Identification and regulation of a stage-specific stem cell niche enriched by Nanog-positive spermatogonial stem cells in the mouse testis

The ability of spermatogonial stem cells to acquire embryonic stem cell (ESC) properties in vitro has recently been of great interest. However, studies focused on the in vivo regulation of testicular stem cells have been hampered because the exact anatomical location of these cells is unknown. Moreover, no specialized stem cell niche substructure has been identified in the mammalian testis thus far. It has also been unclear whether the adult mammalian testis houses pluripotent stem cells or whether pluripotency can be induced only in vitro. Here, we demonstrate, for the first time, the existence of a Nanog-positive spermatogonial stem cell subpopulation located in stage XII of the mouse seminiferous epithelial cycle. The efficiency of the cells from seminiferous tubules with respect to prolonged pluripotent gene expression was correlated directly with stage-specific expression levels of Nanog and Oct4, demonstrating the previously unknown stage-specific regulation of undifferentiated spermatogonia (SPG). Testicular Nanog expression marked a radioresistant spermatogonial subpopulation, supporting its stem cell nature. Furthermore, we demonstrated that p21 acts as an upstream regulator of Nanog in SPG and mouse ESCs, and our results demonstrate that promyelocytic leukemia zinc finger is a specific marker of progenitor SPG. Additionally, we describe a novel method to cultivate Nanog-positive SPG in vitro. This study demonstrates the existence and location of a previously unknown stage-specific spermatogonial stem cell niche and reports the regulation of radioresistant spermatogonial stem cells.     

N-Cadherin is expressed by putative stem/progenitor cells and melanocytes in the human limbal epithelial stem cell niche

Corneal epithelial stem cells are known to be localized to the basal layer of the limbal epithelium, providing a model system for epithelial stem cell biology; however, the mechanisms regarding the maintenance of these stem cells in their specialized niche remain poorly understood. N-cadherin is a member of the classic cadherin family and has previously been demonstrated to be expressed by hematopoietic stem cells. In the present study, we demonstrate that N-cadherin is expressed by putative stem/progenitor cells, as well as melanocytes, in the human limbal epithelial stem cell niche. In addition, we demonstrate that upon in vitro culture using 3T3 feeder layers, loss of N-cadherin expression occurs with cell proliferation. These results indicate that N-cadherin may be a critical cell-to-cell adhesion molecule between corneal epithelial stem/progenitor cells and their corresponding niche cells in the limbal epithelium.     

Hematopoietic stem cell engraftment by early-stage in utero transplantation in a mouse model

A novel intrauterine transplantation (IUT) approach was developed to improve the efficiency of engraftment of hematopoietic stem cells (HSCs). HSCs with a green fluorescent protein (GFP) reporter gene were transplanted in utero on days 12.5, 13.5 and 14.5 post coitum (p.c.). The degree of chimerism of donor cells in recipient newborn mice was examined using fluorescent microscopy, polymerase chain reaction (PCR), fluorescence-activated cell sorting (FACS), and fluorescence in situ hybridization (FISH) analyses. Microscopic examination revealed the presence of green fluorescent signal in the peripheral blood of the chimeric mice. The highest survival rate (47%) as well as the highest chimerism rate (73%) were achieved by our new approach in the newborn mice that were subjected to in utero transplantation (IUT) on day 12.5 p.c. (E12.5) compared to the conventional IUT method. FACS analysis indicated that 1.55 ± 1.10% of peripheral blood cells from the newborn mice were GFP-positive donor cells. FISH showed that cells containing the donor-specific GFP sequence were present in the bone marrow (BM) of the chimeric mice. Thus, the efficiency of chimera production with this new method of IUT was significantly improved over the existing IUT techniques and instruments.     

