Figure 7.

Induction and Isolation of hPGCLCs from Competent hiPSCs/hESCs

(A) FACS analysis of TNAP and NANOS3-mCherry (top) and TNAP and CD38 (bottom) on day 4 embryoids induced from 4i hESCs after preinduction (left), directly without preinduction (middle) or from conventional hESCs (right, Conv hESC).

(B) FACS analysis of TNAP and CD38 in 4i hiPSCs (top) and day 4 embryoids derived from 4i hiPSCs after direct induction (bottom).

(C) Expression analysis by RT-qPCR on TNAP-positive hiPSCs (iPSC TNAP+), TNAP/CD38 double-negative (TNAP−CD38−) population and TNAP/CD38 double-positive population (TNAP+CD38+) on day 4 after hPGCLC induction. Relative expression levels are shown with normalization to β−ACTIN. Error bars indicate mean ± SD from two independent biological replicates.

(D) Overview of human germline development. hESCs in 4i reversibly attains competence for germ cell fate. Exposure of 4i cells to cytokines containing BMPs results in strong induction of hPGCLCs following expression of SOX17-BLIMP1, which are among the key regulators of germ cell fate. SOX17 and BLIMP1 are detected in in vivo gonadal hPGC and TCam-2 seminoma, indicating a likely progression of early human germ cell lineage. CD38, a cell-surface glycoprotein, is shared by all cells with germ cell characteristics, but not by hESC. Loss of SOX17 or BLIMP1 abrogates hPGCLC specification.