Primordial germ cell-like cells differentiated in vitro from skin-derived stem cells

Abstract

Background: We have previously demonstrated that stem cells isolated from fetal porcine skin have the potential to form oocyte-like cells (OLCs) in vitro. However, primordial germ cells (PGCs), which must also be specified during the stem cell differentiation to give rise to these putative oocytes at more advanced stages of culture, were not systematically characterized. The current study tested the hypothesis that a morphologically distinct population of cells derived from skin stem cells prior to OLC formation corresponds to putative PGCs, which differentiate further into more mature gametes.

Methodology/principal findings: When induced to differentiate in an appropriate microenvironment, a subpopulation of morphologically distinct cells, some of which are alkaline phosphatase (AP)-positive, also express Oct4, Fragilis, Stella, Dazl, and Vasa, which are markers indicative of germ cell formation. A known differentially methylated region (DMR) within the H19 gene locus, which is demethylated in oocytes after establishment of the maternal imprint, is hypomethylated in PGC-like cells compared to undifferentiated skin-derived stem cells, suggesting that the putative germ cell population undergoes imprint erasure. Additional evidence supporting the germ cell identity of in vitro-generated PGC-like cells is that, when labeled with a Dazl-GFP reporter, these cells further differentiate into GFP-positive OLCs.

Significance: The ability to generate germ cell precursors from somatic stem cells may provide an in vitro model to study some of the unanswered questions surrounding early germ cell formation.

Figure 1. Morphology and AP activity of PGC-like cells differentiated from porcine skin-derived stem cells.

(A) Bright field images of (a) undifferentiated skin-derived stem cells maintained as spheres at passage 2 (100X) or (b) dissociated passage 2 skin stem cells after being plated in differentiation media for 24 hr in 60-mm dishes (100X). Loosely adherent or non-adherent putative PGCs at D20 of differentiation (c, d) could be distinguished from the somatic monolayer by their large size, round shape, and blebbing at (c) 100X and (d) 200X, respectively. (B) D20 differentiating cells, including a confluent monolayer with loosely adherent PGC-like cells, were fixed and stained for AP. Approximately 0.25% of the large, round PGC-like cells were AP-positive (a) compared to background levels associated with (b) the supporting somatic cell layer (100X). Size bar = 100 µm (Ad), 200 µm (Aa,b,c and B). (C) AP activity in undifferentiated skin-derived stem cells compared to D20 differentiating cells. A * denotes a statistical difference between the two groups (P<0.05). Absorbance values were normalized against total protein (mg/ml) and expressed as a percent relative to undifferentiated cells.

Figure 2. The detection of germ marker mRNA in PGC-like cells.

The expression of several marker genes known to be associated with PGCs was determined by real time RT-PCR. Relative quantification of (A) Oct4, (B) Fragilis, (C) Stella, (D) Dazl, (E) Vasa, and (F) c-Kit mRNA levels in D0 undifferentiated skin-derived stem cells and D20, D25, and D30 non-adherent PGC-like cells. Levels were normalized for the RpII housekeeper, and results are presented relative to D30 PGC-like cells. Data represent the mean±SEM of four independent experiments, with different letter subscripts denoting statistical differences between groups (P<0.05).

Figure 3. Detection of germ markers in PGC-like cells.

Immunolocalization of A) OCT4, B) VASA, D) STELLA, E) c-KIT, and F) DAZL in D30 PGC-like cells. White arrows indicate cells co-staining for OCT4 and VASA (A and B). Controls in which cells were probed with secondary antibody alone (either anti-rabbit or anti-mouse-FITC/PE) were negative (C and G, respectively). Counter-staining with Hoechst was conducted to detect nuclei. Panels are shown at 400X magnification (bar = 50 µm).

Figure 4. The DNA methylation profile in DMR1 of the porcine H19 gene locus.

Sodium bisulfite sequencing revealed that the percentage of unmethylated CpGs in (A) undifferentiated skin stem cells at passage 2, (B) undifferentiated skin stem cells at passage 5 (cultured for 25 days in the absence of differentiation media), and (C) D25 PGC-like cells was 81%, 76%, and 99%, respectively. (D) Porcine oocytes served as a control for the expected maternal imprint (98% unmethylated). Open and black circles represent unmethylyated and methylated cytosines, respectively.

Figure 5. PGC-like cells lentivirally transduced with the Dazl-GFP reporter give rise to OLCs.

Bright field and fluorescent images of a (A) representative aggregate and (B) an OLC derived from D25 PGC-like cells transduced with a Dazl-GFP lentivirus. (A) A GFP+ putative germ cell within an aggregate. bar = 100 µm. (B) After further differentiation, a GFP+ OLC was obtained (a, upper panel). OLCs generated from non-transduced PGC-like cells were GFP- (b, lower panel). bar = 50 µm. (C) Representative agarose gel images depicting the presence of Gfp mRNA in transduced OLCs (upper panel on the left). GFP+ OLCs also expressed Vasa, Oct4, and Gdf9b (panels on the right), confirming their germ cell identity. M: DNA marker, -ve: a reaction without RT enzyme.