Ontological Differences in First Compared to Third Trimester Human Fetal Placental Chorionic Stem Cells

Human mesenchymal stromal/stem cells (MSC) isolated from fetal tissues hold promise for use in tissue engineering applications and cell-based therapies, but their collection is restricted ethically and technically. In contrast, the placenta is a potential source of readily-obtainable stem cells throughout pregnancy. In fetal tissues, early gestational stem cells are known to have advantageous characteristics over neonatal and adult stem cells. Accordingly, we investigated whether early fetal placental chorionic stem cells (e-CSC) were physiologically superior to their late gestation fetal chorionic counterparts (l-CSC). We showed that e-CSC shared a common phenotype with l-CSC, differentiating down the osteogenic, adipogenic and neurogenic pathways, and containing a subset of cells endogenously expressing NANOG, SOX2, c-MYC, and KLF4, as well as an array of genes expressed in pluripotent stem cells and primordial germ cells, including CD24, NANOG, SSEA4, SSEA3, TRA-1-60, TRA-1-81, STELLA, FRAGILIS, NANOS3, DAZL and SSEA1. However, we showed that e-CSC have characteristics of an earlier state of stemness compared to l-CSC, such as smaller size, faster kinetics, uniquely expressing OCT4A variant 1 and showing higher levels of expression of NANOG, SOX2, c-MYC and KLF4 than l-CSC. Furthermore e-CSC, but not l-CSC, formed embryoid bodies containing cells from the three germ layer lineages. Finally, we showed that e-CSC demonstrate higher tissue repair in vivo; when transplanted in the osteogenesis imperfecta mice, e-CSC, but not l-CSC increased bone quality and plasticity; and when applied to a skin wound, e-CSC, but not l-CSC, accelerated healing compared to controls. Our results provide insight into the ontogeny of the stemness phenotype during fetal development and suggest that the more primitive characteristics of early compared to late gestation fetal chorionic stem cells may be translationally advantageous

Osteogenic, adipogenic and neurogenic differentiation of e-CSC and l-CSC.

<p>(<b>a</b>) Alizarin red staining (calcium deposits) and von kossa staining (mineralisation) of cells grown in osteogenic permissive media for 2 weeks. (<b>b</b>) Quantitative real time PCR of fold increased expression of osteogenic genes after osteogenic differentiation; osteopontin (OP), procollagen endopeptidase enhancer (PCE), osteocalcin (OC), osterix (OSX), bone sialo-protein-II (BSP) and bone morphogenic protein-2 (BMP2). (<b>c</b>) Western blot of COL1 for e-CSC and l-CSC not differentiated (non-diff) and differentiated to bone for 2 weeks (diff). Positive and negative controls shown. Loading control is β-actin. (<b>d</b>) Oil Red O staining (lipid droplets) of e-CSC and l-CSC grown in adipogenic permissive media for 3 weeks. (<b>e</b>) Quantitative real time PCR of fold increased expression of adipocyte differentiation regulator peroxisome proliferator-activated receptor gamma (PPARγ). (<b>f</b>) Morphology and expression of neuronal markers β-TUBULIN, MAP2 and NMDA receptor NR1 shown with FITC (green) of e-CSC and l-CSC after 5 days neurogenic differentiation. Nuclei stained with DAPI (blue). All quantitative real time PCR data normalised to GAPDH and basal expression levels of differentiation genes (i.e. 2^−ΔΔCt). e-CSC (black) and l-CSC (red). Data. * P<0.05; ** P<0.01, Student's <i>t</i> test, <i>n</i> = 3 per cell group. Mean ± s.e.m. All scale bars 100 µm.</p

e-CSC and l-CSC are of fetal origin.

<p>(<b>a</b>) Confocal immuno-fluorescence for c-KIT (<b>b</b>) Cell morphology of early and late gestation chorionic stem cells (e-CSC and l-CSC respectively) passage 4–5. (<b>c</b>) PCR for Y chromosome specific SRY. Positive and negative controls shown. (<b>d</b>) FISH analysis for X (FITC) and Y (Texas Red) chromosomes indicated with green and red arrows respectively. 100 cells were counted. All scale bars 100 µm.</p

e-CSC and l-CSC have a similar immunophenotype.

