Putative Mesenchymal Stem Cells Research Articles

Endoglin and MMP14 Contribute to Ewing Sarcoma Spreading by Modulation of Cell-Matrix Interactions.

Endoglin (ENG) is a mesenchymal stem cell (MSC) marker typically expressed by active endothelium. This transmembrane glycoprotein is shed by matrix metalloproteinase 14 (MMP14). Our previous work demonstrated potent preclinical activity of first-in-class anti-ENG antibody-drug conjugates as a nascent strategy to eradicate Ewing sarcoma (ES), a devastating rare bone/soft tissue cancer with a putative MSC origin. We also defined a correlation between ENG and MMP14 expression in ES. Herein, we show that ENG expression is significantly associated with a dismal prognosis in a large cohort of ES patients. Moreover, both ENG/MMP14 are frequently expressed in primary ES tumors and metastasis. To deepen in their functional relevance in ES, we conducted transcriptomic and proteomic profiling of in vitro ES models that unveiled a key role of ENG and MMP14 in cell mechano-transduction. Migration and adhesion assays confirmed that loss of ENG disrupts actin filament assembly and filopodia formation, with a concomitant effect on cell spreading. Furthermore, we observed that ENG regulates cell–matrix interaction through activation of focal adhesion signaling and protein kinase C expression. In turn, loss of MMP14 contributed to a more adhesive phenotype of ES cells by modulating the transcriptional extracellular matrix dynamics. Overall, these results suggest that ENG and MMP14 exert a significant role in mediating correct spreading machinery of ES cells, impacting the aggressiveness of the disease.

Therapeutic and clinical propositions of Mesenchymal Stem Cells(MSCs) focusing on recent trial updates

The mesenchymal stroma harbours considerable population of stem cell-like cells with differentiation and self-renewal abilities that originate from several sources like amniotic fluid, Wharton’s jelly, umbilical cord etc. Mesenchymal Stem Cells (MSCs) are most commonly found in the perivascular niche. This multipotent progenitor cells have the ability to differentiate into mesodermal cell types such as adipocytes, chondrocytes and osteocytes. MSCs can also exert significant immunosuppressive and anti-inflammatory effects by interacting with lymphocytes from both innate and adaptive immune system. MSCs of fetal origins can go through same processes as MSCs derived from elsewhere. Before senescence, they make more cell divisions than adult MSCs of bone marrow or adipose tissue. The propitious properties of mesenchymal stem cells (MSCs), such as their ability to differentiate into diverse cell lineages and their regenerative properties, have intrigued researchers, whose work has provided fascinating perspectives on cell-based therapies for wide range of diseases. MSCs have a high potential for replication in vitro. International Society for Cellular Therapy (ISCT)-based MSC isolation has resulted in heterogeneous, nonclonal stromal cell cultures containing stem cells with various multipotent characteristics, committed progenitors, and differentiated cells. Nonclonal stromal cultures which are derived from bone marrow and other tissues are presently used as sources of putative MSCs for therapeutic aims. Also, MSCs have potential to endogenously repair as well as reduce the inflammatory responses that may lead to decreased morbidity and mortality rate of COVID-19. Herein, the MSC-based clinical prospects, clarifies, the recent clinical findings, therapeutic effects of MSC and clinical trials.

Ptch2 is a Potential Regulator of Mesenchymal Stem Cells.

Ptch receptors 1 and 2 mediate Hedgehog signaling pivotal for organ development and homeostasis. In contrast to embryonic lethal Ptch1 −/− phenotype, Ptch2 −/− mice display no effect on gross phenotype. In this brief report, we provide evidence of changes in the putative incisor mesenchymal stem cell (MSC) niches that contribute to accelerated incisor growth, as well as intriguing changes in the bones and skin which suggest a role for Ptch2 in the regulation of MSCs and their regenerative potential. We employed histological, immunostaining, and computed tomography (µCT) analyses to analyze morphological differences between Ptch2 −/− and wild-type incisors, long bones, and skins. In vitro CFU and differentiation assays were used to demonstrate the MSC content and differentiation potential of Ptch2 −/− bone marrow stromal cells. Wound healing assay was performed in vivo and in vitro on 8-week-old mice to assess the effect of Ptch2 on the wound closure. Loss of Ptch2 causes increases in the number of putative MSCs in the continuously growing incisor, associated with increased vascularization observed in the tooth mesenchyme and the neurovascular bundle. Increased length and volume of Ptch2 −/− bones is linked with the increased number and augmented in vitro differentiation potential of MSCs in the bone marrow. Dynamic changes in the Ptch2 −/− skin thickness relate to changes in the mesenchymal compartment and impact the wound closure potential. The effects of Ptch2 abrogation on the postnatal MSCs suggest a crucial role for Ptch2 in Hedgehog signaling regulation of the organ regenerative potential.

