Biased competition between Lgr5 intestinal stem cells driven by oncogenic mutation induces clonal expansion

The concept of ‘field cancerization’ describes the clonal expansion of genetically altered, but morphologically normal cells that predisposes a tissue to cancer development. Here, we demonstrate that biased stem cell competition in the mouse small intestine can initiate the expansion of such clones. We quantitatively analyze how the activation of oncogenic K-ras in individual Lgr5⁺ stem cells accelerates their cell division rate and creates a biased drift towards crypt clonality. K-ras mutant crypts then clonally expand within the epithelium through enhanced crypt fission, which distributes the existing Paneth cell niche over the two new crypts. Thus, an unequal competition between wild-type and mutant intestinal stem cells initiates a biased drift that leads to the clonal expansion of crypts carrying oncogenic mutations.     

Placenta-derived multipotent stem cells induced to differentiate into insulin-positive cells

In the present study, we successfully isolated PDMSCs from human placental tissues. The RT-PCR results show that PDMSCs preserved the genetic characteristics of the primitive embryonic stage--Oct-4 and Nanog. By using serum-free medium supplemented essential growth factors and induction medium culture for 4 weeks, a monolayer of spindle-like PDMSCs gradually formed 3D spheroid bodies (SB-PDMSCs). By using real-time RT-PCR, early mRNA expressions of Pdx1, as well as the Sox17 and Foxa2 genes, were observed to be significantly activated in SB-PDMSCs, followed by the expression of mature pancreas-related genes (insulin, glucagon, and somatostatin). The high insulin content of SB-PDMSCs was further confirmed by ELISA assay, and the glucose dependency was demonstrated by the corresponding insulin secretion level. In a transplantation study of streptozotocin-pretreated nude mice, the restoration of normoglycemia in the SB-PDMSC treated group was further observed. In conclusion, these results indicate that PDMSCs are an excellent source for the induced differentiation of well-functioning insulin-positive cells. The potential of these insulin producing cells derived from PDMSCs was also demonstrated functionally by the demonstration of secreted insulin in vitro and effective control of blood glucose levels in vivo.     

Development of a multipotent clonal human periodontal ligament cell line

Abstract The periodontal ligament (PDL) that anchors the tooth root to the alveolar bone influences the lifespan of the tooth, and PDL lost through periodontitis is difficult to regenerate. The development of new PDL-regenerative therapies requires the isolation of PDL stem cells. However, their characteristics are unclear due to the absence of somatic PDL stem cell lines and because PDL is composed of heterogeneous cell populations. Recently, we succeeded in immortalizing human PDL fibroblasts that retained the properties of the primary cells. Therefore, we aimed to establish a human PDL-committed stem cell line and investigate the effects of basic fibroblast growth factor (bFGF) on the osteoblastic differentiation of the cells. Here, we report the development of cell line 1–17, a multipotent clonal human PDL cell line that expresses the embryonic stem cell-related pluripotency genes Oct3/4 and Nanog , as well as the PDL-related molecules periostin and scleraxis. Continuous treatment of cell line 1–17 with bFGF in osteoblastic induction medium inhibited its calcification, with down-regulated expression of FGF-Receptor 1 ( FGF-R1 ), whereas later addition of bFGF potentiated its calcification. Furthermore, bFGF induced calcification of cell line 1–17 when it was co-cultured with osteoblastic cells. These results suggest that cell line 1–17 is a PDL-committed stem cell line and that bFGF exerts dualistic (i.e., promoting and inhibitory) effects on the osteoblastic differentiation of cell line 1–17 based on its differentiation stage.     

