Francisco J. Silva

Generation of multipotent cell lines from a distinct population of male germ line stem cells

Spermatogonial stem cells (SSCs) maintain spermatogenesis by self-renewal and generation of spermatogonia committed to differentiation. Under certain in vitro conditions, SSCs from both neonatal and adult mouse testis can reportedly generate multipotent germ cell (mGC) lines that have characteristics and differentiation potential similar to embryonic stem (ES) cells. However, mGCs generated in different laboratories showed different germ cell characteristics, i.e., some retain their SSC properties and some have lost them completely. This raises an important question: whether mGC lines have been generated from different subpopulations in the mouse testes. To unambiguously identify and track germ line stem cells, we utilized a transgenic mouse model expressing green fluorescence protein under the control of a germ cell-specific Pou5f1 (Oct4) promoter. We found two distinct populations among the germ line stem cells with regard to their expression of transcription factor Pou5f1 and c-Kit receptor. Only the POU5F1+/c-Kit+ subset of mouse germ line stem cells, when isolated from either neonatal or adult testes and cultured in a complex mixture of growth factors, generates cell lines that express pluripotent ES markers, i.e., Pou5f1, Nanog, Sox2, Rex1, Dppa5, SSEA-1, and alkaline phosphatase, exhibit high telomerase activity, and differentiate into multiple lineages, including beating cardiomyocytes, neural cells, and chondrocytes. These data clearly show the existence of two distinct populations within germ line stem cells: one destined to become SSC and the other with the ability to generate multipotent cell lines with some pluripotent characteristics. These findings raise interesting questions about the relativity of pluripotency and the plasticity of germ line stem cells.

An Efficient Approach to Isolation and Characterization of Pre- and Postnatal Umbilical Cord Lining Stem Cells for Clinical Applications:

There have been various forms of mesenchymal stem cell-like (MSC-like) cells isolated from umbilical cords (UCs). The isolation of umbilical cord lining stem cells (ULSCs) may be of great value for those interested in a possible treatment to several disease/disorders. Unlike umbilical cord blood cells, these cells are unique because they can be expanded to therapeutically relevant numbers and cryopreserved for several different uses. Here we efficiently isolate stem cells from a small segment of pre- and postnatal UCs, and obtain therapeutically relevant amounts of ULSCs within 3 weeks. We demonstrate their growth potential and characterize them using immunocytochemistry, flow cytometry, and RT-PCR. In addition, we differentiate ULSCs into multiple lineages. Pre- and postnatal ULSCs are morphologically similar to mesenchymal stem cells (MSCs) and easily expand to greater than 70 population doublings. They express pluripotent markers Oct4 and nanog at the protein and RNA level. Flow cytometry demonstrates that they express markers indicative of MSCs in addition to high SSEA-4 expression. ULSCs are easily differentiated into osteogenic, adipogenic, chondrogenic, cardiogenic, and neurogenic cells. Pre- and postnatal ULSCs are characteristically similar in respect to their growth, marker expression, and plasticity, demonstrating they are highly conserved throughout development. ULSCs have phenotypic and genotypic properties of MSCs. These studies demonstrate the therapeutic potential of an otherwise discarded tissue. They are a perfect HLA match for the donor and an excellent match for immediate family members; therefore, they may serve as a therapeutic cell source.