Roles of CD34+ cells and ALK5 signaling in the reconstruction of seminiferous tubule-like structures in 3-D re-aggregate culture of dissociated cells from neonatal mouse testes

Abstract

Tissue reconstruction in vitro can provide, if successful, a refined and simple system to analyze the underlying mechanisms that drive the morphogenesis and maintain the ordered structure. We have recently succeeded in reconstruction of seminiferous cord-like and tubule-like structures using 3-D re-aggregate culture of dissociated testicular cells. In testis formation, endothelial cells that migrated from mesonephroi to embryonic gonads have been shown to be critical for development of testis cords, but how endothelial cells contribute to testis cord formation remains unknown. To decipher the roles of endothelial and peritubular cells in the reconstruction of cord-like and tubule-like structures, we investigated the behavior of CD34+ endothelial and p75+ cells, and peritubular myoid cells (PTMCs) in 3-D re-aggregate cultures of testicular cells. The results showed that these 3 types of cells had the capacity of re-aggregation on their own and with each other, and of segregation into 3 layers in a re-aggregate, which were very similar to interstitial and peritubular tissues in vivo. Observation of behaviors of fluorescent Sertoli cells and other non-fluorescent types of cells using testes from Sox9-EGFP transgenic mice showed dynamic cell movement and segregation in re-aggregate cultures. Cultures of testicular cells deprived of interstitial and peritubular cells resulted in dysmorphic structures, but re-addition of them restored tubule-like structures. Purified CD34+ cells in culture differentiated into p75+ cells and PTMCs. These results indicate that CD34+ cells differentiate into p75+ cells, which then differentiate into PTMCs. TGFβ signaling inhibitors, SB431542 and ALK5i, disturbed the reconstruction of cord-like and tubule-like structures, and the latter compromised re-construction of interstitial-like and peritubular-like structures, as well as the proliferation of CD34+, p75+, PTMCs, and Sertoli cells, and their movement and differentiation. These results indicate that CD34+ cells and signaling through ALK5 play pivotal roles in the morphogenesis of interstitial-like, peritubular-like and cord-like structures.

VCAM1-α4β1 integrin interaction mediates interstitial tissue reconstruction in 3-D re-aggregate culture of dissociated prepubertal mouse testicular cells

“…The first is that the proliferative capacity of ALCs was significantly higher than that of FLCs. Given that CD34 + cells show high proliferative activity and similar proliferative pattern to that of ALCs during re-aggregate culture 12 , these results indicate that our in vitro re-aggregate culture system reflects in vivo situations well; the proliferative activity of FLCs is high during the late fetal stage, but declines after birth 26 , 27 . On the other hand, ALCs arise from stem cells in the neonatal testis, and the precursor cells of ALCs proliferate and differentiate to 3β-HSD-positive LC progenitors (PLCs) 13 , 28 , 29 .…”

“…Dissociated testicular cells cultured inside a collagen matrix form within a week seminiferous tubule-like structures with lumen within which germ cells reside, and peritubular myoid cells (PMCs) attach around the SC-re-aggregates; outside the seminiferous tubule-like structures CD34 + cells form interstitial tissues within which LCs are localized. We have also shown the important roles of the interstitial tissues of the testis: testicular cells deprived of interstitial cells form dysmorphic structures, while re-addition of the interstitial cells restored tubule-like structures 12 .…”

“…Testes from 3 to 8 mice at 9- or 10-dpp, which contain comparable numbers of FLCs and ALCs, were used per experiment. Dissociation of the testes, re-aggregation, and culture in a 3-D system were performed as previously reported 12 with some modifications. Briefly, after the tunica albuginea was removed, whole testes were diced, and treated with 0.08% collagenase (type IV, Worthington) and 0.1% hyaluronidase (type I-S, Sigma) for 1 h at room temperature (RT), and then with 0.01% DNase I (Sigma) for the final 5 min, followed by thorough dispersal by pipetting and filtering through a nylon mesh filter (70 μm, Milteny 130-098-462).…”

“… 9 , 10 . We have recently succeeded in the reconstruction of seminiferous tubule-like structures in vitro using a 3-D culture of dissociated testicular cells from prepubertal mouse 11 , 12 . Dissociated testicular cells cultured inside a collagen matrix form within a week seminiferous tubule-like structures with lumen within which germ cells reside, and peritubular myoid cells (PMCs) attach around the SC-re-aggregates; outside the seminiferous tubule-like structures CD34 + cells form interstitial tissues within which LCs are localized.…”

Behavior and Functional Roles of CD34+ Mesenchymal Cells in Mammalian Testes

“…In order to investigate the behaviors and roles of CD34 + cells and PMCs in the reconstruction of seminiferous tubule-like structures, Abe et al [ 39 ] observed the behavior of CD34 + cells, p75 + cells, and PMCs in re-aggregate cultures. CD34 + cells, p75 + cells, and Sertoli cells initially exist as re-aggregates on their own, while most PMCs are distributed as single cells.…”