Primate spermatogonial stem cells colonize mouse testes

In mice, transplantation of spermatogonial stem cells from a fertile male to the seminiferous tubules of an infertile recipient male results in progeny with donor-derived haplotype. Attempts to extend this approach by transplanting human testis cells to mice have led to conflicting claims that no donor germ cells persisted or that human spermatozoa were produced in the recipient. To examine this issue we used the baboon, a primate in which testis cell populations of several ages could be obtained for transplantation, and demonstrate that donor spermatogonial stem cells readily establish germ cell colonies in recipient mice, which exist for periods of at least 6 mo. However, differentiation of germ cells toward the lumen of the tubule and production of spermatozoa did not occur. The presence of baboon spermatogonial stem cells and undifferentiated spermatogonia in mouse seminiferous tubules for long periods after transplantation indicates that antigens, growth factors, and signaling molecules that are necessary for interaction of these cells and the testis environment have been preserved for 100 million years of evolutionary separation. Because germ cell differentiation and spermatogenesis did not occur, the molecules necessary for this process appear to have undergone greater divergence between baboon and mouse.     

Effect of a neutrophilia-inducing tumor on hemopoietic stem cells in mice

The influence of neutrophilic stimulation on hemopoietic stem cells was studied in mice with tumor-induced neutrophilia. Transfusions of marrow cells from normal and neutrophilic tumor-bearing mice into lethally irradiated normal and tumor-bearing mice were performed. The number and the erythroid:granuloid (E:G) ratio of day 7 colonies in the recipient spleens and bones as well as the size of spleen colonies of recipient animals were determined. The E:G ratio of spleen and bone marrow colonies between normal and tumor-bearing mouse recipients and the number of spleen colonies did not differ significantly in either experiment. However, spleen colonies which developed in tumor-bearing irradiated mice were significantly larger than those which developed in normal recipients in both experiments. These studies indicated that while the line of differentiation taken by hemopoietic stem cells was not affected by the neutrophilic influence of the tumor, the tumor-bearing host environment appeared to enhance proliferation of transfused stem cells and/or their descendants. The stimulators of granulocytopoiesis in this model of neutrophilia appear to act on a population of progenitor cells more mature than the stem cells capable of forming 7-day colonies in the spleen and bone marrow of irradiated recipient mice.     

Efficiency of adult mouse spermatogonial stem cell colony formation under several culture conditions

The aim of this study was to compare the in vitro effects of glial cell line-derived neurotrophic factor, stem cell factor, granulocyte macrophage-colony stimulating factor, and co-culture with Sertoli cells on the efficiency of adult mouse spermatogonial stem cells colony formation. For these purpose, both Sertoli and spermatogonial cells were isolated from adult mouse testes. The identity of the cells was confirmed through analysis of alkaline phosphatase activity, immunocytochemistry against OCT-4, c-kit, and vimentin, and also by transplantation of these cells in the recipient testes. The isolated spermatogonial cells were treated either with various concentrations of the above mentioned factors or co-cultured with Sertoli cells for 3 wk. The spermatogonial cells of the resulting colonies were transplanted via rete testis into the mouse testes, which were irradiated with 14 Gy. The results indicated that glial cell line-derived neurotrophic factor is the most appropriate factor for in vitro colonization of adult mice spermatogonial cells compared with other cytokines and growth factors. A short-term co-culture with Sertoli cells showed a significant increase in the number and diameter of the colonies compared with the treated growth factors and the control group. We have also demonstrated that mouse spermatogonial stem cells in the colonies after co-culturing with Sertoli cells could induce spermatogenesis in the recipient testes after transplantation.     