Identification of label-retaining cells in nasopharyngeal epithelia and nasopharyngeal carcinoma tissues

Adult stem cells can be identified by label-retaining cell (LRC) approach based on their ability to retain nucleoside analog, such as bromodeoxyuridine (BrdU). We hypothesized that mouse nasopharynx contains a small population of epithelial stem/progenitor cells that may be detected by the LRC technique. To identify LRCs in mice nasopharyngeal epithelia, neonatal mice were intraperitoneally injected with BrdU twice daily for 3 consecutive days. After an 8-week chase, long-term BrdU-labeled LRCs (∼2% of cells) were detected in the adult mice nasopharyngeal epithelia by immunostaining with BrdU antibody and some of LRCs (∼12% of cells) were found to be recruited into the S phase of cell cycle with an additional radioactive thymidine-labeling technique, indicating that the stem cells also divide, most likely asymmetrically. To further investigate whether the LRCs existed in human nasopharyngeal carcinoma (NPC) tissues, three NPC cell lines (5-8F, 6-10B and TMNE) were labeled with BrdU in vitro and then individually engrafted into the back of nude mice, which developed tumors. Again, label-retaining stem cells were found in all the three kinds of NPC xenograft tumors (∼0.3% of cells), around 16% of which were also labeled with radioactive thymidine. Thus, this study has demonstrated for the first time the presence of epithelial LRCs in mouse nasopharyngx and human NPC tissues and these stem-like LRCs are not completely quiescent, as they will be recruited into the cell cycle to participate physiological or pathological process at any moment. More importantly, our data showed that NPC also contained stem cells, which are most likely the cause for NPC spread, metastasis and recurrence.     

p53 in stem cells

p53 is well known as a "guardian of the genome" for differentiated cells,in which it induces cell cycle arrest and cell death after DNA damage and thus contributes to the maintenance of genomic stability.In addition to this tumor suppressor function for differentiated cells,p53 also plays an important role in stem cells.In this cell type,p53 not only ensures genomic integrity after genotoxic insults but also controls their proliferation and differentiation.Additionally,p53 provides an effective barrier for the generation of pluripotent stem celllike cells from terminally differentiated cells.In this review,we summarize our current knowledge about p53 activities in embryonic,adult and induced pluripotent stem cells.     

Mesenchymal stem cells as a therapeutic tool to treat sepsis

Sepsis is a clinical syndrome caused by a deregulated host response to an infection. Sepsis is the most frequent cause of death in hospitalized patients. Although knowledge of the pathogenesis of sepsis has increased substantially during the last decades, attempts to design effective and specific therapiestargeting components of the derailed host response have failed. Therefore, there is a dramatic need for new and mechanistically alternative therapies to treat this syndrome. Based on their immunomodulatory properties, adult mesenchymal stem or stromal cells(MSCs) can be a novel therapeutic tool to treat sepsis. Indeed, MSCs reduce mortality in experimental models of sepsis by modulating the deregulated inflammatory response against bacteria through the regulation of multiple inflammatory networks, the reprogramming of macrophages and neutrophils towards a more antiinflammatory phenotype and the release of antimicrobial peptides. This report will review the current knowledge on the effects of MSC treatment in preclinical experimental small animal models of sepsis.     

The commonality of plasticity underlying multipotent tumor cells and embryonic stem cells

Aggressive cancer cells and pluripotent stem cells converge in their capacity for self-renewal, proliferation and plasticity. Recent studies have capitalized on these similarities by demonstrating that tumors arise from specific cancer stem cell populations that, in a manner reminiscent of normal stem cells, are able to both self-renew and give rise to a heterogeneous tumor population. This stem cell like function of aggressive cancer cells is likely attributable to the ectopic expression of embryonic factors such as Nodal and Cancer Testis Specific Antigens (CTAs), which maintain a functional plasticity by promoting pluripotency and immortality. During development, the expression of these embryonic factors is tightly regulated by a dynamic array of mediators, including the spatial and temporal expression of inhibitors such as Lefty, and the epigenetic modulation of the genome. In aggressive cancer cells, particularly melanoma, this balance of regulatory mediators is disrupted, leading to the aberrant expression of pluripotency-associated genes. By exposing aggressive cancer cells to embryonic microenvironments, this balance of regulatory mediators is restored, thereby reprogramming tumor cells to a more benign phenotype. These stem cell-derived mediators, as well as the genes they regulate, provide therapeutic targets designed to specifically differentiate and eradicate aggressive cancers.     

