A new gamboge derivative Compound 2 inhibits cancer stem‐like cells via suppressing EGFR tyrosine phosphorylation in head and neck squamous cell carcinoma

Cancer stem‐like cells represent a population of tumour‐initiating cells that lead to the relapse and metastasis of cancer. Conventional anti‐cancer therapeutic drugs are usually ineffective in eliminating the cancer stem‐like cells. Therefore, new drugs or therapeutic methods effectively targeting cancer stem‐like cells are in urgent need to successfully cure cancer. Gamboge is a natural anti‐cancer medicine whose pharmacological effects are different from those of conventional chemotherapeutical drugs and they can kill some kinds of cancer cells selectively. In this study, we identified a new gamboge derivative, Compound 2 (C2), which presents eminent suppression effects on cancer cells. Interestingly, when compared with cisplatin (CDDP), C2 effectively suppresses the growth of both cancer stem‐like cells and non‐cancer stem‐like cells derived from head and neck squamous cell carcinoma (HNSCC), inhibiting the formation of tumour spheres and colony in vitro, resulting in the loss of expression of multiple cancer stem cell (CSC)‐related molecules in HNSCC. Treating with C2 effectively inhibited the growth of HNSCC in BALB/C nude mice. Further investigation found that C2 notably inhibits the activation of epithelial growth factor receptor and the phosphorylation of its downstream protein kinase homo sapiens v‐akt murine thymoma viral oncogene homolog (AKT) in HNSCC, resulting in down‐regulation of multiple CSC‐related molecules in HNSCC. Our study has demonstrated that C2 effectively inhibits the stem‐like property of cancer stem‐like cells in HNSCC and may be a hopeful targeting drug in cancer therapy.     

Angiogenic and Restorative Abilities of Human Mesenchymal Stem Cells Were Reduced Following Treatment With Serum From Diabetes Mellitus Type 2 Patients

This experiment investigated the impact of serum from patients with type 2 diabetes mellitus on the angiogenic behavior of human mesenchymal stem cells in vitro. Changes in the level of Ang‐1, Ang‐2, cell migration, and trans‐differentiation into pericytes and endothelial lineage were monitored after 7 days. The interaction of mesenchymal stem cells with endothelial cells were evaluated using surface plasmon resonance technique. Paracrine restorative effect of diabetic stem cells was tested on pancreatic β cells. Compared to data from FBS and normal serum, diabetic serum reduced the stem cell survival and chemotaxis toward VEGF and SDF‐1α (P < 0.05). Diabetic condition were found to decline cell migration rate and the activity of MMP‐2 and ‐9 (P < 0.05). The down‐regulation of VEGFR‐2 and CXCR‐4 was observed with an increase in the level of miR‐1‐3p and miR‐15b‐5p at the same time. The paracrine angiogenic potential of diabetic stem cells was disturbed via the changes in the dynamic of Ang‐1, Ang‐2, and VEGF. Surface plasmon resonance analysis showed that diabetes could induce an aberrant increase in the interaction of stem cells with endothelial cells. After treatment with diabetic serum, the expression of VE‐cadherin and NG2 and ability for uptake of Dil‐Ac‐LDL were reduced (P < 0.01). Conditioned media prepared from diabetic stem cells were unable to decrease fatty acid accumulation in β‐cells (P < 0.05). The level of insulin secreted by β‐cells was not affected after exposure to supernatant from diabetic or non‐diabetic mesenchymal stem cells. Data suggest diabetes could decrease angiogenic and restorative effect of stem cells in vitro. J. Cell. Biochem. 119: 524–535, 2018. © 2017 Wiley Periodicals, Inc.     

