GP130 stimulation and the maintenance of stem cells

Recent advantages with the cultivation of adult and embryonic stem cells have raised hopes for therapeutic applications of such cells in the treatment of neurodegenerative diseases and cancer. Cultivation of stem cells on feeder cells or treatment of the cells with cytokines is necessary to maintain stem cells in an undifferentiated state and to keep their pluripotency. In particular, the cytokine leukemia inhibitory factor (LIF) has been used to cultivate murine embryonic stem (ES) cells in the absence of feeder cells. For unknown reasons, LIF does not evoke the same effect on rat or human stem cells. This article summarizes what is known about, and the problems associated with, the cultivation of stem cells and suggests experimental strategies that might help to overcome these difficulties.     

Organization of haemopoietic stem cells: the generation-age hypothesis.

This paper proposes that the previous division history of each stem cell is one determinant of the functional organization of the haemopoietic stem cell population. Stem cells from a lineage of stem cells which have generated many stem cells (older stem cells) are used in the animal to form blood before stem cells which have generated few stem cells (younger stem cells). The stem cell generating capacity of a lineage of stem cells is finite. After a given number of generations a stem cell is lost to the stem cell compartment by forming two committed precursors of the cell lines. Its part in blood formation is taken by the next oldest stem cell. We have called this proposal the generation-age hypothesis. Experimental evidence in support of the proposal is presented. We stripped away older stem cells from normal bone marrow and 13 day foetal liver with phase-specific drugs and revealed a younger population of stem cells whose capacity for stem cell generation was three- to four-fold greater than that of the average normal, untreated population. We aged normal stem cells by continuous irradiation and serial retransplantation and found that their stem cell generative capacity had declined eight-fold. We measured the stem cell generative capacity of stem cells in the bloodstream. It was a half to a quarter that of normal bone marrow stem cells and we found a subpopulation of circulating stem cells whose capacity for stem cell generation was an eighth to a fortieth that of normal femoral stem cells. This subpopulation was identified by its failure to express the brain-associated antigen which was present on 75% of normal femoral stem cells but was not found on their progeny, the committed precursors of granulocytes.     

The bulge area is the origin of nestin-expressing pluripotent stem cells of the hair follicle

Nestin-expressing pluripotent stem cells have been found both in the bulge area (BA) as well as the dermal papilla (DP). Nestin-expressing stem cells of both the BA and DP have been previously shown to be able to form neurons and other non-follicle cell types. The nestin-expressing stem cells from the DP have been termed skin precursor or SKP cells. Both nestin-expressing DP and BA cells have been previously shown to effect repair of the injured spinal cord and peripheral nerve, with the BA being the greater and more constant source of the stem cells. The BA contains nestin-expressing stem cells throughout the hair cycle, whereas nestin-expressing dermal papillae stem cells were found in early and mid-anagen only. Our previous studies have shown that the nestin-expressing stem cells in the BA and DP have similar morphological features. The cells from both regions have a small body diameter of approximately 7 μm with long extrusions, as shown by 2-photon imaging. In the present study, using 2-photon imaging of whisker follicles from transgenic mice expressing nestin-driven green fluorescent protein (ND-GFP), we demonstrate that the BA is the source of the nestin-expressing stem cells of the hair follicle. The nestin-expressing stem cells migrate from the BA to the DP as well as into the surrounding skin tissues including the epidermis, and during wound healing, suggesting that the BA may be the source of the stem cells of the skin itself.     

Targeted delivery of neural stem cells to the brain using MRI-guided focused ultrasound to disrupt the blood-brain barrier