Revealing the glioma cancer stem cell interactome,one niche at a time

Glioblastoma (GBM) cancer stem cells (CSCs) are insidious. They extensively infiltrate brain tissue, resist radiotherapy and chemotherapy, and are thought to represent the ultimate drivers of disease progression. New research has identified CD109, a GPI‐anchored protein, on a population of perivascular CSCs. Investigation of primary human tumour tissue suggests a role for CD109‐expressing CSCs in the progression from low‐grade to high‐grade glioma, and animal modelling reveals a critical role for CD109 in the maintenance of the GBM CSC phenotype. Furthermore, CD109‐expressing CSCs appear to drive the proliferation of adjacent non‐stem tumour cells (NSTCs) in a rare example of CSC–NSTC cooperative interaction. With this Commentary, we highlight the newly revealed biology of CD109, and offer a synthesis of the published information on glioma CSCs in a variety of anatomical growth zones. We also discuss the landscape of interacting cells within GBM tumours, emphasizing the few reported examples of pro‐tumourigenic, interactive tumour cell partnerships, as well as a variety of tumour cell–non‐transformed neural cell interactions. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Hematopoietic stem cells regulate mesenchymal stromal cell induction into osteoblasts thereby participating in the formation of the stem cell niche

Crosstalk between hematopoietic stem cells (HSCs) and the cells comprising the niche is critical for maintaining stem cell activities. Yet little evidence supports the concept that HSCs regulate development of the niche. Here, the ability of HSCs to directly regulate endosteal development was examined. Marrow was isolated 48 hours after "stressing" mice with a single acute bleed or from control nonstressed animals. "Stressed" and "nonstressed" HSCs were cocultured with bone marrow stromal cells to map mesenchymal fate. The data suggest that HSCs are able to guide mesenchymal differentiation toward the osteoblastic lineage under basal conditions. HSCs isolated from animals subjected to an acute stress were significantly better at inducing osteoblastic differentiation in vitro and in vivo than those from control animals. Importantly, HSC-derived bone morphogenic protein 2 (BMP-2) and BMP-6 were responsible for these activities. Furthermore, significant differences in the ability of HSCs to generate a BMP response following stress were noted in aged and in osteoporotic animals. Together these data suggest a coupling between HSC functions and bone turnover as in aging and in osteoporosis. For the first time, these results demonstrate that HSCs do not rest passively in their niche. Instead, they directly participate in bone formation and niche activities. Disclosure of potential conflicts of interest is found at the end of this article.     

3D co-culture of hematopoietic stem and progenitor cells and mesenchymal stem cells in collagen scaffolds as a model of the hematopoietic niche

Here, we propose a collagen-based three-dimensional (3D) environment for hematopoietic stem and progenitor cells (HPC) with mesenchymal stem cells (MSC) derived either from bone marrow (BM) or umbilical cord (UC), to recapitulate the main components of the BM niche. Mechanisms described for HPC homeostasis were systematically analyzed in comparison to the conventional liquid HPC culture. The 3D-cultivation allows dissecting two sub-populations of HPC: (I) HPC in suspension above the collagen gel and (II) migratory HPC in the collagen fibres of the collagen gel. The different sites represent distinct microenvironments with significant impact on HPC fate. HPC in niche I (suspension) are proliferative and a dynamic culture containing HPC (CD34⁺/CD38^-), maturing myeloid cells (CD38⁺, CD13⁺, CAE⁺) and natural killer (NK) cells (CD56⁺). In contrast, HPC in niche II showed clonal growth with significant high levels of the primitive CD34⁺/CD38^- phenotype with starting myeloid (CD13⁺, CAE⁺) differentiation, resembling the endosteal part of the BM niche. In contrast, UC-MSC are not adequate for HSC expansion as they significantly enhance HPC proliferation and lineage commitment. In conclusion, the 3D-culture system using collagen and BM-MSC enables HPC expansion and provides a potential platform to dissect regulatory mechanisms in hematopoiesis.     

The porcine limbal epithelial stem cell niche as a new model for the study of transplanted tissue-engineered human limbal epithelial cells