3164 – A CELLULAR TAXONOMY OF MURINE BONE MARROW STROMAL STEM AND PROGENITOR CELLS UPON DEVELOPMENT

Mesenchymal stem cells (MSCs) in the bone marrow (BM) are thought to play pivotal roles in tissue (re)generation and provide a supportive microenvironment for hematopoietic stem cells (HSCs). MSCs have been well characterized in murine models and isolated using different combinations of markers. Yet, these markers define a broader and more heterogeneous population, of which MSCs are only a minority. Although several studies indicate that bona fide MSCs reside in the BM, further investigations are needed to define their identity and allow their prospective and effective isolation. Here, using a single cell RNA sequencing approach, we aimed to delineate the cellular heterogeneity of the MSC-enriched Sca-1+ CD51+ population throughout development. At the fetal stage (E17), the BMSC population appeared relatively homogeneous with subsets predicted to contain candidate MSCs based on marker expression and computational modeling. The frequency of putative MSC subsets declined upon aging. After birth, the BMSC population was more heterogeneous, and characterized by the emergence of clusters showing transcriptional wiring consistent with specialization towards differentiated (progenitor) subsets. This included the postnatal emergence of a Lepr+ population, expressing HSC regulatory genes and previously established as HSC niches. In the fetal BM, subsets of endothelial cells expressed HSC factors and may thus represent an initial supportive microenvironment for emerging hematopoiesis. Our results shed light on the cellular diversity of BMSCs from different developmental phases, providing a cellular taxonomy of this population at single cell resolution. The exploration of our dataset, and the comparison of transcriptional landscapes from different developmental stages, may provide a resource to identify and further validate factors with a predicted role in BM regeneration. Mesenchymal stem cells (MSCs) in the bone marrow (BM) are thought to play pivotal roles in tissue (re)generation and provide a supportive microenvironment for hematopoietic stem cells (HSCs). MSCs have been well characterized in murine models and isolated using different combinations of markers. Yet, these markers define a broader and more heterogeneous population, of which MSCs are only a minority. Although several studies indicate that bona fide MSCs reside in the BM, further investigations are needed to define their identity and allow their prospective and effective isolation. Here, using a single cell RNA sequencing approach, we aimed to delineate the cellular heterogeneity of the MSC-enriched Sca-1+ CD51+ population throughout development. At the fetal stage (E17), the BMSC population appeared relatively homogeneous with subsets predicted to contain candidate MSCs based on marker expression and computational modeling. The frequency of putative MSC subsets declined upon aging. After birth, the BMSC population was more heterogeneous, and characterized by the emergence of clusters showing transcriptional wiring consistent with specialization towards differentiated (progenitor) subsets. This included the postnatal emergence of a Lepr+ population, expressing HSC regulatory genes and previously established as HSC niches. In the fetal BM, subsets of endothelial cells expressed HSC factors and may thus represent an initial supportive microenvironment for emerging hematopoiesis. Our results shed light on the cellular diversity of BMSCs from different developmental phases, providing a cellular taxonomy of this population at single cell resolution. The exploration of our dataset, and the comparison of transcriptional landscapes from different developmental stages, may provide a resource to identify and further validate factors with a predicted role in BM regeneration.

Search for Novel Plasma Membrane Proteins as Potential Biomarkers in Human Mesenchymal Stem Cells Derived from Dental Pulp, Adipose Tissue, Bone Marrow, and Hair Follicle.

