Distribution and role of CD34-positive stromal cells and myofibroblasts in human normal testicular stroma

CD34-positive stromal cells are distributed in various organs including breast, Fallopian tubes, thyroid gland, colon, pancreas, and uterine cervix. To elucidate the distribution of CD34-positive stromal cells, smooth muscle cells, and myofibroblasts in normal human testis, we examined 48 testes obtained by autopsy and operation, including five fetal, one neonatal, and 42 adult cases without evident testicular lesions, using a streptavidin-biotin immunoperoxidase technique. The expression of alpha-smooth muscle actin (ASMA), h-caldesmon, CD34, and CD31 were immunohistochemically examined in all cases. The tunica albuginea and the inner layer of seminiferous tubules in adult testis were predominantly composed of myofibroblasts. Smooth muscle cells were also scattered throughout these sites in some cases. CD34-positive stromal cells were abundant, and they formed a reticular network around the seminiferous tubules and Leydig cells as well as the outer layer of seminiferous tubules. Moreover, myofibroblasts and the CD34 reticular network were already present in the testicular stroma during fetal or neonatal development. Double immunostaining of fetal, neonatal and adult testes using ASMA and CD34 confirmed that myofibroblasts and CD34-positive stromal cells were present in the inner and outer layers of peritubular tissue, respectively. This distribution and cytological identification was also confirmed by an ultrastructural study of four cases. Finally, CD34-positive stromal cells and myofibroblasts are major components of human testicular stroma.     

The distribution and role of myofibroblasts and CD34-positive stromal cells in normal pancreas and various pancreatic lesions

To elucidate the distribution and role of myofibroblasts and CD34-positive stromal cells in various pancreatic lesions, we performed an immunohistochemical study using a streptoavidin-biotin immunoperoxidase technique. We selected 43 pancreatic lesions from 1 biopsied, 22 surgically resected and 12 autopsied specimens: acute pancreatitis (n=3), chronic non-obstructive pancreatitis (n=4), obstructive pancreatitis (n=7), islet cell tumor (n=4), serous cystadenoma (n=7), mucinous cystadenoma (n=6), and invasive ductal carcinoma (n=12). In normal pancreas, myofibroblasts and CD34-positive stromal cells were predominantly present in the peridcutal and periacinar areas, respectively. Both myofibroblasts and CD34-positive cells were observed in the stroma of chronic pancreatitis. In four islet cell tumors, myofibroblasts were present in the stroma of the tumor center, but no CD34-positive stromal cells were identified. Additionally, myofibroblasts and CD34-positive stromal cells were located in the inner layer and the outer layer of the capsule of three islet cell tumors, respectively. In nine of the thirteen cystadenomas, only myofibroblasts were recognized in the cyst wall. In the remaining four cystadenomas, a small number of CD34-positive cells were observed in the cyst wall. In 12 invasive ductal carcinomas, the stroma possessed a lot of myofibroblasts, but there were no or few CD34-positive stromal cells. In conclusion, it seems that the abundant amount of CD34-stromal cells in the main lesions is characteristic of chronic inflammatory lesions. Myofibroblasts and CD34-positive stromal cells may play a role in regulating the tumor growth in the capsule of islet cell tumors of the pancreas.     

The distribution of CD34-positive stromal cells and myofibroblasts in colorectal carcinoid tumors

In order to understand the stromal reaction associated with colorectal neoplasms, we examined specimens from 26 patients including normal colorectal tissues (n=15), carcinoid tumors (n=12), well differentiated adenocarcinomas (n=10), and poorly differentiated adenocarcinomas (n=4), using an immunohistochemical method. Myofibroblasts and CD34-positive stromal cells were distributed in the mucosa and in the area between the submucosal and subserosal layers, respectively. However, the distribution of these cells markedly changed with the invasion of neoplasms. Namely, myofibroblasts were abundant in the invasive stroma of all colorectal neoplasms. CD34-positive stromal cells were completely absent from the invasive stroma of colorectal cancers. On the other hand, CD34-positive stromal cells were absent from four out of five carcinoid tumor cases with lesions measuring less than 2 mm in size, but were present in all seven cases of carcinoid tumors measuring more than 2 mm. Double-immunostaining identified stromal cells expressing both ASMA and CD34 in several carcinoid tumor cases. Finally, no CD34-positive stromal cells were observed in the invasive stroma of colorectal cancers. However, the distribution of these cells in carcinoid tumors may depend on the lesion size. Namely, CD34-positive stromal cells existed between neoplastic nests in large-sized carcinoid tumors. Myofibroblasts in the stroma of colorectal neoplasms may originate from CD34-positive stromal cells.     