Transparency of the human cornea is dependent upon the integrity of its epithelium and hence a population of limbal epithelial stem cells (LESCs). We have previously shown that LESCs reside in limbal epithelial crypts at the periphery of the human cornea. In this study the anatomy and functionality of the porcine limbus was evaluated for the first time as a novel model of the human limbus. Scanning electron microscopy, confocal microscopy, and histology revealed common structures in the porcine and human limbus in terms of the location and topography of palisades of Vogt and limbal epithelial crypts. Epithelial cells harvested from crypt regions achieved higher colony forming efficiency than cultures established from the noncrypt regions and central cornea. Also, expression of the putative SC markers p63α and integrin β1 brightness was higher in the basal layer of the crypt regions, as shown by immunocytochemistry. De-epithelialized porcine corneas were used as an in vitro organ culture model to study the fate of transplanted human epithelium cultured from the limbus. Multilayered epithelium was observed after ～1 week. Subsequently, wounds were inflicted on the central corneal epithelium. The wounded tissue healed within 5-7 days, and multilayering of the central corneal epithelium was re-established. The transplanted epithelia were repeatedly wounded at least four times and the wounds healed by 1 week. Putative SC marker expression of the transplanted epithelia was confirmed using immunohistochemistry. These results demonstrate that the porcine limbus shares features with the human limbus and as such provides a suitable model for the study of cultured limbal epithelial cell transplantation. These data have significant clinical value as this model can provide information on LESC fate post-transplantation and their ability to respond to injury, which is not possible to study in patients.     

Differentiation of human embryonic stem cells into corneal epithelial-like cells by in vitro replication of the corneal epithelial stem cell niche

Human embryonic stem cells (hESCs) are pluripotent cells capable of differentiating into any cell type of the body. It has long been known that the adult stem cell niche is vital for the maintenance of adult stem cells. The cornea at the front of the eye is covered by a stratified epithelium that is renewed by stem cells located at its periphery in a region known as the limbus. These so-called limbal stem cells are maintained by factors within the limbal microenvironment, including collagen IV in basement membrane and limbal fibroblasts in the stroma. Because this niche is very specific to the stem cells (rather than to the more differentiated cells) of the corneal epithelium, it was hypothesized that replication of these factors in vitro would result in hESC differentiation into corneal epithelial-like cells. Indeed, here we show that culturing of hESC on collagen IV using medium conditioned by the limbal fibroblasts results in the loss of pluripotency and differentiation into epithelial-like cells. Further differentiation results in the formation of terminally differentiated epithelial-like cells not only of the cornea but also of skin. Scanning electron microscopy shows that some differences exist between hESC-derived and adult limbal epithelial-like cells, necessitating further investigation using in vivo animal models of limbal stem cell deficiency. Such a model of hESC differentiation is useful for understanding the early events of epithelial lineage specification and to the eventual potential application of epithelium differentiated from hESC for clinical conditions of epithelial stem cell loss. Disclosure of potential conflicts of interest is found at the end of this article.     

Non-invasive longitudinal tracking of human amniotic fluid stem cells in the mouse heart

Human stem cells from various sources have potential therapeutic applications. The clinical implementation of these therapies introduces the need for methods of noninvasive tracking of cells. The purpose of this study was to evaluate a high resolution magnetic resonance imaging (MRI) technique for in vivo detection and tracking of superparamagnetic micron sized iron oxide particle (MPIO)-labeled human amniotic fluid stem (hAFS) cells injected in the mouse heart. Because of the small subject size, MR signal and resolution of the in vivo MRI were increased using strong gradients, a 7.0 Tesla magnet, and an ECG and respiratory gated gradient echo sequence. MRI images of mouse heart were acquired during a 4 week course of this longitudinal study. At the end of the study, histological analysis was used to correlate cell localization with the MRI results. Introduction of MPIOs into hAFS had no significant effect upon cell proliferation and differentiation. Results of flow cytometry analysis indicated that hAFS cells remained labeled for up to 4 weeks. MRI of MPIO-labeled hAFS cells injected in agarose gels resulted in significant hypointense regions. Labeled hAFS cells injected into mouse hearts produced hypointense regions in the MR images that could be detected 24 hours and 7, 14, 21 and 28 days post injection. The co-localization of labeled cells within the hypointense regions was confirmed by histological analysis. These results indicate that high resolution MRI can be used successfully for noninvasive longitudinal tracking of hAFS cells injected in the mouse heart. The potential utility of this finding is that injected stem cells can be tracked in vivo and might serve to monitor cell survival, proliferation and integration into myocardial tissue.     