Expansion of murine spermatogonial stem cells through serial transplantation

Mammalian male germ cells might be generally thought to have infinite proliferative potential based on their life-long production of huge numbers of sperm. However, there has been little substantial evidence that supports this assumption. In the present study, we performed serial transplantation of spermatogonial stem cells to investigate if they expand by self-renewing division following transplantation. The transgenic mouse carrying the Green fluorescent protein gene was used as the donor cell source that facilitated identification and recollection of colonized donor germ cells in the recipient testes. The established colonies of germ cells in the recipient testes were collected and transplanted to new recipients. This serial transplantation of spermatogonial stem cells repopulated the recipient testes, which were successfully performed sequentially up to four times from one recipient to the next. The incubation periods between two sequential transplantations ranged from 55 to 373 days. During these passages, the spermatogonial stem cells showed constant activity to form spermatogenic colonies in the recipient testis. They continued to increase in number for more than a year following transplantation. Colonization efficiency of spermatogonial stem cells was determined to be 4.25% by using Sl/Sl(d) mice as recipients that propagated only undifferentiated type A spermatogonia in their testes. Based on the colonization efficiency, one colony-forming activity was assessed to equate to about 20 spermatogonial stem cells. The spermatogonial stem cells were estimated to expand over 50-fold in 100 days in this experiment.     

Differentiation of embryonic haemopoietic stem cells from mouse blastocysts grown in vitro

Embryonic haemopoietic stem cells can differentiate from mouse blastocysts grown in vitro. Mouse blastocysts were cultured for 3 or 4 days and the resultant cells were injected intravenously into lethally X-irradiated or genetically anaemic recipient mice. Blastocysts grown in vitro did not maintain normal embryonic morphology. The presence of donor haemoglobin and donor lymphocytic glucose phosphate isomerase in grafted recipients, demonstrates the presence of embryonic haemopoietic stem cells. Recipients of embryonic haemopoietic stem cells, obtained from growth in vitro, were haematologically stable with no evidence of neoplasia. Pluripotent embryonic cells, maintained on fibroblast feeder layers, were unable to colonize X-irradiated or genetically anaemic mice. Recipients of pluripotent cells died at the same time as saline-injected controls.     

同种异体宫内移植小鼠嵌合模型的建立

干细胞宫内移植是一种很有前途的产前治疗方式。为深入研究干细胞移植后的细胞行为,采用宫内移植的方法建立同种异体的嵌合小鼠模型。将雄鼠骨髓单核细胞宫内注射到胎鼠腹腔,在受体鼠出生后检测雌性受体鼠外周血细胞。应用PCR检测外周血是否存在雄性鼠的DNA,并采用定量PCR技术确定其嵌合量;同时用荧光原位杂交（FISH）技术直观观察外周血中雄性来源的细胞。结果表明：共获得4只阳性外周血嵌合小鼠,其中3只稳定嵌合达到6个月以上。应用宫内移植成功建立了外周血中存在异源细胞的小鼠嵌合模型。     

Limiting dilution analysis of the stem cells for T cell lineage

Stem cell activities of bone marrow, spleen, thymus, and fetal liver cells for T cell lineage were studied comparatively by transferring the cells from these organs through i.v. or intrathymus (i.t.) route into right leg- and tail-shielded (L-T-shielded) and 900 R-irradiated recipient mice, which were able to survive without supplying hemopoietic stem cells. Cells from B10.Thy-1.1 (H-2b, Thy-1.1) mice were serially diluted and were transferred into L-T-shielded and irradiated C57BL/6 (H-2b, Thy-1.2) mice, and 21 days later the thymus cells of recipient mice were assayed for Thy-1.1+ cells by flow cytofluorometry. The percentage of recipient mice possessing donor-type T cells was plotted against the number of cells transferred, and the stem cell activity in each cell source was expressed as the 50% positive value, the number of donor cells required for generating donor-type T cells in the thymuses of 50% of recipient mice. In i.v. transfer experiments, the activity of bone marrow cells was similar to that of fetal liver cells, and about 100 times and nearly 1000 times higher than those of spleen cells and thymus cells, respectively. In i.t. transfer experiments, the number of cells required for generating donor-type T cells was much lower than that in i.v. transfer experiments, although the ratio in 50% positive values between i.v. and i.t. transfers differed among cell sources. In i.t. transfers, the 50% positive value of bone marrow cells was five times, 400 times, and 500 times higher than that of fetal liver cells, spleen cells, and thymus cells, respectively. Our previous finding that stem cells are enriched in the spleens of mice which were whole body-irradiated and marrow-reconstituted 7 days earlier was confirmed also by the present limiting dilution assay carried out in i.v. as well as i.t. transfers.     

