated hMSC at each growth phase in B: differentiated Adipogenic cultures and C: Osteogenic cultures. GAPDH was used as the loading control. Scale bar represents 60μm.
Fig 5.
Gene expression changes of MSC cell surface markers and transcription factors.
Several specific cell surface markers (CD14, CD26, CD54, ITGA3) and transcription factors (ZFX, PPARG, MOSPD1, NKX3-2) examined in extended hMSCs demonstrate increased expression in late growth phases.
Fig 6.
Relative gene expression of MSC lineage markers in hMSC populations throughout growth phases.
Relative gene expression changes for A: Stemness markers (CD44); lineage specific B-E: adipogenic (C/EBPa, C/EBPd, PPARG1/2); F-H: Fibroblastic (ACTA2, FN1, FSP1); and I-L: osteogenic (AP, COL1A1, BSPII, OCN) in hMSCs (n = 3) at each passage. The majority of genes examined (lineage and stemness) increased expression at Phase B followed by decreased expression by Phase C (ACTA2, CD44, C/EBPd, COL1A1, FN1, FSP1). In contrast, lineage specific markers osteocalcin; OCN, Alkaline Phosphatase; AP and PPARG1 demonstrated decreased expression throughout expansion with bone sialoprotein II; BSPII, C/EBPa and PPARG2 demonstrated increased expression throughout expansion.
Fig 7.
Neural transcription factors and cell surface CD marker expression.
Undifferentiated hMSCs express transcription factors and cell surface CD markers including stemness (CD73, CD271, HES1, KLF4, SOX5/6/9) and neuronal (CD24, CD56, CD200, CD304, MEF2C, PAX3/9) markers reported to influence neural differentiation. With the exception of CD73 and CD304, expressed at levels equal to CD44 (a cell surface marker of MSCs), these neural markers were expressed at levels 10-10000X lower than CD44.
Fig 8.
hMSCs are positive for neural markers throughout expansion.
Representative ICC images showing positive neural marker staining for A-F: neural markers in each of the three MSC populations examined at each of the three passages investigated. Cells stained positive for: A-C: neural stem cell markers, donkey anti-mouse-Nestin (1/300); and D-F: donkey anti-rabbit-Sox2 (1/1000); as well as G-I: lineage markers donkey anti-rabbit-glial fibrillary acidic protein (astrocyte; GFAP; 1/500); J-L: donkey anti-mouse-microtubule associated protein 2 (neuronal; MAP2; 1/300); and M-O: donkey anti-mouse IgM-O1 (oligodendrocyte; 1/500). Secondary antibodies anti-mouse-FITC (green; 1/1000), anti-rabbit-Cy3 (yellow; 1/1000), anti-mouse IgM-AlexaFluor594 (red; 1/500). Cells were mounted in mounting media containing DAPI (blue) to counterstain nuclei. Scale bar represents 70μm.
Fig 9.
Neural marker proteins examined by FACS and WB in MSCs during expansion.
Positive expression of neural markers is demonstrated. A-C: Populations average more than 30% positive for the neural stem cell marker, Nestin; and D-F: more than 50% positive for Sox2. Variation was observed between populations in percentages of the population positive for each maker, with the greatest variation seen at Phase B. Dark grey histogram is marker of interest. Light grey histogram is secondary antibody only control. G: Neural proteins TUBB3 and SOX2 were detected in undifferentiated hMSCs by WB during expansion.
Table 4.
Summary of NSC markers examined by FACS.
Percentage of the population positive for each NSC maker. Average percentage was calculated between the hMSC populations examined.
Fig 10.
Relative gene expression of neural stem cell and lineage specific markers in hMSC populations throughout growth phases.
A-D: Nestin, Nanog, OCT3/4 and SOX1 are markers of pluripotency in neural stem cells. Expression of these markers at each passage suggests the cells maintain pluripotency necessary for differentiating into different neural cell types. Expression of Nestin increased between Phase A and Phase B with a subsequent decrease in expression to Phase C. In contrast, both Nanog and OCT3/4 reduced expression at Phase B compared to Phase A and Phase C. Expression of each of these markers suggests the hMSC cultures maintain their ability to differentiate down each of the three neural lineages. Lineage specific markers examined include E-F: astrocyte markers GFAP and S100B; G-I: neuronal markers ENO2, MAP2 and TUBB3; J-L: Oligodendrocyte markers GalC, Olig1/2. Expression of MAP2 decreases over time, suggesting reduced ability to differentiate down a neural lineage. Expression of GFAP shows decreased expression at Phase B followed by a subsequent increase by Phase C. In contrast, TUBB3 expression increased between Phase A and Phase B cultures followed reduced expression at Phase C. These results may indicate optimal time points for differentiation to each of the neural lineages.
Fig 11.
Characterisation of Phase A neurospheres.
A: Phase contrast image (20x magnification, scale bar = 90 uM) of hMSC Phase A neurospheres B: FDA/PI stain indicating the presence of live (= green) and dead (= red) cells in hMSC neurospheres (40x magnification, scale bar = 90 uM). Bar graphs indicating C: the average sphere diameter for each hMSC population and D: the ratio of live/dead cells indicated by FDA and PI stain signal intensity. E: Q-PCR expression of self-renewal, neural and MSC lineage markers in undifferentiated Phase A hMSCs compared to neurospheres with significant differences in expression observed in multiple markers (*** p<0.001). F: ICC (40x magnification, scale bar = 90 uM) of Nestin, SOX2, CD44 and TUBB3 in neurospheres along with a bar graph indicating relative signal intensity of each of the markers when compared to DAPI.