Stem cell therapy is a promising strategy to treat neurodegenerative diseases, traumatic brain injury, and stroke. For stem cells to progress towards clinical use, the risks associated with invasive intracranial surgery used to deliver the cells to the brain, needs to be reduced. Here, we show that MRI-guided focused ultrasound (MRIgFUS) is a novel method for non-invasive delivery of stem cells from the blood to the brain by opening the blood brain barrier (BBB) in specific brain regions. We used MRI guidance to target the ultrasound beam thereby delivering the iron-labeled, green fluorescent protein (GFP)-expressing neural stem cells specifically to the striatum and the hippocampus of the rat brain. Detection of cellular iron using MRI established that the cells crossed the BBB to enter the brain. After sacrifice, 24 hours later, immunohistochemical analysis confirmed the presence of GFP-positive cells in the targeted brain regions. We determined that the neural stem cells expressed common stem cell markers (nestin and polysialic acid) suggesting they survived after transplantation with MRIgFUS. Furthermore, delivered stem cells expressed doublecortin in vivo indicating the stem cells were capable of differentiating into neurons. Together, we demonstrate that transient opening of the BBB with MRIgFUS is sufficient for transplantation of stem cells from the blood to targeted brain structures. These results suggest that MRIgFUS may be an effective alternative to invasive intracranial surgery for stem cell transplantation.     

Harnessing the therapeutic potential of myogenic stem cells

The potential clinical use of stem cells for cell transplantation therapies to replace defective genes in myopathies is an area of intense investigation. Precursor cells derived from non-muscle tissue with myogenic potential have been identified in many tissues, including bone marrow and dermis, although the status of these putative stem cells requires clarification. The incorporation of circulating bone-marrow derived stem cells into regenerating adult skeletal muscle has been demonstrated in mice but the contribution of donor cells is so minimal that it would appear clinically irrelevant at this stage. The possibility of a true stem cell subpopulation within skeletal muscle that replenishes the satellite cells (conventional muscle precursors on the surface of myofibres) is also very attractive as a superior source of myoblasts for muscle construction. A full understanding of the intrinsic factors (i.e. gene expression within the stem cell) and extrinsic factors (i.e. signals from the external environment) which control the commitment of stem cells to the myogenic lineage, and the conditions which favour stem cell expansion in vivo is required before stem cells can be seriously considered for clinical cell therapy. This revised version was published online in July 2006 with corrections to the Cover Date.     

Hedgehog signaling regulates the generation of ameloblast progenitors in the continuously growing mouse incisor

In many organ systems such as the skin, gastrointestinal tract and hematopoietic system, homeostasis is dependent on the continuous generation of differentiated progeny from stem cells. The rodent incisor, unlike human teeth, grows throughout the life of the animal and provides a prime example of an organ that rapidly deteriorates if newly differentiated cells cease to form from adult stem cells. Hedgehog (Hh) signaling has been proposed to regulate self-renewal, survival, proliferation and/or differentiation of stem cells in several systems, but to date there is little evidence supporting a role for Hh signaling in adult stem cells. We used in vivo genetic lineage tracing to identify Hh-responsive stem cells in the mouse incisor and we show that sonic hedgehog (SHH), which is produced by the differentiating progeny of the stem cells, signals to several regions of the incisor. Using a hedgehog pathway inhibitor (HPI), we demonstrate that Hh signaling is not required for stem cell survival but is essential for the generation of ameloblasts, one of the major differentiated cell types in the tooth, from the stem cells. These results therefore reveal the existence of a positive-feedback loop in which differentiating progeny produce the signal that in turn allows them to be generated from stem cells.     

Identification and origin of the germline stem cells as revealed by the expression of nanos-related gene in planarians

The planarian's remarkable regenerative ability is thought to be supported by the stem cells (neoblasts) found throughout its body. Here we report the identification of a subpopulation of neoblasts, which was revealed by the expression of the nanos-related gene of the planarian Dugesia japonica, termed Djnos. Djnos-expressing cells in the asexual planarian were distributed to the prospective ovary or testes forming region in the sexual planarian. During sexualization, Djnos-expressing cells produce germ cells, suggesting that in the asexual state these cells were kept as germline stem cells for the oogonia and spermatogonia. Interestingly, the germline stem cells were indistinguishable from the neoblasts by morphology and X-ray sensitivity and did not seem to contribute to the regeneration at all. Germline stem cells initially appear in the growing infant planarian, suggesting that germline stem cells are separated from somatic stem cells in the planarian. Thus, planarian neoblasts can be classified into two groups; somatic stem cells for regeneration and tissue renewal, and germline stem cells for production of germ cells during sexualization. However, Djnos-positive cells appeared in the newly formed trunk region from the head piece, suggesting that somatic stem cells can convert to germline stem cells.     