乳腺癌细胞株MCF-7/ADM中肿瘤干细胞的研究

目的： 通过对阿霉素耐药乳腺癌细胞系MCF-7/ADM中乳腺癌干细胞（breast cancer stem cells,BCSCs）成分分析，观察化疗耐药处理的MCF-7乳腺癌细胞株是否可高效富集乳腺癌干细胞，为研究乳腺癌的发病机制提供思路。方法: MTT法分别测定阿霉素耐药乳腺癌细胞系MCF-7/ADM及其亲本细胞株MCF-7对阿霉素的IC50，计算其耐药倍数。通过细胞侧群（side population,SP）分析、球囊培养、流式细胞仪检测MCF-7/ADM及MCF-7中CD+44CD-24细胞比例三方面鉴定MCF-7/ADM和MCF-7中乳腺癌干细胞比例。结果: 阿霉素耐药乳腺癌细胞系MCF-7/ADM相对于MCF-7对阿霉素的耐药倍数为37.1；MCF-7/ADM中SP细胞比例为（9.60±0.66）%，MCF-7细胞的SP细胞比例为（0.39±0.11）%；两者球囊形成率分别为 （10.27±0.64）%和（1.03±0.15）%；两者的CD+44CD-24细胞比例分别为（64.87±3.87）%和（3.70±0.53）%,差异显著（P<0.05）。结论: 阿霉素耐药乳腺癌细胞系MCF-7/ADM中乳腺癌干细胞比例明显高于MCF-7细胞。化疗耐药处理的MCF-7乳腺癌细胞株可高效富集乳腺癌干细胞，这对于乳腺癌发病机制的研究具有重要意义。     

Spatial domains of progenitor-like cells and functional complexity of a stem cell niche in the neonatal rat spinal cord

During spinal cord development, progenitors in the neural tube are arranged within spatial domains that generate specific cell types. The ependyma of the postnatal spinal cord seems to retain cells with properties of the primitive neural stem cells, some of which are able to react to injury with active proliferation. However, the functional complexity and organization of this stem cell niche in mammals remains poorly understood. Here, we combined immunohistochemistry for cell-specific markers with patch-clamp recordings to test the hypothesis that the ependyma of the neonatal rat spinal cord contains progenitor-like cells functionally segregated within specific domains. Cells on the lateral aspects of the ependyma combined morphological and molecular traits of ependymocytes and radial glia (RG) expressing S100β and vimentin, displayed passive membrane properties and were electrically coupled via Cx43. Cells contacting the ventral and dorsal poles expressed the neural stem cell markers nestin and/or vimentin, had the typical morphology of RG, and appeared uncoupled displaying various combinations of K(+) and Ca(2+) voltage-gated currents. Although progenitor-like cells were mitotically active around the entire ependyma, the proliferative capacity seemed higher on lateral domains. Our findings represent the first evidence that the ependyma of the rat harbors progenitor-like cells with heterogeneous electrophysiological phenotypes organized in spatial domains. The manipulation of specific functional properties in the heterogeneous population of progenitor-like cells contacting the ependyma may in future help to regulate their behavior and lineage potential, providing the cell types required for the endogenous repair of the injured spinal cord. Stem Cells2012;30:2020-2031.     

Determination of “settling factor” (F) by cloning of hematopoietic stem cells in the mouse spleen and bone marrow

The effects of distribution of injected colony-forming units (CFU) in the recipient were studied by the use of the settling factor (F) as the criterion. This factor was found from the number of colonies discovered in histological sections of the spleen and femoral bone marrow compared with the number of colonies distinguishable on the surface of the spleen. It was shown that the value of F both for the whole volume of the spleen and for the femoral marrow can be estimated by the method of macroscopic counting of colonies on the spleen. The value of F in this case was independent of the character of differentiation of the CFU. It was shown that the value of F can vary depending on the physiological state of the CFU population. After irradiation of bone marrow CFU in doses of 200, 400 and 600 R the value of F in the spleen was reduced, whereas in the bone marrow it was unchanged compared with the control.Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 86, No. 11, pp. 601–604, November, 1978.     

