Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture

146

108

Stem cells represent a unique population of cells with self-renewal capacity. Although they are important therapeutic targets, the genetic manipulation of tissue-specific stem cells has been limited, which complicates the study and practical application of these cells. Here, we demonstrate successful gene trapping and homologous recombination in spermatogonial stem cells. Cultured spermatogonial stem cells were transfected with gene trap or gene targeting vectors. Mutagenized stem cells were expanded clonally by drug selection. These cells underwent spermatogenesis and produced heterozygous offspring after transplantation into the seminiferous tubules of infertile mouse testes. Heterozygous mutant mice were intercrossed to produce homozygous gene knockouts. Using this strategy, the efficiency of homologous recombination for the occludin gene locus was 1.7% using a nonisogenic DNA construct. These results demonstrate the feasibility of altering genes in tissue-specific stem cells in a manner similar to embryonic stem cells and have important implications for gene therapy and animal transgenesis.spermatogenesis ͉ germ cell ͉ testis ͉ transplantation S tem cells represent a unique cell population with self-renewal potential (1). Although stem cells are low in number, these cells proliferate extensively to sustain the various self-renewing tissues, such as bone marrow and intestine. Although these tissue-specific stem cells normally divide very slowly, stem cells are the last cell type to be destroyed after cytotoxic damage, and they regenerate the entire tissue in a relatively short time. In addition, stem cells often have migratory activities, and they can be transplanted between animals; transplanted stem cells migrate to a specific niche and regenerate the self-renewing tissue. Because of their unique properties, stem cells have become the attractive target of cell and gene therapies.Among the many types of tissue-specific stem cells, spermatogonial stem cells are unique in that they have germ-line potential (2, 3). Genetic modification of spermatogonial stem cells creates permanent changes in the germ line, which are transmitted to the offspring by means of fertilization. In contrast to female germ-line cells, which cease to divide after birth, male germ-line cells proliferate continuously and produce sperm throughout the life of the animal. If these stem cells could be cultured and manipulated in a manner similar to embryonic stem (ES) cells (4, 5), they could be used to create knockout animals. As a first step toward this goal, a germ cell transplantation technique was developed in 1994 (6, 7), in which dissociated donor testis cells colonized the seminiferous tubules of infertile recipient testis and produced donor-derived spermatogenesis and offspring. Although this technique was an opportunity to produce offspring from manipulated spermatogonial stem cells, it has been difficult to produce transgenic animals using spermatogonial stem cells, because their number is very low in the testis, and the lack of me...

Section: Discussionmentioning

confidence: 94%

Production of knockout mice by random or targeted mutagenesis in spermatogonial stem cells

Kanatsu-Shinohara

Ikawa

Takehashi

et al. 2006

146

108

“…It recently has been shown that bFGF strongly promotes self-renewal of undifferentiated human ES cells in vitro (23), and bFGF is a potent growth factor for proliferation of mouse PGCs (24,25). However, when MACS EpCAM ϩ cells were cultured with bFGF alone, the culture supported only small germ cell clumps.…”

Section: Discussionmentioning

confidence: 99%

Conservation of spermatogonial stem cell self-renewal signaling between mouse and rat

Ryu

Kubota

Avarbock

et al. 2005

252

236

Self-renewal of spermatogonial stem cells (SSCs) is the foundation for maintenance of spermatogenesis throughout life in males and for continuation of a species. The molecular mechanism underlying stem cell self-renewal is a fundamental question in stem cell biology. Recently, we identified growth factors necessary for self-renewal of mouse SSCs and established a serum-free culture system for their proliferation in vitro. To determine whether the stimulatory signals for SSC replication are conserved among different species, we extended the culture system to rat SSCs. Initially, a method to assess in vitro expansion of SSCs was developed by using flow cytometric analysis, and, subsequently, we found that a combination of glial cell line-derived neurotrophic factor, soluble glial cell line-derived neurotrophic factor-family receptor ␣-1 and basic fibroblast growth factor supports proliferation of rat SSCs. When cultured with the three factors, stem cells proliferated continuously for >7 months, and transplantation of the cultured SSCs to recipient rats generated donor stem cell-derived progeny, demonstrating that the cultured stem cells are normal. The growth factor requirement for replication of rat SSCs is identical to that of mouse; therefore, the signaling factors for SSC self-renewal are conserved in these two species. Because SSCs from many mammals, including human, can replicate in mouse seminiferous tubules after transplantation, the growth factors required for SSC self-renewal may be conserved among many different species. Furthermore, development of a long-term culture system for rat SSCs has established a foundation for germ-line modification of the rat by gene targeting technology.germ-line stem cell ͉ spermatogenesis ͉ testis ͉ glial cell line-derived neurotrophic factor

“…Although it has been shown that mouse spermatogonia express GFR␣1 (10, 11), we reasoned that adding soluble GFR␣1 molecules might potentiate the stimulatory signal through the c-Ret receptor or modulate the signaling pathways (19). In addition, bFGF, a critical growth factor for primordial germ cells (PGCs) in vitro (20,21), was examined. MACS Thy-1 cells from C57 ϫ ROSA pups were cultured with GDNF alone or in the combination with soluble GFR␣1 (GFR␣1-Fc), bFGF, or both.…”

Section: Sscs Derived From Dba͞2j-background Mice Can Be Cultured Inmentioning

confidence: 99%

Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells

Kubota

Avarbock²,

Brinster³

2004

847

944

Spermatogonial stem cells (SSCs) self-renew and produce large numbers of committed progenitors that are destined to differentiate into spermatozoa throughout life. However, the growth factors essential for self-renewal of SSCs remain unclear. In this study, a serum-free culture system and a transplantation assay for SSCs were used to identify exogenous soluble factors that promote proliferation of SSCs. Mouse pup testis cells were enriched for SSCs by selection with an anti-Thy-1 antibody and cultured on STO (SIM mouse embryo-derived thioguanine and ouabain resistant) feeders in a serum-free defined medium. In the presence of glial cell line-derived neurotrophic factor (GDNF), SSCs from DBA͞2J strain mice formed densely packed clumps of cells and continuously proliferated. However, other strains of mice required the addition of soluble GDNF-family receptor ␣-1 and basic fibroblast growth factor to support replication. The functional transplantation assay proved that the clump-forming cells are indeed SSCs. Thus, GDNFinduced cell signaling plays a central role in SSC self-renewal. The number of SSCs in culture doubled every 5.6 days, and the clumpforming cells strongly expressed Oct-4. Under these conditions, SSCs proliferated over 6 months, reconstituted long-term spermatogenesis after transplantation into recipient testes, and restored fertility to infertile recipients. The identification of exogenous factors that allow continuous proliferation of SSCs in vitro establishes the foundation to study the basic biology of SSCs and makes possible germ-line modification by sophisticated technologies. Moreover, the ability to recover, culture indefinitely, and transplant SSCs will make the germ-line of individual males available for periods extending beyond a normal lifetime.