One of the drawbacks preventing the use of mesenchymal stem cells (MSCs) in clinical practice is the heterogeneous nature of their cultures. MSC cultures are not homogeneously formed by the MSCs and may contain non-mesenchymal cell types. Therefore, prior to use in clinics or research, complete characterization of MSCs should be performed to demonstrate the existence or absence of proper stem cell markers, many of which are happened to be cell-surface proteins. Unfortunately, the success of MSC characterization studies is limited due to the low specificity of the currently available cell-surface markers. Therefore, in this study, we aimed to investigate the plasma membrane (PM) proteins of MSCs isolated from human dental pulp (DP), adipose tissue (AT), bone marrow (BM), and hair follicle (HF) with the hope of proposing novel putative specific MSC markers. Differential-velocity centrifugation was used to enrich PM proteins. The isolated proteins were then identified by nLC-MS/MS and subjected to bioinformatics analysis. Proteins that were unique to each MSC type (CD9, CD10, CD63 for DP-MSCs; CD26, CD81, CD201, CD364 for AT-MSCs; Cd49a, CD49d for HF-MSCs; CD49e, CD56, CD92, CD97, CD156b, CD156c, CD220, CD221, CD298, CD315 for BM-MSCs) and common to all four MSC types (CD13, CD29, CD44, CD51, CD59, CD73, CD90) were identified. Uncharacterized proteins that have transmembrane (TM) domains were also detected. Some of the proteins identified in this study were the putative cell-surface markers that might be used for characterization of MSCs.

Human endometrial perivascular stem cells exhibit a limited potential to regenerate endometrium after xenotransplantation.

What are the localization, characteristics and potential for tissue regeneration of two perivascular stem cells, namely CD34+ adventitial cells and CD146+ pericytes, in human endometrium? Human endometrial CD34+ adventitial cells (located in the outermost layer of blood vessels and mainly in the basal layer) and CD146+ pericytes showed mesenchymal stem cell (MSC) phenotypes in in vitro culture, but presented limited potential to regenerate endometrium. Periodic endometrial regeneration is considered to be maintained by MSCs. Blood vessel wall, regarded as stem cell niche, harbors a large reserve of progenitor cells that may be integral to the origin of MSCs. However, a lack of validated markers has hampered the isolation of putative endometrial MSCs. Currently, CD146+ pericytes and Sushi Domain Containing 2 (SUSD2) positive cells have been identified in the endometrial perivascular region as sharing MSCs characteristics. The locations of adventitial cells and pericytes in the human endometrium were identified by immunofluorescence staining (n = 4). After CD34+CD146-CD45-CD56-CD144- adventitial cells and CD146+CD34-CD45-CD56-CD144- pericytes were isolated from the endometrium of normal women (n = 6) by fluorescence-activated cell sorting, their characteristics were investigated in culture. Adventitial cells and pericytes were induced to differentiate, respectively, into vascular endothelial-like cells or endometrial stromal-like cells in vitro, with their potential explored by in vivo xenotransplantation (n = 2 in each group) and eutopic transplantation (n = 2 in each group). CD34+ adventitial cells and CD146+ pericytes were cultured in the inducing medium to differentiate into endothelial-like cells in vitro, and then analyzed for CD31, von Willebrand factor immunofluorescent staining and tube formation. They were also cultured to differentiate into endometrial stromal cells in vitro, with the expression of vimentin and CD13 being detected by western blot before and after induction, and the expression of prolactin and insulin-like growth factor-binding protein 1 being determined as well. Single dispersed CD34+ adventitial cells and CD146+ pericytes were respectively transplanted under the kidney capsule of NOG mice to investigate their differentiation potential in vivo. A eutopic transplantation model was constructed by grafting recellularized uterine matrix loaded up with CM-Dil labeled adventitial cells or pericytes into the injury region of nude rat's uterus. CD34+ adventitial cells were mainly located at the outmost layer of endometrial large vessels, while CD146+ pericytes were found surrounding the inner endothelial cells of microvessels. A small proportion of CD34+ adventitial cells expressed SUSD2. The number of adventitial cells was ∼40 times higher than that of pericytes in the endometrium. Both adventitial cells and pericytes showed MSC phenotypes after in vitro culture. After in vitro induction into endometrial endothelial-like cells and stromal-like cells, adventitial cells showed higher plasticity than pericytes and a closer correlation with stromal-like cells. In the mouse xenotransplantation model, vimentin+ cells, CD31+ endothelial-like cells and CD146+ pericyte-like cells could be observed after adventitial cells were transplanted. CM-Dil-labeled adventitial cells or pericytes could survive in the immunocompromised nude rats after eutopic transplantation, and vimentin+ cells were detected. In addition, CM-Dil-labeled adventitial cells or pericytes did not express α-smooth muscle actin or E-cadherin after transplantation. N/A. CD34 was chosen as a novel marker to isolate adventitial cells from human endometrium according to previous literature. The association of endometrial CD34+ adventitial cells and SUSD2+ MSCs should be further investigated. The decellularized uterine matrix model might be useful in endometrial stem cell therapy. L.D. is supported by grants from National Key Research and Development Program of China (2018YFC1004700), Nature Science Foundation of China (81871128, 81571391) and Nanjing Medical Science Development Project (ZKX16042). H.S. is supported by a grant from Jiangsu Province Social Development Project (BE2018602). X.Z. was supported by grants from the Postgraduate Innovative Project of Jiangsu Province (KYCX19-1177). The authors declare no conflict of interest.

