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In-depth understanding of human spermatogenesis requires studying specific molecular signatures and interactions of spermatogonia with other testicular cell populations, for which isolation of pure populations of different cell types is crucial. Here, we describe a technique to simultaneously enrich pure, multiple testicular cell populations, including spermatogonia, endothelial (TECs), and perivascular mesenchymal stem/stromal cells (TMSCs), from testicular tissue by flow cytometry using a combination of defined markers. Immunohistochemical studies, multicolor staining, and cell sorting followed by multiplex quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that spermatogonia were highly enriched in the CD49f + CD49a - HLA-ABC - SSEA-4 + fraction of primary testicular cells. In contrast to spermatogonia, TMSCs and TECs were highly enriched in the CD49f + CD49a + HLA-ABC + CD144 - and CD49f + CD49a + HLA-ABC + CD144 + subsets, respectively. The delineation was confirmed by the expression of specific stromal and endothelial key markers as well as by the differentiation and angiogenic capacity of the sorted populations. In this article, for the first time, we performed transcriptome profiling of highly enriched, freshly isolated human spermatogonia and compared their expression profile with that of TMSCs. Our RNA sequencing data favor the hypothesis that TMSCs are candidate niche components for spermatogonia. The composite genotype and phenotype of defined testicular cell populations combined with a robust isolation procedure from small biopsies contributes to a better understanding of cellular interactions and for the establishment of efficient culture techniques to maintain spermatogonial progenitors.

Tissue injury is inevitably accompanied by disruption of the endothelium and exposure of the subendothelial matrix. To generate a guidance molecule directing progenitor cells to sites of vascular lesions, we designed a bifunctional protein. The protein consists of the soluble platelet collagen receptor glycoprotein VI and an antibody to CD133 (hereafter called GPVI-CD133). In vitro and in vivo, this construct substantially mediates endothelial progenitor cell (EPC) homing to vascular lesions. Exposure of EPCs to GPVI-CD133 did not impair their capability to differentiate toward mature endothelial cells as verified by the formation of colony-forming units, the upregulation of endothelial markers CD31 and CD146 analyzed by flow cytometry or von Willebrand factor and endoglin assessed by immunofluorescence microscopy, as well as the presence of Weibel-Palade bodies using transmission electron microscopy. In vivo, GPVI-CD133 augments reendothelialization of vascular lesions. Thus, this bifunctional protein could be a potential new therapeutic option for cardiovascular diseases.

Stem cells in human interfollicular epidermis are still difficult to identify, mainly because of a lack of definitive markers and the inability to label human beings for label-retaining cells (LRCs). Here, we report that LRCs could be identified and localized in organotypic cultures (OTCs) made with human cells. Labeling cultures for 2 weeks with iododeoxyuridine (IdU) and then chasing for 6-10 weeks left <1% of basal cells retaining IdU label. Whole mounts demonstrated that LRCs were individually dispersed in the epidermal basal layer. Some LRCs, but not all, colocalized with cells expressing melanoma chondroitin sulfate proteoglycan, a putative stem cell marker. Although we found LRCs in both collagen- and scaffold-based OTCs, only the scaffold-OTCs supported long-term survival and regeneration. LRCs ' short survival in collagen-OTCs was not due to loss of appropriate growth factors from fibroblasts. Instead, it was due to expression of metalloproteinases, especially matrix metalloproteinase (MMP)-2 and MMP-14, which caused collagen fragmentation, matrix degradation, and dislocation of specific basement membrane components bound to epidermal integrins. Blocking MMP activation not only abrogated MMP-dependent matrix degradation but also increased longevity of the epidermis and the LRCs in these cultures. Such findings indicate that the stem cell niche, the microenvironment surrounding and influencing the stem cell, is essential for stem cell survival and function, including long-term tissue regeneration. Disclosure of potential conflicts of interest is found at the end of this article.