The distribution of myofibroblasts and CD34-positive stromal cells in normal renal pelvis and ureter and their cancers

In this article, we examined the distribution of myofibroblasts and CD34-positive stromal cells in normal renal pelvis and ureter and their cancers using immunohistochemistry. Eighteen tumors and normal tissues apart from the main tumor were examined. In the wall of normal renal pelvis and ureter, no myofibroblasts were observed through all layers, but CD34-positive stromal cells were observed in the deep area of lamina propria, muscular layer and adventitia. In the stroma of renal pelvic and ureteral cancers, myofibroblasts were distributed in fifteen tumors and were absent in three tumors. All three tumors containing no myofibroblasts in the stroma were non-invasive type and all invasive cancers contained myofibroblasts in the stroma. CD34-positive stromal cells were consistently absent in the stroma of cancers, irrespective of the invasiveness. Finally, myofibroblasts are major stromal components in renal pelvic and ureteral cancers, particularly in invasive cancers, and CD34-positive stromal cells are consistently absent or lost in the stroma of their cancers. These findings suggest that the invasion of renal pelvic and ureteral cancers may cause the phenotypic change of stromal cells.     

The disappearance of CD34-positive and alpha-smooth muscle actin-positive stromal cells associated with human intra-uterine and tubal pregnancies

In order to elucidate the change in alpha-smooth muscle actin (ASMA)-positive and CD34-positive stromal cells associated with pregnancy, we examined endometrial and Fallopian tube tissues from 40 patients including normal endometrium (n=10), intra-uterine pregnancy (n=10), normal Fallopian tube (n=10), and tubal pregnancy (n=10), using immunohistochemistry. In normal endometrium, only a few ASMA-positive cells were focally observed. Additionally, a wide range of CD34-positive stromal cell abundance was observed. In normal Fallopian tube mucosa, a small to moderate number of both ASMA-positive and CD34-positive stromal cells was observed. Neither ASMA-positive nor CD34-positive stromal cells were observed anywhere in the decidual stroma during both intra-uterine and tubal pregnancies. Likewise, a varying abundance of ASMA-positive cells but no CD34-positive stromal cells were observed at the fetal side during both intra-uterine and tubal pregnancies. In conclusion, the disappearance of CD34-positive and ASMA-positive stromal cells may be an indicator of decidualisation induced change in the stroma during both intra-uterine and tubal pregnancies. ASMA-positive stromal cells at the fetal side associated with pregnancy may play a role in the production of villous extracellular matrix or regulation of blood flow.     

The appearance of myofibroblasts and the disappearance of CD34-positive stromal cells in the area adjacent to xanthogranulomatous foci of chronic cholecystitis

We investigated the distribution of myofibroblasts and CD34-positive stromal cells in normal gallbladder and its pathological conditions (cholecystitis, n=25) using immunohistochemistry and in situ hybridization. In the wall of normal gallbladder, myofibroblasts were generally absent from all layers, but many CD34-positive stromal cells were observed in the connective tissue layer. In chronic cholecystitis with mild perimuscular fibrosis, a small to moderate number of myofibroblasts appeared in the mucosal layer. In chronic cholecystitis with marked perimuscular fibrosis, a small to large number of myofibroblasts appeared predominantly in the connective tissue layer, whereas the number of CD34-positive stromal cells decreased at the same location, although the number of myofibroblasts increased. In chronic cholecystitis with xanthogranulomatous foci, a small to large number of myofibroblasts were observed in the periphery of the xanthogranulomatous reaction and adjacent area. In contrast, CD34-positive stromal cells were completely absent or were limited to the area just around the xanthogranulomatous reaction. Induction of collagen type I and III mRNA was predominantly observed in the cytoplasm of myofibroblasts associated with the marked fibrosis, which consisted primarily of mature collagen fibers, and in the cytoplasm of myofibroblasts around the xanthogranulomatous reaction, respectively. Finally, myofibroblasts were observed in all subtypes. The increased number of myofibroblasts was most prominent in the connective tissue layer of chronic cholecystitis with marked perimuscular fibrosis or in the area adjacent to xanthogranulomatous foci of chronic cholecystitis. Under these conditions, CD34-positive stromal cells tended to disappear from the connective tissue layer, which exhibited an increase in myofibroblasts.     

