The therapeutic potential of multipotent stromal cells (MSCs) largely depends on the isolation and expansion methods used. In this study, we propose a laminin-based technique to select and enrich for MSCs isolated from the mouse testis. Primary cell cultures were prepared from juvenile mouse testes and the capacity to generate colony forming units together with population doubling time (PDT) during expansion were determined. The identity of MSCs was assayed using reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry for the active expression of cell surface markers, such as CD44, CD73, and CD29; absence of the CD45 hematopoietic cell marker; and in vitro differentiation of the cells into osteoblasts and adipocytes. Testis-derived MSCs (tMSCs) displayed self-renewal properties and in the early passages, exhibited high proliferation patterns with an average PDT of 44.1 hours. The lack of Vasa expression implied that the tMSCs were not of germ cell origin. The RT-PCR data, which were confirmed by immunophenotyping, revealed high expression of CD44 and the absence of CD45 expression in tMSCs. The strong Alizarin Red stain in tMSCs that were stimulated into making bone cells was indicative of the presence of calcium-producing cells (osteoblasts). Likewise, the adipogenic potential of tMSCs was demonstrated based on Oil Red O staining of lipid vacuoles in differentiated cells. Loss of fibroblast-like morphology in late passage cells along with the increase in PDT and the decrease in the mRNA levels of CD73 and CD29 suggested that the tMSCs developmental program is reformed at this stage.

In the vertebrate retina, an interplay between retinal ganglion cells (RGCs), amacrine (AC), and bipolar (BP) cells establishes a synaptic layer called the inner plexiform layer (IPL). This circuit conveys signals from photoreceptors to visual centers in the brain. However, the molecular mechanisms involved in its development remain poorly understood. Striatin-interacting protein 1 (Strip1) is a core component of the striatin-interacting phosphatases and kinases (STRIPAK) complex, and it has shown emerging roles in embryonic morphogenesis. Here, we uncover the importance of Strip1 in inner retina development. Using zebrafish, we show that loss of Strip1 causes defects in IPL formation. In strip1 mutants, RGCs undergo dramatic cell death shortly after birth. AC and BP cells subsequently invade the degenerating RGC layer, leading to a disorganized IPL. Mechanistically, zebrafish Strip1 interacts with its STRIPAK partner, Striatin 3 (Strn3), and both show overlapping functions in RGC survival. Furthermore, loss of Strip1 or Strn3 leads to activation of the proapoptotic marker, Jun, within RGCs, and Jun knockdown rescues RGC survival in strip1 mutants. In addition to its function in RGC maintenance, Strip1 is required for RGC dendritic patterning, which likely contributes to proper IPL formation. Taken together, we propose that a series of Strip1-mediated regulatory events coordinates inner retinal circuit formation by maintaining RGCs during development, which ensures proper positioning and neurite patterning of inner retinal neurons.

In the vertebrate retina, an interplay between retinal ganglion cells (RGCs), amacrine and bipolar cells establishes a synaptic layer called the inner plexiform layer (IPL). This circuit conveys signals from photoreceptors to visual centers in the brain. However, the molecular mechanisms involved in its development remain poorly understood. Striatin-interacting protein 1 (Strip1) is a core component of the STRIPAK complex, and it has shown emerging roles in embryonic morphogenesis. Here, we uncover the importance of Strip1 in inner retina development. Using zebrafish, we show that loss of Strip1 causes defects in IPL formation. In strip1 mutants, RGCs undergo dramatic cell death shortly after birth. Amacrine and bipolar cells subsequently invade the degenerating RGC layer, leading to a disorganized IPL. Thus, Strip1 promotes IPL formation through RGC maintenance. Mechanistically, zebrafish Strip1 interacts with its STRIPAK partner, Striatin3, to promote RGC survival by suppressing Jun-mediated apoptosis. In addition to its function in RGC maintenance, Strip1 is required for RGC dendritic patterning, which likely contributes to proper IPL formation. Taken together, we propose that a series of Strip1-mediated regulatory events coordinates inner retinal circuit formation by maintaining RGCs during development, which ensures proper positioning and neurite patterning of inner retinal neurons.

No abstract