Here we describe the in vitro generation of a novel adherent cell fraction derived from highly enriched, mobilized CD133(+) peripheral blood cells after their culture with Flt3/Flk2 ligand and interleukin-6 for 3 to 5 weeks. These cells lack markers of hematopoietic stem cells, endothelial cells, mesenchymal cells, dendritic cells, and stromal fibroblasts. However, all adherent cells expressed the adhesion molecules VE-cadherin, CD54, and CD44. They were also positive for CD164 and CD172a (signal regulatory protein-alpha) and for a stem cell antigen defined by the recently described antibody W7C5. Adherent cells can either spontaneously or upon stimulation with stem cell factor give rise to a transplantable, nonadherent CD133(+)CD34(-) stem cell subset. These cells do not generate in vitro hematopoietic colonies. However, their transplantation into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice induced substantially higher long-term multilineage engraftment compared with that of freshly isolated CD34(+) cells, suggesting that these cells are highly enriched in SCID-repopulating cells. In addition to cells of the myeloid lineage, nonadherent CD34(-) cells were able to give rise to human cells with B-, T-, and natural killer-cell phenotype. Hence, these cells possess a distinct in vivo differentiation potential compared with that of CD34(+) stem cells and may therefore provide an alternative to CD34(+) progenitor cells for transplantation.

Background: Connective tissue progenitors (CTPs) from native bone marrow (BM) or their culture-expanded progeny, often referred to as mesenchymal stem/stromal cells, represents a promising strategy for treatment of cartilage injuries. But the cartilage regeneration capacity of these cells remains unpredictable because of cell heterogeneity. Hypothesis: The harvest technique of BM may highly influence stem cell heterogeneity and, thus, cartilage formation because these cells have distinct spatial localization within BM from the same bone. Study Design: Controlled laboratory study. Methods: CTPs obtained from the femur of patients undergoing total hip replacement by 2 harvest techniques—BM aspiration and BM collection—after bone rasping were immunophenotyped by flow cytometry and evaluated for chondrogenic ability. The spatial localization of different CTP subsets in BM was verified by immunohistochemistry. Results: Cells from the BM after rasping were significantly more chondrogenic than the donor-matched aspirate, whereas no notable difference in their osteogenic or adipogenic potential was observed. The authors then assessed whether distinct immunophenotypically defined CTP subsets were responsible for the different chondrogenic capacity. Cells directly isolated from BM after rasping contained a higher percentage (mean, 7.2-fold) of CD45–CD271+CD56+ CTPs as compared with BM aspirates. The presence of this subset in the harvested BM strongly correlated with chondrogenic ability, showing that CD271+CD56+ cells are enriched in chondroprogenitors. Furthermore, evaluation of these CTP subsets in BM revealed that CD271+CD56+ cells were localized in the bone-lining regions whereas CD271+CD56– cells were found in the perivascular regions. Since the iliac crest remains a frequent site of BM harvest for musculoskeletal regeneration, the authors also compared the spatial distribution of these subsets in trabeculae of femoral head and iliac crest and found CD271+CD56+ bone-lining cells in both tissues. Conclusion: Chondrogenically distinct CTP subsets have distinct spatial localization in BM; hence, the harvest technique of BM determines the efficiency of cartilage formation. Clinical Relevance: The harvest technique of BM may be of major importance in determining the clinical success of BM mesenchymal stem/stromal cells in cartilage repair. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) are self-renewing cells with the ability to differentiate into various mesodermal-derived tissues. Recently, we have identified in adult human periodontal ligament (PDL) a population of stem cells (PDL-MSCs) with the ability to differentiate into osteoblasts and adipocytes. The aim of the present work was to further characterize this population and the expression profile of its cells. To achieve our objective we have used flow cytometry, magnetic cell sorting, cytokine antibody array, and light and electron microscope immunostaining. Our results show that the PDL-MSCs contain a subpopulation of frizzled-9 (CD349) positive cells expressing a panel of key mesenchymal and embryonic markers including CD10, CD26, CD29, CD44, CD73, CD90, CD105, CD166, SSEA-1, and SSEA-4. They are additionally positive for nanog and Oct-4; two critical transcription factors directing self-renewal and pluripotency of embryonic stem cells, and they also express the cytokines EGF and IP-10. The presence of nanog, Oct-4, SSEA-1, and SSEA-4 suggests that PDL-MSCs are less differentiated than bone marrow-derived MSCs. Taken together, these data indicate the presence of immature MSCs in PDL and suggest that the frizzled-9/Wnt pathway plays an important role in regulating proliferation and differentiation of these cells. J. Cell. Physiol. 225: 123–131, 2010. © 2010 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