Spontaneous transformation of adult mesenchymal stem cells from cynomolgus macaques in vitro

Mesenchymal stem cells (MSCs) have shown potential clinical utility in cell therapy and tissue engineering, due to their ability to proliferate as well as to differentiate into multiple lineages, including osteogenic, adipogenic, and chondrogenic specifications. Therefore, it is crucial to assess the safety of MSCs while extensive expansion ex vivo is a prerequisite to obtain the cell numbers for cell transplantation. Here we show that MSCs derived from adult cynomolgus monkey can undergo spontaneous transformation following in vitro culture. In comparison with MSCs, the spontaneously transformed mesenchymal cells (TMCs) display significantly different growth pattern and morphology, reminiscent of the characteristics of tumor cells. Importantly, TMCs are highly tumorigenic, causing subcutaneous tumors when injected into NOD/SCID mice. Moreover, no multiple differentiation potential of TMCs is observed in vitro or in vivo, suggesting that spontaneously transformed adult stem cells may not necessarily turn into cancer stem cells. These data indicate a direct transformation of cynomolgus monkey MSCs into tumor cells following long-term expansion in vitro. The spontaneous transformation of the cultured cynomolgus monkey MSCs may have important implications for ongoing clinical trials and for models of oncogenesis, thus warranting a more strict assessment of MSCs prior to cell therapy.     

Translational regulation in growing clonal human astrocytoma cells in culture

The in vivo and in vitro protein synthesis by polysomes prepared from Cox astrocytoma cells grown in the presence of 100 mM ethanol were examined during transition from exponential to stationary growth phase. A sharp decline of translational activities of Cox poly (A)+messenger RNAs (mRNAs) occurred during this transition. This decline was accentuated when cells were grown in the presence of ethanol. The observed decline in mRNA translational activity was investigated in vitro in a micrococcal nuclease treated, mRNA depleted postmitochondrial supernatant (PMS) fraction containing [³⁵S]methionine. The formation of the³⁵S-labeled 40S ternary complex in the absence of mRNA and of the³⁵S-labeled 80S initiation complex in the presence of Cox or brain poly (A)+mRNAs were reduced substantially when the source of PMS was from stationary phase or ethanol exposed cells. The sedimentation of peaks containing 40S ternary and 80S initiation complexes following sucrose density gradient analysis showed marked reductions in [³⁵S] methionine labeling during the transition to stationary phase and also following ethanol exposure. The reduced formation of initiation complexes suggests possible functional modifications of eukaryotic initiation factor-2 (eIF-2) present in the PMS fraction and of mRNAs under these conditions. Data suggest that cells initiate adaptive or protective mechanisms by reducing the rate of the initiation reaction following environmental alterations produced by ethanol.     

Isolation and characterization of porcine adult muscle-derived progenitor cells

Here, we report the isolation of progenitor cells from pig skeletal muscle tissue fragments. Muscle progenitor cells were stimulated to migrate from protease-digested tissue fragments and cultured in the presence of 5 ng/ml basic fibroblast growth factor. The cells showed a sustained long-term expansion capacity (>120 population doublings) while maintaining a normal karyotype. The proliferating progenitor cells expressed PAX3, DESMIN, SMOOTH MUSCLE ACTIN, VIMENTIN, CD31, NANOG and THY-1, while MYF5 and OCT3/4 were only expressed in the lower or higher cell passages. Myogenic differentiation of porcine progenitor cells was induced in a coculture system with murine C2C12 myoblasts resulting in the formation of myotubes. Further, the cells showed adipogenic and osteogenic lineage commitment when exposed to specific differentiation conditions. These observations were determined by Von Kossa and Oil-Red-O staining and confirmed by quantitative RT-PCR analysis. In conclusion, the porcine muscle-derived progenitor cells possess long-term expansion capacity and a multilineage differentiation capacity.     