Two subpopulations of stem cells for T cell lineage

An assay system for the stem cell that colonizes the thymus and differentiates into T cells was developed, and by using this assay system the existence of two subpopulations of stem cells for T cell lineage was clarified. Part-body-shielded and 900-R-irradiated C57BL/6 (H-2b, Thy-1.2) recipient mice, which do not require the transfer of pluripotent stem cells for their survival, were transferred with cells from B10 X Thy-1.1 (H-2b, Thy-1.1) donor mice. The reconstitution of the recipient's thymus lymphocytes was accomplished by stem cells in the donor cells and those spared in the shielded portion of the recipient that competitively colonize the thymus. Thus, the stem cell activity of donor cells can be evaluated by determining the proportion of donor-type (Thy-1.1+) cells in the recipient's thymus. Bone marrow cells were the most potent source of stem cells, the generation of donor-derived T cells being observed in two out of 14 recipients transferred with as few as 1.5 X 10(4) cells. The stem cell activity of spleen cells was estimated to be about 1% of that of bone marrow cells, and no activity was found in thymus cells. By contrast, when the stem cell activity was compared between spleen and bone marrow cells of whole-body-irradiated (800 R) C57BL/6 mice reconstituted with B10 X Thy-1.1 bone marrow cells by assaying in part-body-shielded and irradiated C57BL/6 mice, the activity of these two organs showed quite a different time course of development. Spleen cells showed a markedly high level of activity 7 days after the reconstitution, followed by a decline, whereas the activity of bone marrow cells was very low on day 7 and increased crosswise. The results strongly suggest that the stem cells for T cell lineage in the bone marrow comprise at least two subpopulations, spleen-seeking and bone marrow-seeking cells. Such patterns of compartmentalization of stem cells in the spleen and bone marrow of irradiated recipients completely conform to the general scheme of the relationship between restricted stem cells and less mature stem cells, including pluripotent stem cells, which became evident in other systems such as in the differentiation of spleen colony-forming cells or of stem cells for B cell lineage.     

Long-term proliferation in culture and germline transmission of mouse male germline stem cells

Spermatogenesis is a complex process that originates in a small population of spermatogonial stem cells. Here we report the in vitro culture of spermatogonial stem cells that proliferate for long periods of time. In the presence of glial cell line-derived neurotrophic factor, epidermal growth factor, basic fibroblast growth factor, and leukemia inhibitory factor, gonocytes isolated from neonatal mouse testis proliferated over a 5-month period (>10(14)-fold) and restored fertility to congenitally infertile recipient mice following transplantation into seminiferous tubules. Long-term spermatogonial stem cell culture will be useful for studying spermatogenesis mechanism and has important implications for developing new technology in transgenesis or medicine.     

造血干细胞移植条件对小鼠造血重建的影响

通过同种基因型小鼠构建造血干细胞移植模型,将预处理的全骨髓单个核细胞或c-Kit⁺造血干细胞移植至致死剂量照射的受体小鼠体内,动态监测移植2～16周后受体小鼠体内供体来源细胞造血重建以及嵌合情况,以期揭示不同群体的供体细胞以及预处理等因素对小鼠造血干细胞移植后造血重建的影响。实验结果显示,移植后早期(2周)全骨髓单个核细胞组髓系比例要高于c-Kit⁺细胞移植组,但全骨髓移植组受体小鼠呈现出较大的移植后不良反应,出现脱毛、食欲不振以及体重减轻的症状。c-Kit⁺细胞移植组在淋系重建上要早于全骨髓移植组,供体细胞的嵌合植入也早于全骨髓移植组,但两组实验组最终均能完成造血重建过程。实验结果表明c-Kit⁺细胞移植组在移植后能够较快地实现供体细胞植入,进而开始造血重建,且c-Kit⁺细胞移植组的不良反应要低于全骨髓移植组。结果说明在整体造血重建效果上c-Kit⁺细胞移植组要优于全骨髓移植组。     