Comparison of Effects of Curcumin and Nano-curcumin on the Survival of Human-Derived Mesenchymal Stem Cells: An Experimental Study

Aim: To evaluate and compare the effect of curcumin (CUR) and Nano-curcumin (N-CUR) on human-derived mesenchymal stem cells (MSCs) in a dose-dependent manner. Materials and Methods: An experimental study performed with putative MSCs from a total of five systemically healthy subjects with chronic periodontitis. These putative MSCs were isolated by cell culture and were further characterized and identified by colony-forming unit assay and immunocytochemical analysis using cell surface markers CD105, CD146, CD45 and CD73. The identified MSCs were treated with different doses of CUR and N-CUR, and compared with α-minimum essential medium (α -MEM) for its cell viability by performing MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay for 48 and 72 hr. The statistically analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test and Bonferroni’s post hoc test. Results: Compared to the α-MEM group, both CUR and N-CUR treated cells have shown significantly ( P = .029) higher survival rate at lower concentration (0.1 and 0.5 µM/L), at 48 hr incubation. However, there was no statistically significant difference between the CUR and N-CUR groups on cell survival rate at both 48 and 72 hr incubation. When compared between the concentrations of the same group, significantly higher cell viability ( P = .001) was observed at lower concentrations (0.1, 0.5 µM/L) in both test groups after incubation for 48 and 72 hr. Conclusion: Both CUR and N-CUR have a dose-dependent effect on human derived MSCs survival when incubated for 48 hr, whereas N-CUR shows increased cell survival rate even at 72 hr of incubation. Although, the cautious use of CUR and N-CUR at higher concentrations is recommended.

220 Exploring the use of mesenchymal stem cells for treatment of mastitis and metritis in cattle

The present study was carried out to isolate mesenchymal stem cells (MSCs) from adipose tissue of cattle (Bos indicus), characterise them, and apply them for the treatment of mastitis and metritis in the cow. Cattle MSCs were isolated from adipose tissue near the loin region of cow. Isolated adipose tissue was subjected to enzymatic digestion using 2% collagenase with agitation at regular intervals. The cells obtained after digestion were resuspended in cell culture flasks containing growth enriched medium and cultured under standard culture conditions. Alkaline phosphatase staining was used as one of the parameters to confirm cultured putative MSCs. Bovine Ad-MSCs were further characterised using real time-PCR by amplification of MSC-specific markers: CD73, CD90, and CD105 as positive markers and CD34, CD45, and CD79a as negative markers. Immunocytochemistry showed the presence of CD73, CD90, and CD105 on the cell surface. Three groups-control (C), local (L), and intravenous (IV)-with 6 cows suffering from mastitis were taken in each group and subjected to MSC transplantation through local and intravenous routes. Control group animals were subjected to antibiotic treatment only. Similarly, another three groups were taken with 6 cows in each group suffering from metritis. Post-transplantation wound healing, tissue repair, and reduction in inflammation were monitored for 26 days, at different time intervals; that is, after Days 1, 3, 7, and 15. Blood samples were also collected from animals at the same time intervals for real time-PCR. A similar examination was also done in metritis groups along with the analysis of the reduction in turbidity of cervical fluid at the abovementioned time intervals. Real time-PCR was performed to determine relative expression of genes for proliferative factors, anti-inflammatory cytokines, and antimicrobial peptides on cells isolated from blood collected at different time intervals. Gene expression in the local group of mastitis subjected to MSC injection was significantly higher than that of the IV and control group. The somatic cell count declined in both local and IV groups compared with the control group. Whereas the expression of the same genes in the IV group of metritis was significantly higher than that of the local and control groups of cows. The turbidity of cervical fluid and mucus was reduced in the IV group compared with the local group. In conclusion, we demonstrated the healing potential of MSCs in a cow model via MSC injection. Promising results were obtained in curing mastitis in both local and IV groups, whereas healing in the case of metritis was significantly higher in the IV group compared with both the control and local groups of cows. The study indicates the potential use of MSc for treatment of mastitis and metritis in cattle through wound healing and decreasing microbial infection.