TWEAK/Fn14信号调控干细胞增殖与分化的作用与机制

肿瘤坏死因子样弱凋亡诱导蛋白(TWEAK)是肿瘤坏死因子(TNF)超家族成员,通过作用于唯一受体成纤维细胞生长因子14(Fn14)调控细胞的增殖、分化和迁移等多种生命活动。近来研究表明,TWEAK/Fn14信号可以作用于多种干细胞,如肝干细胞、神经干细胞和间充质干细胞等,通过影响其增殖与分化的能力,干预组织的修复与再生。对该领域的研究进行综述,将有助于揭示TWEAK/Fn14信号调控干细胞增殖与分化的作用与机制,并为干细胞在疾病发生机制等基础研究、细胞治疗和组织工程等临床医学研究提供新的方向。     

Involvement of ERK phosphorylation in the prevention of ischemia-induced ovarian follicular depletion by stem cells

Ovarian failure is commonly caused by aging, autoimmune disease, menopause and cancer therapy. We used an ischemic model in the ovary to test the hypothesis that stem cells are helpful for ovarian regeneration after injury. Three treatment regimes were employed: sham-operated control, ligation plus PBS, and ligation plus immortalized human bone marrow stromal cells (stem cells) groups. After ligation-induced ischemia, stem cells or PBS were injected into rat ovaries. Then, pregnant mare serum gonadotropin was given intra-peritoneally to initiate folliculogenesis. The animals were then sacrificed. The ovary gland was weighed, and ovarian folliculogenesis, stem cell differentiation and vascular neogenesis were evaluated. In order to study improvement of folliculogenesis after ovarian ischemia, steroidogenic acute regulatory protein (StAR), p44/p42 MAPK (T-ERK1/2), and phospho-p44/ p42 MAPK (P-ERK1/2) expression were specifically evaluated. Results indicated that ovarian size was smaller and that the rate of folliculogenesis was lower in ovarian ischemic-reperfusion animals, but both recovered after stem cell treatment. The stem cells migrated into the ovary and differentiated into theca cells, granulosa cells, corona radiata cells and vascular endothelial cells. In addition, von Willebrand factor (vWF) expression was increased; 17beta-estradiol (E2), progesterone (P4), P-ERK1/2 and StAR protein expression was recovered by stem cells treatment in the ischemic ovaries. The serum LH was significantly increased in ovaries of ischemia-reperfusion animals, but the stem cell treatment restored the effects. These results suggest that stem cells might be helpful for ovarian regeneration after injuries by promoting vascular neogenesis and steroidogenesis through the MAPK pathway.     

Investigating the mincing method for isolation of adipose-derived stem cells from pregnant women fat

The success of stem cell application in regenerative medicine, usually require a stable source of stem or progenitor cells. Fat tissue represents a good source of stem cells because it is rich in stem cells and there are fewer ethical issues related to the use of such stem cells, unlike embryonic stem cells. Therefore, there has been increased interest in adipose-derived stem cells (ADSCs) for tissue engineering applications. Here, we aim to provide an easy processing method for isolating adult stem cells from human adipose tissue harvested from the subcutaneous fat of the abdominal wall during gynecologic surgery. We used a homogenizer to mince fat and compared the results with those obtained from the traditional cut method involving a sterile scalpel and forceps. Our results showed that our method provides another stable and quality source of stem cells that could be used in cases with a large quantity of fat. Furthermore, we found that pregnancy adipose-derived stem cells (P-ADSCs) could be maintained in vitro for extended periods with a stable population doubling and low senescence levels. P-ADSCs could also differentiate in vitro into adipogenic, osteogenic, chondrogenic, and insulin-producing cells in the presence of lineage-specific induction factors. In conclusion, like human lipoaspirates, adipose tissues obtained from pregnant women contain multipotent cells with better proliferation and showed great promise for use in both stem cell banking studies as well as in stem cell therapy.     