Effect of the GnRH-agonist leuprolide on colonization of recipient testes by donor spermatogonial stem cells after transplantation in mice

Gonadotropin-releasing hormone (GnRH)-agonist or antagonist treatment supports recovery of spermatogenesis after irradiation damage in the rat and appears to be beneficial to colonization of recipient testes after spermatogonial transplantation from fertile donors to the testes of infertile recipients in rats and mice. In the present study, we quantified the effect of treatment of recipient mice with the GnRH-agonist leuprolide acetate on the extent of colonization by donor spermatogonial stem cells in the recipient testis. Testis cells from mice carrying transgenes, which produce beta-galactosidase in spermatogenic cells, were used as donor cells for transplantation to allow for quantification of donor spermatogenesis in the recipient testis by staining for enzyme activity. Donor cell colonization 3 months after transplantation was compared between recipients receiving leuprolide in different treatment protocols and untreated control mice. Two injections of leuprolide 4 weeks apart prior to transplantation with as little as 3.8 mg/kg resulted in a pronounced improvement in the number of donor-derived spermatogenic colonies as well as in the in the area of recipient seminiferous tubules occupied by donor cell spermatogenesis. Improved colonization efficiency by treatment with GnRH-agonist can make the technique of spermatogonial transplantation applicable to situations when only low numbers of donor cells are available.     

Increased initiation and growth of tumor cell lines, cancer stem cells and biopsy material in mice using basement membrane matrix protein (Cultrex or Matrigel) co-injection

This protocol requires 2-4 h and presents a method for injecting tumor cells, cancer stem cells or dispersed biopsy material into subcutaneous or orthotopic locations within recipient mice. The tumor cells or biopsy are mixed with basement membrane matrix proteins (CultrexBME or Matrigel) at 4 °C and then injected into recipient animals at preferred anatomical sites. Tumor cells can also be co-injected with additional cell types, such as fibroblasts, stromal cells, endothelial cells and so on. Details are given on appropriate cell numbers, handling and concentration of the basement membrane proteins, recipient animals, injection location and techniques. This procedure enables the growth of tumors from cells or biopsy material (tumor graft) with greater efficiency of take and growth, and with retention of the primary tumor phenotype based on histology. Co-injection with additional cell types provides more physiological models of human cancers for use in drug screening and studying cancer biology.     

Germ line stem cell competition in postnatal mouse testes

Niche is believed to affect stem cell behavior. In self-renewing systems for which functional transplantation assays are available, it has long been assumed that stem cells are fixed in the niche and that ablative treatments to remove endogenous stem cells are required for successful donor engraftment. Our results demonstrate that enriched populations of donor stem cells can produce long-lasting spermatogenic colonies in testes of immature and mature, nonablated mice, albeit at a lower frequency than in ablated mice. Colonization of nonablated recipient testes by neonate, pup, and cryptorchid adult donor spermatogonial stem cells demonstrates that competition for niche begins soon after birth and that endogenous stem cells influence the degree and pattern of donor cell colonization. Thus, a dynamic relationship between stem cell and niche exists in the testis, as has been suggested for hematopoiesis. Therefore, similar competitive properties of donor stem cells may be characteristic of all self-renewing systems.     

Transplantation of germ cells from rabbits and dogs into mouse testes.

Spermatogonial stem cells of a fertile mouse transplanted into the seminiferous tubules of an infertile mouse can develop spermatogenesis and transmit the donor haplotype to progeny of the recipient mouse. When testis cells from rats or hamsters were transplanted to the testes of immunodeficient mice, complete rat or hamster spermatogenesis occurred in the recipient mouse testes, albeit with lower efficiency for the hamster. The objective of the present study was to investigate the effect of increasing phylogenetic distance between donor and recipient animals on the outcome of spermatogonial transplantation. Testis cells were collected from donor rabbits and dogs and transplanted into testes of immunodeficient recipient mice in which endogenous spermatogenesis had been destroyed. In separate experiments, rabbit or dog testis cells were frozen and stored in liquid nitrogen or cultured for 1 mo before transplantation to mice. Recipient testes were analyzed, using donor-specific polyclonal antibodies, from 1 to >12 mo after transplantation for the presence of donor germ cells. In addition, the presence of canine cells in recipient testes was demonstrated by polymerase chain reaction using primers specific for canine alpha-satellite DNA. Donor germ cells were present in the testes of all but one recipient. Donor germ cells predominantly formed chains and networks of round cells connected by intercellular bridges, but later stages of donor-derived spermatogenesis were not observed. The pattern of colonization after transplantation of cultured cells did not resemble spermatogonial proliferation. These results indicate that fresh and cryopreserved germ cells can colonize the mouse testis but do not differentiate beyond the stage of spermatogonial expansion.     