Interstitial interfaces show marked differences in regenerating tubules, matured tubules, and the renal stem/progenitor cell niche

Stem/progenitor cells are promising candidates for the regeneration of parenchyma in acute and chronic renal failure. After an implantation stem/progenitor cells must migrate through the interstitial space to concentrate at the site of damage. However, information is lacking to what extent the interstitial interface is influencing the development of stem/progenitor cells into nephron structures. In consequence, tubule regeneration within an artificial polyester interstitium was analyzed by electron microscopy in comparison with the interstitial interface of matured tubules and the interstitium within the renal stem/progenitor cell niche. The experiments demonstrate that fixation of specimens with glutaraldehyde (GA) is leading in all cases to inconspicuously looking interstitial interfaces. In contrast, fixation of regenerating tubules in GA containing ruthenium red and tannic acid shows a dense network of fibers lining along the basal lamina. In contrast, matured tubules reveal after ruthenium red label an extremely thickened basal lamina, while only a punctate pattern is obtained after tannic acid treatment. Finally, within the renal stem/progenitor cell niche ruthenium red and tannic acid label reveals large amounts of extracellular matrix spanning through the interstitium. Thus, fixation of tissue in GA containing ruthenium red and tannic acid exhibits an unexpectedly regional heterogeneity of the renal interstitial interface. This fact has to be considered for an optimal therapeutic repair of parenchyma, since contacts between stem/progenitor cells with the interstitial interface influence further development.     

C57BL/6J×129/J杂交小鼠ES细胞系的建株(英)

 目的：建立C57BL/6J×129/J杂交小鼠ES细胞系。 方法： 收集3.5 d.p.c.的囊胚，培养在预先铺有小鼠成纤维细胞（MEFs）的高糖DMEM培养液中。3-4 d后，挑出内细胞团（ICM），消化后重新种到新鲜的有MEFS培养液中。等到有典型的ES样集落长出，即传代以得到永久ES细胞系。通过分析碱性磷酸酶活性，SSEA-1，Oct-4的表达和形成畸胎瘤的能力来鉴定ES细胞的多向分化能力。 结果： 获得的两个C57BL/6J×129/J杂交小鼠ES细胞系绝大多数细胞具有正常的核型（40，XY），碱性磷酸酶染色阳性，SSEA-1，Oct-4表达阳性， ES细胞注入SCID鼠后可获得来自3个胚层的组织。 结论： 建立了两株具有长期自我更新能力和多向分化潜能的C57BL/6J×129/J杂交小鼠ES细胞系。     

The role of endothelial cells in the retinal stem and progenitor cell niche within a 3D engineered hydrogel matrix

Cell-cell interactions are critical to understanding functional tissues. A number of stem cell populations have been shown to receive key regulatory information from endothelial cells (ECs); however, the role of ECs in the retinal stem and progenitor cell (RSPC) niche has been largely unexplored. To gain greater insight into the role of ECs on RSPC fate, a three-dimensional (3D) co-culture model, incorporating cell-cell interactions, was designed by covalently-modifying agarose hydrogels with growth factors and cell-adhesive peptides in defined volumes. Therein ECs adopted tubular-like morphologies similar to those observed in vivo, but not observed in two-dimensional (2D) cultures. Unexpectedly, ECs inhibited proliferation and differentiation of RSPCs, revealing, for the first time, the possible role of ECs on RSPC fate. This 3D hydrogel scaffold provides a simple, reproducible and versatile method with which to answer biological questions related to the cellular microenvironment.     

Identification of a stem cell niche in the zone of Ranvier within the knee joint

A superficial lesion of the articular cartilage does not spontaneously self-repair and has been suggested to be partly due to lack of progenitor cells within the joint that can reach the site of injury. To study whether progenitor cells are present within the joint, 3-month-old New Zealand white rabbits were exposed to bromodeoxyuridine (BrdU) for 12 consecutive days and were then sacrificed 4, 6, 10, 14, 28 and 56 days after the first BrdU administration. Presence of BrdU and localization of progenitor markers were detected using immunohistochemistry. After 10 days of BrdU exposure, BrdU-positive cells, i.e. proliferating cells, were abundantly detected in the epiphyseal plate, the perichondrial groove of Ranvier, and in all zones of the articular cartilage. After a wash-out period, BrdU-positive cells were still present, i.e. those considered to be progenitor cells, in these regions of the knee except for the proliferative zone of the epiphyseal plate. Cells in the perichondrial groove of Ranvier were further positive for several markers associated with progenitor cells and stem cell niches, including Stro-1, Jagged1, and BMPr1a. Our results demonstrate that a small population of progenitor cells is present in the perichondrial groove of Ranvier as well as within the articular cartilage in the knee. The perichondrial groove of Ranvier also demonstrates the properties of a stem cell niche.