Spontaneous transformation and immortalization of human endothelial cells

Summary A new cell line from the human umbilical vein has been established and maintained for more than 5 yr (180 generations; 900 population doublings). This strain, designated ECV304, is characterized by a cobblestone monolayer growth pattern, high proliferative potential without any specific growth factor requirement, and anchorage dependency with contact inhibition. Karyotype analysis of this cell line reveals it to be of human chromosomal constitution with a high trisomic karyotype (mode 80). Ultrastructurally, endothelium-specific Weibel-Palade bodies were identified. Although one of the endothelial cell markers, Factor VIII-related antigen (VIIIR:Ag) was negative in this cell line, immunocytochemical staining for the lectin Ulex europaeus I (UEA-I), and PHM5 (anti-human endothelium as well as glomerular epithelium monoclonal antibody) was positive, and angiotensin-converting enzyme (ACE) activity was also demonstrated. In addition, ECV304 displayed negativity for alkaline and acid phosphatase and for the epithelial marker keratin. All of these findings suggest that ECV304 cells originated from umbilical vein endothelial cells by spontaneous transformation. Ultrastructurally, no viruslike particles have been detected intracellularly. Nude mouse tumorigenicity and rabbit cornea tests were both positive. This is a report on a novel case of phenotypic alteration of normal venous endothelial cells of human origin in vitro, and generation of a transformant with indefinite life spans. This line may be useful in studies of some physiologically active factors available for medical use.     

A putative mesenchymal stem cells population isolated from adult human testes

Mesenchymal stem cells (MSCs) isolated from several adult human tissues are reported to be a promising tool for regenerative medicine. In order to broaden the array of tools for therapeutic application, we isolated a new population of cells from adult human testis termed gonadal stem cells (GSCs). GSCs express CD105, CD166, CD73, CD90, STRO-1 and lack hematopoietic markers CD34, CD45, and HLA-DR which are characteristic identifiers of MSCs. In addition, GSCs express pluripotent markers Oct4, Nanog, and SSEA-4. GSCs propagated for at least 64 population doublings and exhibited clonogenic capability. GSCs have a broad plasticity and the potential to differentiate into adipogenic, osteogenic, and chondrogenic cells. These studies demonstrate that GSCs are easily obtainable stem cells, have growth kinetics and marker expression similar to MSCs, and differentiate into mesodermal lineage cells. Therefore, GSCs may be a valuable tool for therapeutic applications.     

Clinical trials using dental stem cells: 2022 update

For nearly 20 years, dental stem cells(DSCs) have been successfully isolated from mature/immature teeth and surrounding tissue, including dental pulp of permanent teeth and exfoliated deciduous teeth, periodontal ligaments, dental follicles, and gingival and apical papilla. They have several properties(such as self-renewal, multidirectional differentiation, and immunomodulation) and exhibit enormous potential for clinical applications. To date, many clinical articles and clinical trials using DS...     

Allogenic banking of dental pulp stem cells for innovative therapeutics

Medical research in regenerative medicine and cell-based therapy has brought encouraging perspectives for the use of stem cells in clinical trials. Multiple types of stem cells, from progenitors to pluripotent stem cells, have been investigated. Among these, dental pulp stem cells (DPSCs) are mesenchymal multipotent cells coming from the dental pulp, which is the soft tissue within teeth. They represent an interesting adult stem cell source because they are recovered in large amount in dental pulps with non-invasive techniques compared to other adult stem cell sources. DPSCs can be obtained from discarded teeth, especially wisdom teeth extracted for orthodontic reasons. To shift from promising preclinical results to therapeutic applications to human, DPSCs must be prepared in clinical grade lots and transformed into advanced therapy medicinal products (ATMP). As the production of patient-specific stem cells is costly and time-consuming, allogenic biobanking of clinical grade human leukocyte antigen (HLA)-typed DPSC lines provides efficient innovative therapeutic products. DPSC biobanks represent industrial and therapeutic innovations by using discarded biological tissues (dental pulps) as a source of mesenchymal stem cells to produce and store, in good manufacturing practice (GMP) conditions, DPSC therapeutic batches. In this review, we discuss about the challenges to transfer biological samples from a donor to HLA-typed DPSC therapeutic lots, following regulations, GMP guidelines and ethical principles. We also present some clinical applications, for which there is no efficient therapeutics so far, but that DPSCs-based ATMP could potentially treat.