Pathological ASXL1 Mutations and Protein Variants Impair Neural Crest Development.

SummaryThe neural crest (NC) gives rise to a multitude of fetal tissues, and its misregulation is implicated in congenital malformations. Here, we investigated molecular mechanisms pertaining to NC-related symptoms in Bohring-Opitz syndrome (BOS), a developmental disorder linked to mutations in the Polycomb group factor Additional sex combs-like 1 (ASXL1). Genetically edited human pluripotent stem cell lines that were differentiated to NC progenitors and then xenotransplanted into chicken embryos demonstrated an impairment of NC delamination and emigration. Molecular analysis showed that ASXL1 mutations correlated with reduced activation of the transcription factor ZIC1 and the NC gene regulatory network. These findings were supported by differentiation experiments using BOS patient-derived induced pluripotent stem cell lines. Expression of truncated ASXL1 isoforms (amino acids 1–900) recapitulated the NC phenotypes in vitro and in ovo, raising the possibility that truncated ASXL1 variants contribute to BOS pathology. Collectively, we expand the understanding of truncated ASXL1 in BOS and in the human NC.

Isolation and Differentiation of Mesenchymal Stem Cells From Broiler Chicken Compact Bones

Chicken mesenchymal stem cells (MSCs) can be used as an avian culture model to better understand osteogenic, adipogenic, and myogenic pathways and to identify unique bioactive nutrients and molecules which can promote or inhibit these pathways. MSCs could also be used as a model to study various developmental, physiological, and therapeutic processes in avian and other species. MSCs are multipotent stem cells that are capable of differentiation into bone, muscle, fat, and closely related lineages and express unique and specific cell surface markers. MSCs have been isolated from numerous sources including human, mouse, rabbit, and chicken with potential clinical and agricultural applications. MSCs from chicken compact bones have not been isolated and characterized yet. In this study, MSCs were isolated from compact bones of the femur and tibia of day-old male broiler chicks to investigate the biological characteristics of the isolated cells. Isolated cells took 8–10 days to expand, demonstrated a monolayer growth pattern and were plastic adherent. Putative MSCs were spindle-shaped with elongated ends and showed rapid proliferation. MSCs demonstrated osteoblastic, adipocytic, and myogenic differentiation when induced with specific differentiation media. Cell surface markers for MSCs such as CD90, CD105, CD73, CD44 were detected positive and CD31, CD34, and CD45 cells were detected negative by PCR assay. The results suggest that MSCs isolated from broiler compact bones (cBMSCs) possess similar biological characteristics as MSCs isolated from other chicken tissue sources.

Label-Retaining, Putative Mesenchymal Stem Cells Contribute to Murine Myometrial Repair During Uterine Involution.

Uterine remodeling during pregnancy is a fundamental, dynamic process required for successful propagation of eutherian species. The uterus can increase in size up to 40-fold during pregnancy, which is largely attributed to expansion of the myometrium by hyperplasia and hypertrophy. After pregnancy, the uterus repairs the remodeled or "damaged" tissue during uterine involution (INV). Little is known about this repair process, particularly the role of mesenchymal stem/progenitor cells. The objective of this study was to identify and characterize putative mesenchymal stem/progenitor cells in the murine myometrium using a combination of label retention and mesenchymal stem cell (MSC) marker expression and a pregnancy and uterine INV model. Tet-off transgenic mice with the Cre-lox system were used to specifically label mesenchymal cells (ie, myometrial and endometrial stromal cells) within the uterus while avoiding other cell types (eg, epithelial, immune, and endothelial cells) to identify slowly dividing cells and assess their stem cell qualities. We identified myometrial label-retaining cells (LRCs) that persisted for at least 3 months, expressed CD146 and CD140b (MSC markers), and proliferated at a higher rate during uterine INV compared with nonlabeled cells. The LRCs did not appear to express either estrogen receptor alpha or progesterone receptor, nor did the number of LRCs change at different estrous stages or in response to exogenous estradiol or progesterone administration, suggesting that LRCs were not involved in normal estrous cycling. The results from this study provide important insight into putative stem/progenitor cells in the myometrium and their possible role in uterine physiology.