We have recently shown that frizzled-9 (FZD9, CD349) is expressed on the cell surface of cultured mesenchymal stromal cells (MSC) derived from the human bone marrow (BM) and chorionic placenta (PL). To study whether FZD9 is also a marker for naive mesenchymal stem cells (MSC), we analyzed the expression pattern of FZD9 on freshly isolated PL cells and determined the clonogenic potential of isolated FZD9+ cells using the colony-forming units-fibroblastic (CFU-F) assay. About 0.2% of isolated PL cells were positive for FZD9. Two-color analysis revealed that FZD9+ PL cells uniformly express CD9, CD63, and CD90, but are heterogeneous for CD10, CD13, and CD26 expression. In contrast to BM-derived MSC, PL-derived MSC expressed only low levels of CD271. Colony assays of sorted cells showed that clonogenic CFU-F reside exclusively in the FZD9+ but not in the FZD9− fraction. Further analysis revealed that CFU-F were enriched by 60-fold in the FZD9+CD10+CD26+ fraction but were absent in the FZD9+CD10−CD26− population. Cultured FZD9+ cells expressed the embryonic stem cell makers Oct-4 and nanog as well as SSEA-4 and TRA1-2-49/6E. In addition, they could be differentiated into functional adipocytes and osteoblasts. This report describes for the first time that FZD9 is a novel and specific marker for the prospective isolation of MSC from human term PL. [ABSTRACT FROM AUTHOR]

Placental tissue draws great interest as a source of cells for regenerative medicine because of the phenotypic plasticity of many of the cell types isolated from this tissue. Furthermore, placenta, which is involved in maintaining fetal tolerance, contains cells that display immunomodulatory properties. These two features could prove useful for future cell therapy-based clinical applications. Placental tissue is readily available and easily procured without invasive procedures, and its use does not elicit ethical debate. Numerous reports describing stem cells from different parts of the placenta, using nearly as numerous isolation and characterization procedures, have been published. Considering the complexity of the placenta, an urgent need exists to define, as clearly as possible, the region of origin and methods of isolation of cells derived from this tissue. On March 23-24, 2007, the first international Workshop on Placenta Derived Stem Cells was held in Brescia, Italy. Most of the research published in this area focuses on mesenchymal stromal cells isolated from various parts of the placenta or epithelial cells isolated from amniotic membrane. The aim of this review i~ to summarize and provide the state of the art of research in this field, addressing aspects such as cell isolation protocols and characteristics of these cells, as well as providing preliminary indications of the possibilities for use of these cells in future clinical applications. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is a multifactorial disease strongly correlated with history of joint trauma, joint dysplasia, and advanced age. Mesenchymal stem cells (MSCs) are promising cells for biological cartilage regeneration. Conflicting data have been published concerning the availability of MSCs from the iliac crest, depending on age and overall physical fitness. Here, we analyzed whether the availability and chondrogenic differentiation capacity of MSCs isolated from the femoral shaft as an alternative source is age- or OA etiology-dependent. MSCs were isolated from the bone marrow (BM) of 98 patients, categorized into three OA-etiology groups (age-related, joint trauma, joint dysplasia) at the time of total hip replacement. All BM samples were characterized for cell yield, proliferation capacity, and phenotype. Chondrogenic differentiation was studied using micromass culture and analyzed by histology, immunohistochemistry, and quantitative reverse transcriptasepolymerase chain reaction. Significant volumes of viable BM (up to 25 ml) could be harvested from the femoral shaft without observing donor-site morbidity, typically containing >107 mononuclear cells per milliliter. No correlation of age or OA etiology with the number of mononuclear cells in BM, MSC yield, or cell size was found. Proliferative capacity and cellular spectrum of the harvested cells were independent of age and cause of OA. From all tested donors, MSCs could be differentiated into the chondrogenic lineage. We conclude that, irrespective of age and OA etiology, sufficient numbers of MSCs can be isolated and that these cells possess an adequate chondrogenic differentiation potential. Therefore, a therapeutic application of MSCs for cartilage regeneration of OA lesions seems feasible. [ABSTRACT FROM AUTHOR]