Effect of hepatocyte growth factor on long term hematopoiesis of human progenitor cells in transgenic-severe combined immunodeficiency mice

Hepatocyte growth factor (HGF), which was originally isolated as a liver generating factor, enhances hematopoiesis. To study the effect of HGF on hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs), we generated severe combined immunodeficiency (SCID) mice producing human (h) HGF and/or stem cell factor (SCF) by transferring the relevant genes to fertilized eggs, and then transplanted hematopoietic progenitors from human cord blood into the transgenic (Tg) SCID mice. Six months after transplantation, a significantly larger number of human cells were found in the Tg SCID mice than in non-Tg controls. Characteristically, the recipient SCID mice producing h HGF (HGF-SCID) had a significantly increased number of h CD41+ cells, whereas the SCF-SCID recipients had more CD11b+ cells. Significantly large numbers of CD34+ progenitors were found in the SCID mice transferred with both h HGF and h SCF genes (HGF/SCF-SCID) when compared with HGF-SCID or SCF-SCID mice. These results imply that HGF supports the differentiation of progenitors in megakaryocyte lineage, whereas SCF supports that in myeloid lineage. The results also imply that HGF acts on HSCs/HPCs as a synergistic proliferative factor combined with SCF. We have demonstrated the advantage of the human cytokine-producing animal in the maintenance of human HSCs.     

Cell surface marker phenotypes and gene expression profiles of murine radiation-induced acute myeloid leukemia stem cells are similar to those of common myeloid progenitors

Radiation exposure induces acute myeloid leukemia (AML) in humans and mice. Recent studies postulated that AML stem cells of spontaneous human AML arise from hematopoietic stem cells. However, other studies support the possibility that short-lived committed progenitors transform into AML stem cells, accompanied by a particular gene mutation. It remains unclear whether AML stem cells are present in radiation-induced AML, and information regarding AML-initiating cells is lacking. In this study, we identified and analyzed AML stem cells of mice with radiation-induced AML. The AML stem cells were identified by transplanting 100 bone marrow cells from mice with radiation-induced AML. We injected 100 cells of each of seven cell populations corresponding to different stages of hematopoietic cell differentiation and compared the latencies of AMLs induced in recipient mice. The identified radiation-induced AML stem cells frequently displayed similarities in both CD antigen and gene expression profiles with normal common myeloid progenitors. The number of common myeloid progenitor-like AML stem cells was significantly increased in mice with radiation-induced AML, but the progeny of common myeloid progenitors was decreased. In addition, analysis of radiation effects on the hematopoietic system showed that common myeloid progenitor cells were extremely radiosensitive and that their numbers remained at low levels for more than 2?months after radiation exposure. Our results suggest that murine radiation-induced AML stem cells arise from radiosensitive cells at a common myeloid progenitor stage.     

Developments in modern hematology.