Isolation and culture of putative mesenchymal stem cells from equine umbilical cord Wharton’s jelly

Despite major progress and knowledge related to the application of adult stem cells, finding alternative sources for bone marrow MSCs has remained a challenge in both humans and animals. In the current study, two protocols namely sequential enzymatic tissue digestion and tissue explant techniques were tried for successful establishment of MSC culture. Umbilical tissues were isolated each time of foaling from five sequential foalings of Marwari mares. Total cell yield, their growth potential and cryopreservation potential were studied. Adherent cell colonies could be established using both isolation methods. Both the cell populations yielded from different protocols performed similarly in terms of population doubling and CFU number value. Additionally, the cells proliferated vigourously and displayed a similar morphology of mesenchymal stem cells. The MSCs were plastic adherent, colonogenic and their morphology was polygonal and fibroblast like. During the proliferation, the cells exhibited density dependent inhibition; analysis of microbial contamination from bacteria, mycoplasma and fungi were negative; the population doubling time of the MSCs isolated was 34.8 h and 40.2 h in enzymatic treatment and tissue explant methods respectively, and diploid chromosome number of the cells was 64, and the diploid frequency was higher than 80%. In conclusion, this study reveals that both the techniques proved to be non-invasive, efficient, simple and quick for isolation and establishment of MSC culture of extra embryonic tissues from equines.

Abstract B007: Stemness features of prostate cancer cells induced by carcinoma-associated fibroblasts

Abstract Introduction: Carcinoma-associated fibroblasts (CAFs) play a key role during cancer progression through several mechanisms, including the induction of the epithelial-mesenchymal transition (EMT) program, giving cancer cells the ability to initiate metastasis. As the cells progress to the mesenchymal state they acquire properties of cancer stem cells (CSC) or mesenchymal stem cell (MSC). However, the contribution of the CAFs in the acquisition of the differential stemness is largely unknown. Here, we studied the stemness features of prostate cancer cells (PC3) during the EMT program induced by fibroblasts obtained from patients with nonmetastatic prostate cancer (CAF) and bone metastatic prostate cancer (mCAF). Materials and Methods: PC3 cells were incubated with conditioned medium (CM) of CAF and mCAF for 7,14, and 21 days to induced EMT. The presence of putative CSCs and MSCs was determined by qPCR, immunocytochemistry and Western blot for the expression of CSCs and MSCs markers, respectively, and functionally evaluated by the ability to perform adipogenic, chondrogrenic, and osteogenic differentiation (MSCs) and in vitro spheroids formation and clonogenic assay (CSCs). Results and Discussion: Our results demonstrate that cancer cells treated with CM-CAF undergo to a partial EMT program, exhibiting certain mesenchymal traits and enriched for cancer stem cells (CSCs). On the other hand, tumor cells exposed to CM-mCAF, exhibited a full EMT conversion with the greater abilities to migrate and invade in vitro and in vivo. This suggests that CAFs and mCAFs induce differential EMT programs in prostate cancer cells, which leads them to acquire CSCs or MSC features enhancing the metastatic capabilities. (FONDECYT 1150397.) Citation Format: Muriel A. Nuñez, Javier Cerda-Infante, Marianela Sánchez, Paula Sotomayor, Alejandro S. Godoy, Viviana P. Montecinos. Stemness features of prostate cancer cells induced by carcinoma-associated fibroblasts [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B007.

PO-328 Stemness features of prostate cancer cells induced by carcinoma-associated fibroblasts

IntroductionCarcinoma-associated fibroblasts (CAFs) play a key role during cancer progression through several mechanisms including the induction of the epithelial-mesenchymal transition (EMT) program, giving cancer cells the ability to initiate metastasis. As the cells progress to the mesenchymal state acquire properties of Cancer Stem Cells (CSC) or Mesenchymal Stem Cell (MSC). However, the contribution of the CAFs in the acquisition of the differential stemness is largely unknown. Here, we studied the stemness features of prostate cancer cells (PC3) during the EMT program induced by fibroblasts obtained from patients with non-metastatic prostate cancer (CAF) and bone metastatic prostate cancer (mCAF).Material and methodsPC3 cells were incubated with conditioned medium (CM) of CAF and mCAF for 7,14 and 21 days to induced EMT. The presence of putative CSCs and MSCs was determined by qPCR, immunocytochemistry and western blot for the expression of CSCs and MSCs markers respectively and functionally evaluated by the ability to perform adipogenic, chondrogrenic and osteogenic differentiation (MSCs) and in vitro spheroids formation and clonogenic assay (CSCs).Results and discussionsOur results demonstrate that cancer cells treated with CM-CAF undergo to a partial EMT program, exhibiting certain mesenchymal traits and enriched for cancer stem cells (CSCs). On the other hand, tumour cells exposed to CM-mCAF, exhibited a full EMT conversion with the greater abilities to migrate and invade in vitro and in vivo.ConclusionThis suggests that CAFs and mCAFs induce differential EMT programs in prostate cancer cells, which leads them to acquire CSCs or MSC features enhancing the metastatic capabilities.