Oocyte-like cells induced from CD34-positive mouse hair follicle stem cells in vitro

<正>Adult stem cells have been identified in a variety of mammalian organs including skin,hair follicles,pancreas,and bone marrow(Kruse et al.,2004).These stem cells reside in a specific cellular environment where they remain in an undifferentiated state(Theise,2006).In addition,they are generally considered to be multipotent,possessing the capacity to generate multiple cell types     

In vitro generation of murine myeloid dendritic cells from CD34-positive precursors

Dendritic cells (DCs) link the innate and adaptive immune system. Currently, murine DCs for cell biology investigations are developed from MHC class II-negative bone marrow (BM) precursor cells, non-depleted BM cells or BM monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Here we demonstrate an isolation procedure of functionally intact myeloid CD11c⁺ CD11b⁺ DCs derived from murine CD34-positive precursors. DCs derived from CD34⁺ cells show functional internalization, maturation, cytokine secretion, MHC-restricted antigen presentation, and MHCII retrograde transport of antigens from the lysosomes to the cell surface. In comparison to the established method, the advantages of this isolation procedure are a shorter cultivation period, a superior transfection efficiency, the yield of a purer and more homogeneous population of immature DCs, and less consumption of cell culture medium and GM-CSF. The new isolation procedure and the functional quality of CD34⁺ cell-derived murine myeloid DCs make them ideally suited for immunology and cell biology studies.     

Reconstitution activity of hypoxic cultured human cord blood CD34-positive cells in NOG mice

Hematopoietic stem cells (HSCs) reside in hypoxic areas of the bone marrow. However, the role of hypoxia in the maintenance of HSCs has not been fully characterized. We performed xenotransplantation of human cord blood cells cultured in hypoxic or normoxic conditions into adult NOD/SCID/IL-2Rγ^null (NOG) mice. Hypoxic culture (1% O₂) for 6 days efficiently supported the maintenance of HSCs, although cell proliferation was suppressed compared to the normoxic culture. In contrast, hypoxia did not affect in vitro colony-forming ability. Upregulation of a cell cycle inhibitor, p21, was observed in hypoxic culture. Immunohistochemical analysis of recipient bone marrow revealed that engrafted CD34⁺CD38⁻ cord blood HSCs were hypoxic. Taken together, these results demonstrate the significance of hypoxia in the maintenance of quiescent human cord blood HSCs.     

Characterization of cancer stem cell properties of CD24 and CD26-positive human malignant mesothelioma cells

Malignant mesothelioma (MM) is an asbestos-related malignancy characterized by rapid growth and poor prognosis. In our previous study, we have demonstrated that several cancer stem cell (CSC) markers correlated with CSC properties in MM cells. Among these markers, we focused on two: CD24, the common CSC marker, and CD26, the additional CSC marker. We further analyzed the CSC properties of CD24 and CD26-positve MM cells. We established RNAi-knockdown cells and found that these markers were significantly correlated with chemoresistance, proliferation, and invasion potentials in vitro. Interestingly, while Meso-1 cells expressed both CD24 and CD26, the presence of each of these two markers was correlated with different CSC property. In addition, downstream signaling of these markers was explored by microarray analysis, which revealed that their expressions were correlated with several cancer-related genes. Furthermore, phosphorylation of ERK by EGF stimulation was significantly affected by the expression of CD26, but not CD24. These results suggest that CD24 and CD26 differentially regulate the CSC potentials of MM and could be promising targets for CSC-oriented therapy.     

Non-multipotent stroma inhibit the proliferation and differentiation of mesenchymal stromal cells in vitro

Background aimsThe ability to expand and maintain bone marrow (BM)-derived mesenchymal stem cells (MSC) in vitro is an important aspect of their therapeutic potential. Despite this, the exact composition of stromal cell types within these cultures and the potential effects of non-stem cells on the maintenance of MSC are poorly understood.MethodsC57BL/6J BM stroma was investigated as a model to determine the relationship between MSC and non-multipotent cells in vitro. Whole BM and single-cell derived cultures were characterized using flow cytometry and cell sorting combined with multipotent differentiation. Proliferation of individual stromal populations was evaluated using BrdU.ResultsAt a single-cell level, MSC were distinguished from committed progenitors, and cells lacking differentiation ability, by the expression of CD105 (CD105⁺). A 3-fold reduction in the percentage of CD105⁺ cells was detected after prolonged culture and correlated with loss of MSC. Depletion of CD105⁺ cells coincided with a 10–20% increase in the frequency of proliferating CD105^? cells. Removal of CD105^? stroma caused increased proliferation in CD105⁺ cells, which could be diminished by conditioned media from parent cultures. Comparison of the multipotent differentiation potential in purified and non-purified CD105⁺ cells determined that MSC were detectable for at least 3 weeks longer when cultured in the absence of CD105^? cells.ConclusionsThis work identifies a simple model for characterizing the different cellular components present in BM stromal cultures and demonstrates that stromal cells lacking multipotent differentiating capacity greatly reduce the longevity of MSC.     