<p>(<b>a</b>) Flow cytometry for percent of e-CSC (black) and l-CSC (red) populations positive for surface adhesion markers; CD62P, Integrin β7, CD11a, CD106, CD51/61, CD49b, CXCR4, Integrin β3, CD49f, CD49d, αV Integrin and CD49e. (<b>b</b>) Percentage of e-CSC (black) and l-CSC (red) populations that adhered to fibronectin a-chemotryptic 40 kDa (Fn 40 kDa), fibronectin a-chemotryptic 120 kDa (Fn 120 kDa), whole fibronectin (Fn), collagen I (col I), collagen IV (col IV), human placenta laminin (laminin) and vascular cell adhesion molecule-1 (VCAM-1). Negative control DMEM alone (media). Data. * P<0.05, Student's <i>t</i> test, <i>n</i> = 3 per cell group. Mean ± s.e.m. (<b>c</b>) Confocal immuno-fluorescence for endothelial marker (CD14), hematopoietic markers (CD34 and CD45), MSC-associated markers (CD105, CD73 and CD44), matrix protein (vimentin) and markers found in pluripotent cells as well as MSC (CD29 and CD90) stained with FITC (green). Nuclei stained with DAPI (blue). Scale bar 100 µm. Positive controls are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043395#pone.0043395.s003" target="_blank">Fig. S3a</a>.</p

e-CSC have higher expression of pluripotency genes than l-CSC.

<p>(<b>a</b>) RT-PCR for OCT4A variant 1. GAPDH and positive and negative controls shown. (<b>b–f</b>) Quantitative real time PCR for (<b>b</b>) OCT4A all variants, (<b>c</b>) NANOG, (<b>d</b>) SOX2, (<b>e</b>) CMYC and (<b>f</b>) KLF4 for e-CSC (black) and l-CSC (red) as percentage expression of human embryonic stem cells (hESC). * P<0.05; *** P<0.001, Student's <i>t</i> test, <i>n</i> = 4 per cell group. Mean ± s.e.m.</p

e-CSC showed better tissue repair capacity <i>in vivo</i> compared to l-CSC.

<p>(<b>a</b>) Mechanical 3-point bending data in a mouse model of bone brittleness (<i>oim</i>) for overall bone quality shown by total energy input in millijoules to fracture and for (<b>b</b>) femoral plasticity shown by energy input in millijoules from yield to fracture. Results shown per mouse for non-transplanted <i>oim</i> (blue) and <i>oim</i> transplanted with e-CSC (black) and l-CSC (red). (<b>c</b>) Cross-section of skin wounds 7 days after 4 mm dermal biopsy in mice treated with either PBS alone (Control) or transplanted with e-CSC or l-CSC; stained with haematoxylin and eosin. Indicated is collagen (white arrow), granulated cells (black arrow) and epithelial thickness (dashed line). In the same model of wound repair, (<b>d</b>) number of granulated cells in the wound bed (per 100 µm²) and (<b>e</b>) epithelial thickness across wound in µm. Data. * P<0.05; ** P<0.01, n.s. (not significant), Student's <i>t</i> test. Mean ± s.e.m.</p

e-CSC are smaller in size and grow faster than l-CSC.

<p>(<b>a</b>) Image of cells in suspension used for cell size analysis. (<b>b</b>) Average cell size in µm of e-CSC (black) and l-CSC (red) when in suspension. (<b>c</b>) Average growth rate in hours taken for cell population to double during exponential growth phase; i.e. population doubling time (DT). e-CSC (black), l-CSC (red). (<b>d</b>) Cell expansion capacity over 1500 hours measured by average cumulative population doublings (Cumulative PD) of e-CSC (▴) and l-CSC (▾) when passaged at sub-confluence. (<b>e</b>) Cell kinetics measured by average cumulative population doublings of e-CSC (▴) and l-CSC (▾) when seeded at low density and grown beyond confluence for 288 hours. * P<0.05; ** P<0.01, Student's <i>t</i> test, <i>n</i> = 3 different samples per cell group. Mean ± s.e.m. All scale bars 100 µm.</p

e-CSC and l-CSC contain a subpopulation positive for pluripotency markers.

<p>(<b>a</b>) Representative flow cytometry (<i>n</i> = 3) for percent of cells positive for CD24, OCT4, SOX2, CMYC, SSEA4, SSEA3, TRA-1-60 and TRA-1-81 in e-CSC and l-CSC whole populations (isotype control in black). (<b>b</b>) Representative confocal immunofluorescence images for OCT4, SOX2, KLF4, NANOG, REX1, SSEA4, SSEA3, TRA-1-60 and TRA-1-81 stained with FITC (green). Positive cells indicated with green arrow, negative cells with white arrow. Nuclei stained with DAPI (blue). Scale bar 25 µm. Positive controls are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043395#pone.0043395.s003" target="_blank">Fig. S3b</a>.</p

e-CSC and l-CSC express markers found in primordial germ cells.

<p>(<b>a</b>) RT-PCR for expression of c-KIT, STELLA, FRAGILIS, NANOS3, SSEA1, VASA and GAPDH. (<b>b</b>) Flow cytometry for percent of e-CSC (black) and l-CSC (red) populations positive for STELLA, FRAGILIS, NANOS3, SSEA1, DAZL, PUM2, VASA, TNAP and BLIMP1. (<b>c</b>) DNA methylation status of imprinted gene <i>H19</i> as percent total input DNA hypermethylated (black), unmethylated (light grey) and intermediately methylated (dark grey). <i>n</i> = 3 per cell group.</p