The Role of Stem Cells in Dupuytren's Disease: A Review.

Summary:The pathogenesis of Dupuytren’s disease (DD) remains unclear although there is increasing evidence supporting the role of stem cells in this and other fibrotic conditions. This review examines the role of DD tissue-associated embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs), and circulating fibrocytes and circulating MSCs, in the biology of DD. It is exciting to infer that dysfunction of an upstream ESC-like population within the affected tissue leads to the downstream development and proliferation of aberrant myofibroblasts through a putative MSC intermediate. This ESC-like population may be a potential novel therapeutic target through modulation of the renin-angiotensin system. Furthermore, circulating CD34+ fibrocytes and MSCs either derived from the bone marrow, peripheral blood cells, or DD-associated ESC-like population, may serve as potential additional extra-palmar reservoirs that undergo endothelial-to-mesenchymal transition, eventually giving rise to the aberrant myofibroblasts. Further studies examining the relative roles of these stem cells and the precise regulatory pathways that govern them may lead to novel therapy that targets these populations.

Applications of inflammation-derived gingival stem cells for testing the biocompatibility of dental restorative biomaterials

BackgroundNormal or inflamed gingival tissues are regarded as a source of mesenchymal stem cells (MSCs) abundant and easily accessible through minimally invasive dental procedures. Due to the proximity of dental resin composites to gingival tissues and to the possible local cytotoxic effect of the eluted components, gingiva-derived MSCs could be used to investigate the biocompatibility of dental biomaterials. PurposeThe present research aimed to isolate (MSCs) from inflamed and normal gingiva, to fully characterize them and to observe their behavior in relation with some commercial resin composite materials and one experimental material. Material and methodsFollowing their isolation, putative MSCs from both gingival sources were grown under the same culture conditions and characterized by immunophenotyping of cell surface antigens by flow-cytometry and transcription factors by immunocytochemical staining. Moreover, stemness gene expression was evaluated by RT-PCR analysis. Multipotent mesenchymal differentiation potential was investigated. Osteogenic and neurogenic differentiated cells were highlighted by immunocytochemical staining, chondrogenic cells by cytochemical staining, and adipocytes by cytochemical staining and spectrophotometry, respectively. Resin composite cytotoxicity was evaluated by cell membrane fluorescent labeling with PKH 26 and MTT assay. The results of PKH labeling were statistically analysed using two-way RM ANOVA with Bonferroni post-tests. For MTT assay, two-way RM ANOVA with Bonferroni post-tests and unpaired t test with Welch’s correction were used. ResultsA similar expression pattern of surface markers was observed. The cells were positive for CD105, CD73, CD90, CD49e, CD29, CD44 and CD166 and negative for CD45, CD34, CD14, CD79, HLA-DR and CD117 indicating a mesenchymal stem cell phenotype. The qRT-PCR analysis revealed a low gene expression for NOG, BMP4 and Oct3/4 and an increased expression for Nanog in both cells lines. Immunocytochemical analysis highlighted a more intense protein expression for Nanog, Oct3/4 and Sox-2 in MSCs derived from normal gingiva than from inflamed gingiva. Multipotent differentiation capacity of MSCs isolated from both sources was highlighted. The tested materials had no hazardous effect on MSCs as the two cell lines developed well onto resin composite substrates. Cell counting revealed some significant differences in the number of PKH-labeled MSCs at some experimental moments. Also, some differences in cell viability were recorded indicating better developmental conditions offered by some of the tested biomaterials. ConclusionsThe experimental resin composite behaved like the most biocompatible commercial material. Inflamed gingiva-derived MSCs retain their stem cell properties and could be used as a valuable cell line for testing dental biomaterials.

Host cell recruitment patterns by bone morphogenetic protein-2 releasing hyaluronic acid hydrogels in a mouse subcutaneous environment.

