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Spermatogonial stem cells (SSCs) are unipotent germ cells that are at the foundation of spermatogenesis and male fertility. However, the underlying molecular mechanisms governing SSC stemness and growth properties remain elusive. We have recently identified chromodomain helicase/ATPase DNA binding protein 1-like (Chd1l) as a novel regulator for SSC survival and self-renewal, but how these functions are controlled by Chd1l remains to be resolved. Here, we applied high-throughput small RNA sequencing to uncover the microRNA (miRNA) expression profiles controlled by Chd1l and showed that the expression levels of 124 miRNA transcripts were differentially regulated by Chd1l in SSCs. KEGG pathway analysis shows that the miRNAs that are differentially expressed upon Chd1l repression are significantly enriched in the pathways associated with stem cell pluripotency and proliferation. As a proof of concept, we demonstrate that one of the most highly upregulated miRNAs, miR-486, controls SSC stemness gene expression and growth properties. The matrix metalloproteinase 2 (MMP2) gene has been identified as a novel miR-486 target gene in the context of SSC stemness gene regulation and growth properties. Data from cotransfection experiments showed that Chd1l, miR-486, and MMP2 work in concert in regulating SSC stemness gene expression and growth properties. Finally, our data also revealed that MMP2 regulates SSC stemness gene expression and growth properties through activating β-catenin signaling by cleaving N-cadherin and increasing β-catenin nuclear translocation. Our data demonstrate that Chd1l-miR-486-MMP2 is a novel regulatory axis governing SSC stemness gene expression and growth properties, offering a novel therapeutic opportunity for treating male infertility.

Embryonic stem cells (ESCs) differentiation via embryoid body (EB) formation is an established method that generates the three germ layers. However, EB differentiation poses several problems including formation of heterogeneous cell populations. Herein, we described a differentiation protocol on enhancing mesoderm derivation from murine ESCs (mESCs) using conditioned medium (CM) from HepG2 cells. We used this technique to direct hematopoiesis by generating "embryoid-like" colonies (ELCs) from murine (m) ESCs without following standard formation of EBs. Our CM-mESCs group yielded an almost fivefold increase in ELC formation (p ≤ 0.05) and higher expression of mesoderm genes;-Brachyury-T, Goosecoid, and Flk-1 compared with control mESCs group. Hematopoietic colony formation from CM-mESCs was also enhanced by twofold at days 7 and 14 with earlier colony commitment compared to control mESCs (p ≤ 0.05). This early clonogenic capacity was confirmed morphologically by the presence of nucleated erythrocytes and macrophages as early as day 7 in culture using standard 14-day colony-forming assay. Early expression of hematopoietic primitive (ζ-globin) and definitive (β-globin) erythroid genes and proteins was also observed by day 7 in the CM-treated culture. These data indicate that hematopoietic cells more quickly differentiate from CM-treated, compared with those using standard EB approaches, and provide an efficient bioprocess platform for erythroid-specific differentiation of ESCs.

Embryonic stem cells (ESCs) differentiation via embryoid body (EB) formation is an established method that generates the three germ layers. However, EB differentiation poses several problems including formation of heterogeneous cell populations. Herein, we described a differentiation protocol on enhancing mesoderm derivation from murine ESCs (mESCs) using conditioned medium (CM) from HepG2 cells. We used this technique to direct hematopoiesis by generating "embryoid-like" colonies (ELCs) from murine (m) ESCs without following standard formation of EBs. Our CM-mESCs group yielded an almost fivefold increase in ELC formation (p ≤ 0.05) and higher expression of mesoderm genes;-Brachyury-T, Goosecoid, and Flk-1 compared with control mESCs group. Hematopoietic colony formation from CM-mESCs was also enhanced by twofold at days 7 and 14 with earlier colony commitment compared to control mESCs (p ≤ 0.05). This early clonogenic capacity was confirmed morphologically by the presence of nucleated erythrocytes and macrophages as early as day 7 in culture using standard 14-day colony-forming assay. Early expression of hematopoietic primitive (ζ-globin) and definitive (β-globin) erythroid genes and proteins was also observed by day 7 in the CM-treated culture. These data indicate that hematopoietic cells more quickly differentiate from CM-treated, compared with those using standard EB approaches, and provide an efficient bioprocess platform for erythroid-specific differentiation of ESCs.