Characterizing the Radioresponse of Pluripotent and Multipotent Human Stem Cells

The potential capability of stem cells to restore functionality to diseased or aged tissues has prompted a surge of research, but much work remains to elucidate the response of these cells to genotoxic agents. To more fully understand the impact of irradiation on different stem cell types, the present study has analyzed the radioresponse of human pluripotent and multipotent stem cells. Human embryonic stem (ES) cells, human induced pluripotent (iPS) cells, and iPS-derived human neural stem cells (iPS-hNSCs) cells were irradiated and analyzed for cell survival parameters, differentiation, DNA damage and repair and oxidative stress at various times after exposure. While irradiation led to dose-dependent reductions in survival, the fraction of surviving cells exhibited dose-dependent increases in metabolic activity. Irradiation did not preclude germ layer commitment of ES cells, but did promote neuronal differentiation. ES cells subjected to irradiation exhibited early apoptosis and inhibition of cell cycle progression, but otherwise showed normal repair of DNA double-strand breaks. Cells surviving irradiation also showed acute and persistent increases in reactive oxygen and nitrogen species that were significant at nearly all post-irradiation times analyzed. We suggest that stem cells alter their redox homeostasis to adapt to adverse conditions and that radiation-induced oxidative stress plays a role in regulating the function and fate of stem cells within tissues compromised by radiation injury.     

人胚胎干细胞向生殖细胞分化的研究进展

小鼠胚胎干细胞体外已成功诱导分化为配子细胞,人胚胎干细胞理论上也具备分化为生殖细胞的潜能。本文从影响人胚胎干细胞体外向生殖系分化的基因调控和干细胞小生境（niche）方面进行综述,并指出胚胎干细胞在生殖医学及不孕治疗中的研究方向和应用前景。     

Genetic ablation of caveolin-1 increases neural stem cell proliferation in the subventricular zone (SVZ) of the adult mouse brain

Adult neural stem cells are self-renewing multipotent cells that have the potential to replace dysfunctional and/or dying neuronal cells at the site of brain injury or degeneration. Caveolins are well-known tumor-suppressor genes that were recently found to be involved in the regulation of stem cell proliferation. For instance, ablation of the caveolin-1 (Cav-1) gene in mice markedly increases the proliferation of intestinal and mammary stem cells. However, the roles of caveolins in the proliferation of adult neural stem cells still remain unknown. In this study, dual-label immunofluorescence analysis of the proliferation marker, Ki67, and the stem cell markers, nestin and Sox2, was performed on brains of 8 week-old wild-type (WT) and Cav-1 knockout (KO) mice. Our results demonstrate an increased number of Ki67-positive nuclei in the subventricular zone (SVZ) of Cav-1 KO brains. Importantly, our dual-label immunofluorescence analyses demonstrate increased co-localization of Ki67 with both nestin and Sox2 in the SVZ of Cav-1 KO brains. Remarkably similar results were also obtained with Cav-2 and Cav-3 KO mouse brains as well, with increased proliferation of adult neural stem cells. Thus, the SVZ of caveolin KO mouse brains displays an increased proliferation of adult neural stem cells. Caveolin proteins might represent new crucial regulators of adult neural stem cell proliferation.     

不同防冻剂对兔胚胎干细胞慢速冷冻保存的影响

干细胞冷冻保存是干细胞研究和临床应用中的必需技术。为提高兔胚胎干细胞在慢速冻存过程中的保存效果,比较了二甲基亚砜(DMSO)和乙二醇(ethylene glycol,EG)对兔胚胎干细胞冷冻保护效果。对冷冻复苏后的细胞进行台盼蓝染色,并研究其胚胎干细胞分子特性,结果表明DMSO比EG具有更好的冷冻保护效果。再在以10%DMSO为基础的防冻液中添加膜稳定剂海藻糖(trehalose)或谷氨酰胺(glutamine),细胞冷冻复苏后结果显示,谷氨酰胺对兔胚胎干细胞有明显的冷冻保护作用,使细胞存活率从71%提高到83.7%。当谷氨酰胺浓度为0、5、10、20、40mmol/L分别加入防冻液中后,20mmol/L的谷氨酰胺具有最佳的冷冻保护效果。以上结果得出兔胚胎干细胞慢速冷冻的防冻液改进配方为:在胚胎干细胞培养液中添加10%DMSO 20mmol/L谷氨酰胺。     

Factors that control the differentiation of stem cells. I. The change in direction of hematopoietic stem cell differentiation under the effect of differentiating T-lymphocytes]