Why the stroma matters in breast cancer: Insights into breast cancer patient outcomes through the examination of stromal biomarkers

Survival and recurrence rates in breast cancer are variable for common diagnoses, and therefore the biological underpinnings of the disease that determine those outcomes are yet to be fully understood. As a result, translational medicine is one of the fastest growing arenas of study in tumor biology. With advancements in genetic and imaging techniques, archived biopsies can be examined for purposes other than diagnosis. There is a great deal of evidence that points to the stroma as the major regulator of tumor progression following the initial stages of tumor formation, and the stroma may also contribute to risk factors determining tumor formation. Therefore, aspects of stromal biology are well-suited to be a focus for studies of patient outcome, where statistical differences in survival among patients provide evidence as to whether that stromal component is a signpost for tumor progression. In this review we summarize the latest research done where breast cancer patient survival was correlated with aspects of stromal biology, which have been put into four categories: reorganization of the extracellular matrix (ECM) to promote invasion, changes in the expression of stromal cell types, changes in stromal gene expression, and changes in cell biology signaling cascades to and from the stroma.     

CD34+ -derived Langerhans cell-like cells are different from epidermal Langerhans cells in their response to thymic stromal lymphopoietin

Thymic stromal lymphopoietin (TSLP) endows human blood‐derived CD11c⁺ dendritic cells (DCs) and Langerhans cells (LCs) obtained from human epidermis with the capacity to induce pro‐allergic T cells. In this study, we investigated the effect of TSLP on umbilical cord blood CD34⁺‐derived LC‐like cells. These cells are often used as model cells for LCs obtained from epidermis. Under the influence of TSLP, both cell types differed in several ways. As defined by CD83, CD80 and CD86, TSLP did not increase maturation of LC‐like cells when compared with freshly isolated LCs and epidermal émigrés. Differences were also found in the production of chemokine (C‐C motif) ligand (CCL)17. LCs made this chemokine only when primed by TSLP and further stimulated by CD40 ligation. In contrast, LC‐like cells released CCL17 in response to CD40 ligation, irrespective of a prior treatment with TSLP. Moreover, the CCL17 levels secreted by LC‐like cells were at least five times higher than those from migratory LCs. After maturation with a cytokine cocktail consisting of tumour necrosis factor‐α, interleukin (IL)‐1β, IL‐6 and prostaglandin (PG)E₂ LC‐like cells released IL‐12p70 in response to CD40 ligation. Most importantly and in contrast to LC, TSLP‐treated LC‐like cells did not induce a pro‐allergic cytokine pattern in helper T cells. Due to their different cytokine secretion and the different cytokine production they induce in naïve T cells, we conclude that one has to be cautious to take LC‐like cells as a paradigm for ‘real’ LCs from the epidermis.     

Enhanced tumor responses to dendritic cells in the absence of CD8-positive cells

Wild-type mice immunized with MART-1 melanoma Ag-engineered dendritic cells (DC) generate strong Ag-specific immunity that has an absolute requirement for both CD8(+) and CD4(+) T cells. DC administration to CD8 alpha knockout mice displayed unexpectedly enhanced levels of protection to tumor challenge despite this deficiency in CD8(+) T cells and the inability to mount MHC class I-restricted immune responses. This model has the following features: 1) antitumor protection is Ag independent; 2) had an absolute requirement for CD4(+) and NK1.1(+) cells; 3) CD4(+) splenocytes are responsible for cytokine production; 4) lytic cells in microcytotoxicity assays express NK, but lack T cell markers (NK1.1(+) alpha beta TCR(-) CD3(-)); and 5) the lytic phenotype can be transferred to naive CD8 alpha knockout mice by NK1.1(+) splenocytes. Elucidation of the signaling events that activate these effective cytotoxic cells and the putative suppressive mechanisms in a wild-type environment may provide means to enhance the clinical activity of DC-based approaches.