In the past 40 years our concepts about hemopoiesis have been changed dramatically. The results of bone marrow transplantation into lethally irradiated mice since the mid-fifties suggested the existence of a hemopoietic stem cell, which was initially identified as a spleen colony forming cell (CFU-S). Later experiments showed that the stem cell compartment is rather heterogeneous and that the most primitive stem cell, unlike the CFU-S, has the ability for long-term engraftment of an irradiated recipient. Daughter cells of such primitive quiescent stem cells lose their capacity for self-generation gradually with each mitosis and become more and more committed to a specific differentiation lineage. In vitro culture techniques in a serum-free semi-solid medium enabled the establishment and analysis of specific hemopoietic growth factors. Such factors, which are essential for the maintenance, proliferation and differentiation of progenitor cells and the functional activity of mature cells can now be produced with recombinant DNA techniques in pure form and large quantities. Hemopoiesis requires an appropriate microenvironment, consisting of various stromal cell types and an extracellular matrix. Intercellular contacts, adhesion of cells and growth factors to the matrix molecules seem essential in the regulating action of this hemopoietic microenvironment. In long-term bone marrow cultures the development of a stromal hemopoietic microenvironment can facilitate long-term maintenance of stem cells and hemopoietic differentiation. For bone marrow transplantation and infusion of hemopoietic growth factors many clinical indications are well established and our possibilities to interfere in the regulation of hemopoiesis are still growing.     

Magnetic field exposure of marrow donor mice can increase the number of spleen colonies (CFU-S 7d) in marrow recipient mice

Transplantation of bone marrow cells of magnetic-field-exposed mice led to increased numbers of spleen colonies (CFU-S 7d) in conditioned recipient mice (Peterson et al. 1986). Here we report on the dependence of this phenomenon on body temperature, field strength and exposure time. It was found that the effect can only be seen when the body temperature is 27 degrees C, the field strength not less than 1.4 T and the exposure time at least 15 min. It is suggested that the magnetic field increases the number of spleen colonies either directly by affecting membrane components (receptors) responsible for the seeding of the transplanted stem cells to the recipient spleens or indirectly affecting radical/redox-systems that may have a regulatory function in the stem cells.     

胎肝造血干细胞的移植特性

胚胎发育中,肝脏是一个重要的造血器官。近年来胎肝移植的临床应用重新引起了人们的关注。本文应用染色体的 C-带染色法研究了小鼠骨髓和胎肝造血干细胞在照射受体小鼠中的增殖能力与相互间的竞争作用。实验结果表明胎肝造血干细胞在成年骨髓中的植入率比较同样条件下的成年骨髓造血干细胞低,但胎肝造血干细胞比较成年骨髓造血干细胞具有更强的自我更新或增殖能力。在同种胎肝造血干细胞移植中,为了降低同种移植抗力,提高移植的胎肝造血干细胞在受体中的耐受性,移植前对受体作适当的免疫抑制处理是必要的。因此,克服个体发育屏障和移植免疫屏障是提高同种胎肝造血干细胞移植效果中两个重要的研究课题。     

Germ cell transplantation and testis tissue xenografting in domestic animals

Transplantation of germ cells from fertile donor mice to the testes of infertile recipient mice results in donor-derived spermatogenesis and transmission of the donor's genetic material to the offspring of recipient animals. Germ cell transplantation provides a bioassay to study the biology of male germ line stem cells, develop systems to isolate and culture spermatogonial stem cells, examine defects in spermatogenesis and treat male infertility. Although most widely studied in rodents, germ cell transplantation has been applied to larger mammals. In domestic animals including pigs, goats and cattle, as well as in primates, germ cells can be transplanted to a recipient testis by ultrasonographic-guided cannulation of the rete testis. Germ cell transplantation was successful between unrelated, immuno-competent pigs and goats, whereas transplantation in rodents requires syngeneic or immuno-compromised recipients. Genetic manipulation of isolated germ line stem cells and subsequent transplantation will result in the production of transgenic sperm. Transgenesis through the male germ line has tremendous potential in domestic animal species where embryonic stem cell technology is not available and current options to generate transgenic animals are inefficient. As an alternative to transplantation of isolated germ cells to a recipient testis, ectopic grafting of testis tissue from diverse mammalian donor species, including horses and primates, into a mouse host represents a novel possibility to study spermatogenesis, to investigate the effects of drugs with the potential to enhance or suppress male fertility, and to produce fertile sperm from immature donors. Therefore, transplantation of germ cells or xenografting of testis tissue are uniquely valuable approaches for the study, preservation and manipulation of male fertility in domestic animals.