A mixed transplantation of bone marrow cells, and lymph nodes or thymic cells of mice CBA strain into lethally irradiated hybrid recipients (CBAXC57B1)F1 is accompanied with changes in the differentiation pattern from a mainly erythroid to a mainly granuloid way. Thymectomy of either donor of bone marrow cells or recipients, or both, destroys the stem cell differentiation in the direction of granulopoieseis. Intact syngeneic lymphocytes normalize differentiation of the stem cells, but in the presence of tissue antigens these provide for the stem cell differentiation mainly in the direction of granulopoiesis. The differentiation of stem haemopoietic cells is accomplished under the thymic and lymphocyte control. T-differentiating lymphocytes (Td) are the lymphocytes controlling the stem cell differentiation.     

The discovery of the matrix cell, the identification of the multipotent neural stem cell and the development of the central nervous system

In the early 1960s I applied 3H-thymidine autoradiography to the study of the cells constituting the neural tube, and found that its wall was composed solely of one kind of single-layered epithelial cell, which perform an elevator movement between the mitotic and DNA-synthetic zones in the wall in accord with the cell cycle. They were identified as multipotent stem cells of the central nervous sytem (CNS) to which I gave the name of matrix cells. (3)H-thymidine autoradiography also revealed the chronology of development of these matrix cells: At first they proliferate only to expand the population (stage I), then switch to differentiate specific neuroblasts in given sequences (stage II), and finally change themselves into ependymoglioblasts, common progenitors of ependymal cells and neuroglia (stage III). Based on these findings, I proposed a monophyletic view of cytogenesis of the central nervous sytem. This matrix cell theory claiming the existence of multipotent stem cells has long been the target of severe criticism and not been accepted among neuro-embryologists for a long time. Recent findings by experimental and clinical neuroscientists on the importance of stem cells have renewed interest in the nature and biology of the multipotent neural stem cells. The present paper describes how the concept of the matrix cell (multipotent neural stem cells in vivo) emerged and what has come out from this view over the last 45 years, and how the basic concept of the matrix cell theory has recently been reconfirmed after a long period of controversy and neglect.     

Variation in the sensitivity of the mouse spermatogonial stem cell population to fission neutron irradiation during the cycle of the seminiferous epithelium

Dose-response studies of the radiosensitivity of spermatogonial stem cells in various epithelial stages after irradiation with graded doses of fission neutrons of 1 MeV mean energy were carried out in the Cpb-N mouse. These studies on the stem cell population in stages IX-XI yielded simple exponential lines characterized by an average D0 value of 0.76 +/- 0.02 Gy. In the subsequent epithelial stages XII-III, a significantly lower D0 value of 0.55 +/- 0.02 Gy was found. In contrast to the curves obtained for stem cells in stages IX-III, the curves obtained in stages IV-VIII indicated the presence of a mixture of radioresistant and radiosensitive stem cells. In stage VII, almost no radioresistant stem cells appeared to be present and a D0 value for the radiosensitive stem cells of 0.22 +/- 0.01 Gy was derived. Previously, data were obtained on the size of colonies (in number of spermatogonia) derived from surviving stem cells. Combining these data with data from the newly obtained dose-response curves yielded the number of stem cells, per stage and with the specific radiosensitivities, present in the control epithelium. In stages IX-XI, there are approximately 6 stem cells per 1000 Sertoli cells with a radiosensitivity characterized by a D0 of 0.76 Gy, which corresponds to one-third of the As population in these stages. (The As spermatogonia are presumed to be the stem cells of spermatogenesis.) IN stages XII-III, there are approximately 12 stem cells per 1000 Sertoli cells with a radiosensitivity characterized by a D0 of 0.55 Gy, which roughly equals the number of A single spermatogonia in these stages. These calculations could not be made for stages IV-VIII since no simple exponential lines were obtained for these stages. In view of the pattern of the proliferative activity of the spermatogonial stem cells during the epithelial cycle, it appears that the stem cell population is most radiosensitive during the period when the majority of these cells are in G0 phase, most resistant when the cells are stimulated again into proliferation, and of intermediate sensitivity during active proliferation.