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

Abstract

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.

Potential of Human Fetal Chorionic Stem Cells for the Treatment of Osteogenesis Imperfecta

“…Proteins were precipitated with 10% TCA, resuspended in RIPAE buffer (1 · TBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and 0.004% sodium azide; Sigma) containing PMSF (Sigma) and protease inhibitor cocktail, run on an 8% SDS-PAGE, transferred to nitrocellulose, blocked with milk, and stained with a COL1A2 (129 kDa) primary antibody (Abcam), then with an HRPlinked anti-rabbit IgG secondary antibody (GE Healthcare), followed by enhanced chemiluminescence detection (Thermo Scientific). The loading control used was b-ACTIN (43 kDa) (Santa Cruz) [23]. Detection of COL1A2 in transplanted oim bones was confirmed using wild-type positive controls and specificity confirmed using nontransplanted oim negative controls (n = 4 per group).…”

“…Collection of human fetal early chorionic stem cells (e-CSC) was as previously described [23] from first trimester chorionic villous tissue sampled during pregnancy termination (9-10 weeks gestation age) as approved by the Research Ethics Committee of Hammersmith and Queen Charlotte's Hospital. Isolated cells were plastic adherent and cultured in Dulbecco's modified Eagle's medium high glucose (DMEM-HG) (Sigma) supplemented with 10% fetal bovine serum (BioSera), 2 mM l-glutamine, 50 IU/mL penicillin, and 50 mg/mL streptomycin (Gibco-BRL) (D10 medium).…”

“…For unconjugated antibodies, cells were subsequently washed with 1% BSA and incubated with secondary goat anti-murine IgM PE (Santa Cruz) [23]. Otherwise cells were analyzed by FACS calibur flow cytometry (Becton Dickinson) using hESC as positive and antibody-specific isotypes as negative controls.…”

“…Cells were differentiated along the adipocyte lineage over 2 weeks in DMEM supplemented with 0.5 mM hydrocortisone, 0.5 mM isobutyl methylxanthine, and 60 mM indomethacin, then fixed and stained with oil red O [23]. Cells were differentiated along the chondrocyte lineage over 2 weeks in DMEM-LG supplemented with 0.01 mg/mL TGF-b3, 0.1 mM dexamethasone, 0.17 mM ascorbic acid, 1 mM sodium pyruvate, 0.35 mM l-proline, 1% ITSS, and 50 mg/mL linoleic acid (reagents from Sigma), then cells were fixed in and stained with alcian blue (2%).…”

“…Recently, we have shown human fetal early chorionic stem cells (e-CSC) isolated from human placental tissue accelerated tissue repair in dermal excision skin wounds and improved bone quality and plasticity in oim mice. This tissue repair capacity of e-CSC was greater than its late gestation counterparts in vivo, as was the osteogenic differentiation and cell expansion potential in vitro [23]. This may be due to the more primitive characteristics of e-CSC compared to term isolated CSC, which showed an intermediate phenotype between human embryonic stem cells (hESCs) and MSC [23].…”