This study aimed to identify host cell recruitment patterns in a mouse model in response to rhBMP-2 releasing hyaluronic acid hydrogels and influence of added nano-hydroxyapatite particles on rhBMP-2 release and pattern of bone formation. Implanted gels were retrieved after implantation and cells were enzymatically dissociated for flow cytometric analysis. Percentages of macrophages, progenitor endothelial cells and putative mesenchymal stem cells were measured. Implants were evaluated for BMP-2 release by ELISA and by histology to monitor tissue formation. Hyaluronic acid+BMP-2 gels influenced the inflammatory response in the bone healing microenvironment. Host-derived putative mesenchymal stem cells were major contributors. Addition of hydroxyapatite nanoparticles modified the release pattern of rhBMP-2, resulting in enhanced bone formation.

Isolation and characterization of putative mesenchymal stem cells from mammalian gut.

Here, we provide a protocol for reliable isolation and subculture of putative mesenchymal stem cells from mice colons. This method provides a good approach to cultivate and characterize putative colonic mesenchymal stem cells (cMSCs). A high purity of cMSCs can be obtained according to this protocol. The whole isolation processes of cMSCs take about 2h with two important digestion steps involved. Only with common culture medium, maturation of cMSCs in culture proceeds approximately 2 weeks to allow relevant researches to be conducted. This protocol sheds light on better cultivation of MSCs in vitro from post-natal colon tissues. These putative cMSCs share common phenotypic property with those in vivo reported, and contain potential lineage differentiation capacity. The successful culture of cMSCs in vitro provides an ideal model for study of MSCs biology in the intestine.

Clinical Trials With Mesenchymal Stem Cells: An Update.

In the last year, the promising features of mesenchymal stem cells (MSCs), including their regenerative properties and ability to differentiate into diverse cell lineages, have generated great interest among researchers whose work has offered intriguing perspectives on cell-based therapies for various diseases. Currently the most commonly used adult stem cells in regenerative medicine, MSCs, can be isolated from several tissues, exhibit a strong capacity for replication in vitro, and can differentiate into osteoblasts, chondrocytes, and adipocytes. However, heterogeneous procedures for isolating and cultivating MSCs among laboratories have prompted the International Society for Cellular Therapy (ISCT) to issue criteria for identifying unique populations of these cells. Consequently, the isolation of MSCs according to ISCT criteria has produced heterogeneous, nonclonal cultures of stromal cells containing stem cells with different multipotent properties, committed progenitors, and differentiated cells. Though the nature and functions of MSCs remain unclear, nonclonal stromal cultures obtained from bone marrow and other tissues currently serve as sources of putative MSCs for therapeutic purposes, and several findings underscore their effectiveness in treating different diseases. To date, 493 MSC-based clinical trials, either complete or ongoing, appear in the database of the US National Institutes of Health. In the present article, we provide a comprehensive review of MSC-based clinical trials conducted worldwide that scrutinizes biological properties of MSCs, elucidates recent clinical findings and clinical trial phases of investigation, highlights therapeutic effects of MSCs, and identifies principal criticisms of the use of these cells. In particular, we analyze clinical trials using MSCs for representative diseases, including hematological disease, graft-versus-host disease, organ transplantation, diabetes, inflammatory diseases, and diseases in the liver, kidney, and lung, as well as cardiovascular, bone and cartilage, neurological, and autoimmune diseases.

Concise Review: When Colonies Are Not Clones: Evidence and Implications of Intracolony Heterogeneity in Mesenchymal Stem Cells.

The emergence of heterogeneity in putative mesenchymal stem cell (MSC) populations during in vitro expansion is not appreciated fully by the various communities who study, engineer, and use such stem cells. However, this functional diversity holds direct implications for basic research and therapeutic applications of MSCs that require predictable phenotypic function and efficacy. Despite numerous clinical trials pursuing MSC therapies, the in vitro expansion of homogeneous populations to therapeutically relevant quantities remains an elusive goal. Variation in MSC cultures has been noted not only among donors and within populations expanded from the same donor, but also debatably within single-cell-derived colonies. The potential for even intracolony heterogeneity suggests that any purified subpopulation will inevitably become heterogeneous upon further expansion under current culture conditions. Here, we review the noted or retrospective evidence of intracolony MSC heterogeneity, to facilitate discussion of its possible causes and potential solutions to its mitigation. This analysis suggests that functional diversity within an MSC colony must be considered in design of experiments and trials for even nonclonal stem cell populations, and can be mitigated or even exploited when the mechanisms of onset are better understood. Stem Cells 2016;34:1135-1141.