Bone morphogenetic protein 4 mediates human embryonic germ cell derivation

Abstract

Human primordial germ cells (PGCs) have proven to be a source of pluripotent stem cells called embryonic germ cells (EGCs). Unlike embryonic stem cells, virtually little is known regarding the factors that regulate EGC survival and maintenance. In mice, the growth factor bone morphogenetic protein 4 (BMP4) has been shown to be required for maintaining mouse embryonic stem cells, and disruptions in this gene lead to defects in mouse PGC specification. Here, we sought to determine whether recombinant human BMP4 could influence EGC derivation and/or human PGC survival. We found that the addition of recombinant BMP4 increased the number of human PGCs after 1 week of culture in a dose-responsive manner. The efficiency of EGC derivation and maintenance in culture was also enhanced by the presence of recombinant BMP4 based on alkaline phosphatase and OCT4 staining. In addition, an antagonist of the BMP4 pathway, Noggin, decreased PGC proliferation and led to an increase in cystic embryoid body formation. Quantitative real-time (qRT)-polymerase chain reaction analyses and immunostaining confirmed that the constituents of the BMP4 pathway were upregulated in EGCs versus PGCs. Downstream activators of the BMP4 pathway such as ID1 and phosphorylated SMADs 1 and 5 were also expressed, suggesting a role of this growth factor in EGC pluripotency.

FIG. 1.

(A) Dose response in PGC number following 1 week in culture with either increasing doses of BMP4, equimolar concentrations of Noggin (50 ng/mL) plus BMP4 (20 ng/mL), or 50 ng/mL Noggin alone. PGCs were isolated by stage specific embryonic antigen (SSEA1) expression and equal numbers were seeded in a 96-well plate with irradiated SIM 6-thioguanine resistant ouabain (STO) mouse feeder cells. After 7 days, PGCs were counted live using SSEA1 and Alexa 488-secondary antibodies. Dose response in EGCs generated (B) after 1 week from PGCs in A and (C) after 3 weeks in subculture. Lower-case letters denote statistically significant differences among treatments (t-tests, P < 0.05; n = 8). (D) Indirect immunofluorescence demonstrating OCT4 expression (green) and (E) phase-contrast alkaline phosphatase expression colocalized in an undifferentiated human EGC colony. Scale bars: 150 μM. PGC, primordial germ cell; BMP, bone morphogenetic protein; EGC, embryonic germ cell. Color images available online at www.liebertonline.com/scd.

FIG. 2.

(A) The incident of cystic EB formation (the number of EBs per total number of colonies) decreases in the presence of BMP4 and significantly increases with the addition of Noggin after 1 week in culture. Dose response in cystic EB formation following 1 week in culture with either increasing doses of BMP4 or equimolar concentrations of Noggin (50 ng/mL) plus BMP4 (20 ng/mL). EGC differentiation is enhanced in the presence of BMP4 inhibitor Noggin. Cystic EB formation is enhanced after (B) 24 h and (C) 7 days following exposure to 50 ng/mL Noggin in culture compared with controls (insets D and E, respectively). Inset figures D and E represent a single colony grown in the presence of 20 ng/mL BMP4 alone after 24 h and 7 days. Lower-case letters denote statistically significant differences among treatments (t-tests, P < 0.05; n = 8). Indirect immunofluorescence of cryosectioned EBs demonstrate a fluid-filled center surrounded by cells labeled by markers of the (F) ectoderm (Nestin: red) and (G) endoderm (AFP: green) lineage but not mesoderm. STO feeder cells surrounding EBs do not express these markers. DAPI (blue) stains the nuclei. Scale bars: 150 μM. EB, embryoid body. Color images available online at www.liebertonline.com/scd.

FIG. 3.

Quantitative real-time qRT–polymerase chain reaction of BMP signaling components in human EGCs, ESCs, PGCs, and gonadal stromal cells (STR) cultured without BMP4. BMP receptors (Bmpr1A, Acvr1, Bmpr2), Smad1, Smad4, and Smad5 were enriched in EGCs compared with PGCs and stromal cells. In contrast, the activin receptor ACVR2A and the nodal receptor ACR2B were differentially expressed in these cells. Id1, a known BMP downstream activator, was also highly expressed in EGCs and undetectable in stromal cells. Samples were tested from 6 biological samples, 3 male and 3 female age-matched specimens, in triplicate. Data are represented as relative quantity log 10 with standard error of the mean; lower-case letters denote statistically significant differences between cell lines for each gene; P < 0.05. ESC, embryonic stem cell. Color images available online at www.liebertonline.com/scd.

FIG. 4.

(A–J) Indirect immunofluorescence detection of BMP4 pathway constituents in human EGCs cultured on mouse feeders without BMP4. A and B demonstrate expression of BMP4 type 2 but not the activin/nodal type 1 receptor ACVR1B in the same EGC colony. Colocalization is shown for 2 BMP4 type 1 receptors, (C) ACVR1A (alk2) and (D) ACVRL1 (alk1), which are utilized by BMP4 to maintain mouse ESC pluripotency [34], (E) BMP4 type 1 receptor, BMPR1A (alk3), and (F) SMAD1. Colocalization of (G) SMAD4, (H) SMAD5, (I) SMAD8, and (J) ID1 are demonstrated. (K–Q) Indirect immunofluorescence detection of BMP4 pathway constituents in human ESCs cultured on matrigel without BMP4. (K) Absence of BMP4 type 2 receptor. (L) Absence of ACVR1A (alk2). (M) Absence of BMP4 type 1 receptor, BMPR1A (alk3). Absence of expression of (N) SMAD1, (O) SMAD4, (P) SMAD5, and (Q) ID1 in ESCs is demonstrated. (R–T) Negative controls in EGCs. (R) Goat IgG. (S) Rabbit IgG. (T) Mouse IgG1. DAPI (blue) stains the nuclei. Scale bars: 50 microns or μm. Color images available online at www.liebertonline.com/scd.

FIG. 5.

Indirect immunofluorescence detection of the BMP pathway SMAD activation in human EGCs, SSEA1+ PGCs, and ESCs cultured in the presence of 20 ng/mL BMP4. A, B, and C demonstrate increased SMAD1P expression in EGCs compared with PGCs and undetectable levels in ESCs. D, E, and F demonstrate a similar pattern for SMAD5P. Moreover, A and D demonstrate that SMAD1P and SMAD5P are colocalized in EGCs, and B and E demonstrate the same in isolated SSEA1+ PGCs. The initiation of ESC differentiation is evident by the loss of tight compacted colony formation that is occurring in these cultures. DAPI (blue) stains the nuclei. Scale bars: 150 μM. Color images available online at www.liebertonline.com/scd.

FIG. 6.

Indirect immunofluorescence detection of markers of trophoblastic differentiation in EGCs and ESCs cultured in the presence of 20 ng/mL BMP4. (A, C) HCG and (B, D) TROMO1 protein expression is not detected in EGCs but clearly shown in trophoblastic cells derived from human ESCs. DAPI (blue) stains the nuclei. Scale bars: (A, B) 150 μM; (C, D) 300 μM. Color images available online at www.liebertonline.com/scd.