Conventionally, mesenchymal stem cells (MSC) are generated by plating cells from bone marrow (BM) or other sources into culture flasks and selecting plastic-adherent cells with fibroblastoid morphology. These cells express CD9, CD10, CD13, CD73, CD105, CD166, and other markers but show only a weak or no expression of the embryonic markers stage-specific embryonic antigen-4 (SSEA-4), Oct-4 and nanog-3. Using a novel protocol we prepared MSC from BM and non-amniotic placenta (PL) by culture of Ficoll-selected cells in gelatin-coated flasks in the presence of a serum-free, basic fibroblast growth factor (b-FGF)-containing medium that was originally designed for the expansion of human embryonic stem cells (ESC). MSC generated in gelatin-coated flasks in the presence of ESC medium revealed a four-to fivefold higher proliferation rate than conventionally prepared MSC which were grown in uncoated flasks in serum-containing medium. In contrast, the colony forming unit fibroblast number was only 1.5- to twofold increased in PL-MSC and not affected in BM-MSC. PL-MSC grown in ESC medium showed an increased surface expression of SSEA-4 and frizzled-9 (FZD-9), an increased Oct-4 and nestin mRNA expression, and an induced expression of nanog-3. BM-MSC showed an induced expression of FZD-9, nanog-3, and Oct-4. In contrast to PL-MSC, only BM-MSC expressed the MSC-specific W8B2 antigen. When cultured under appropriate conditions, these MSC gave rise to functional adipocytes and osteoblast-like cells (mesoderm), glucagon and insulin expressing pancreatic-like cells (endoderm), as well as cells expressing the neuronal markers neuron-specific enolase, glutamic acid decarboxylase-67 (GAD), or class III β-tubulin, and the astrocyte marker glial fibrillary acidic protein (ectoderm). In conclusion, using a novel protocol we demonstrate that adult BM-and neonatal PL-derived MSC can be induced to express high levels of FZD-9, Oct-4, nanog-3, and nestin and are able of multi-lineage differentiation. [ABSTRACT FROM AUTHOR]

Background: Mesenchymal stem cells (MSC) are multipotent cells which can differentiate along osteogenic, chondrogenic, and adipogenic lineages. The present study was designed to investigate the influence of mechanical force as a specific physiological stress on the differentiation of (MSC) to osteoblast-like cells. Methods: Human MSC were cultured in osteoinductive medium with or without cyclic uniaxial mechanical stimulation (2000 μstrain, 200 cycles per day, 1 Hz). Cultured cells were analysed for expression of collagen type I, osteocalcin, osteonectin, and CD90. To evaluate the biomineral formation the content of bound calcium in the cultures was determined. Results: After 14 days in culture immunfluorescence staining revealed enhancement of collagen type I and osteonectin expression in response to mechanical stimulation. In contrast, mechanically stimulated cultures stained negative for CD90. In stimulated and unstimulated cultures an increase in the calcium content over time was observed. After 21 days in culture the calcium content in mechanical stimulated cultures was significantly higher compared to unstimulated control cultures. Conclusion: These results demonstrate the influence of mechanical force on the differentiation of human MSC into osteoblast-like cells in vitro. While significant enhancement of the biomineral formation by mechanical stimulation is not detected before 21 days, effects on the extracellular matrix became already obvious after 14 days. The decrease of CD90 expression in mechanically stimulated cultures compared to unstimulated control cultures suggests that CD90 is only transiently expressed expression during the differentiation of MSC to osteoblast-like cells in culture. [ABSTRACT FROM AUTHOR]