LncRNA MALAT1/miR‐129 axis promotes glioma tumorigenesis by targeting SOX2

We aimed to explore the interaction among lncRNA MALAT1, miR‐129 and SOX2. Besides, we would investigate the effect of MALAT1 on the proliferation of glioma stem cells and glioma tumorigenesis. Differentially expressed lncRNAs in glioma cells and glioma stem cells were screened out with microarray analysis. The targeting relationship between miR‐129 and MALAT1 or SOX2 was validated by dual‐luciferase reporter assay. The expressions of MALAT1, miR‐129 and SOX2mRNA in both glioma non‐stem cells and glioma stem cells were examined by qRT‐PCR assay. The impact of MALAT1 and miR‐129 on glioma stem cell proliferation was observed by CCK‐8 assay, EdU assay and sphere formation assay. The protein expression of SOX2 was determined by western blot. The effects of MALAT1 and miR‐129 on glioma tumour growth were further confirmed using xenograft mouse model. The mRNA expression of MALAT1 was significantly up‐regulated in glioma stem cells compared with non‐stem cells, while miR‐129 was significantly down‐regulated in glioma stem cells. MALAT1 knockdown inhibited glioma stem cell proliferation via miR‐129 enhancement. Meanwhile, miR‐129 directly targeted at SOX2 and suppressed cell viability and proliferation of glioma stem cells by suppressing SOX2 expression. The down‐regulation of MALAT1 and miR‐129 overexpression both suppressed glioma tumour growth via SOX2 expression promotion in vivo. MALAT1 enhanced glioma stem cell viability and proliferation abilities and promoted glioma tumorigenesis through suppressing miR‐129 and facilitating SOX2 expressions.     

Identification and expansion of human osteosarcoma‐cancer‐stem cells by long‐term 3‐aminobenzamide treatment

A novel cancer stem‐like cell line (3AB‐OS), expressing a number of pluripotent stem cell markers, was irreversibly selected from human osteosarcoma MG‐63 cells by long‐term treatment (100 days) with 3‐aminobenzamide (3AB). 3AB‐OS cells are a heterogeneous and stable cell population composed by three types of fibroblastoid cells, spindle‐shaped, polygonal‐shaped, and rounded‐shaped. With respect to MG‐63 cells, 3AB‐OS cells are extremely smaller, possess a much greater capacity to form spheres, a stronger self‐renewal ability and much higher levels of cell cycle markers which account for G1‐S/G2‐M phases progression. Differently from MG‐63 cells, 3AB‐OS cells can be reseeded unlimitedly without losing their proliferative potential. They show an ATP‐binding cassette transporter ABCG2‐dependent phenotype with high drug efflux capacity, and a strong positivity for CD133, marker for pluripotent stem cells, which are almost unmeasurable in MG‐63 cells. 3AB‐OS cells are much less committed to osteogenic and adipogenic differentiation than MG‐63 cells and highly express genes required for maintaining stem cell state (Oct3/4, hTERT, nucleostemin, Nanog) and for inhibiting apoptosis (HIF‐1α, FLIP‐L, Bcl‐2, XIAP, IAPs, and survivin). 3AB‐OS may be a novel tumor cell line useful for investigating the mechanisms by which stem cells enrichment may be induced in a tumor cell line. The identification of a subpopulation of cancer stem cells that drives tumorigenesis and chemoresistance in osteosarcoma may lead to prognosis and optimal therapy determination. Expression patterns of stem cell markers, especially CD133 and ABCG2, may indicate the undifferentiated state of osteosarcoma tumors, and may correlate with unfavorable prognosis in the clinical setting. J. Cell. Physiol. 219: 301–313, 2009. © 2009 Wiley‐Liss, Inc.     

A new genetic tool to improve immune‐compromised mouse models: Derivation and CRISPR/Cas9‐mediated targeting of NRG embryonic stem cell lines

Development of human hematopoietic stem cells and differentiation of embryonic stem (ES) cells/induced pluripotent stem (iPS) cells to hematopoietic stem cells are poorly understood. NOD (Non‐obese diabetic)‐derived mouse strains, such as NSG (NOD‐Scid‐il2Rg) or NRG (NOD‐Rag1‐il2Rg), are the best available models for studying the function of fetal and adult human hematopoietic cells as well as ES/iPS cell‐derived hematopoietic stem cells. Unfortunately, engraftment of human hematopoietic stem cells is very variable in these models. Introduction of additional permissive mutations into these complex genetic backgrounds of the NRG/NSG mice by natural breeding is a very demanding task in terms of time and resources. Specifically, since the genetic elements defining the NSG/NRG phenotypes have not yet been fully characterized, intense backcrossing is required to ensure transmission of the full phenotype. Here we describe the derivation of embryonic stem cell (ESC) lines from NRG pre‐implantation embryos generated by in vitro fertilization followed by the CRISPR/CAS9 targeting of the Gata‐2 locus. After injection into morula stage embryos, cells from three tested lines gave rise to chimeric adult mice showing high contribution of the ESCs (70%–100%), assessed by coat color. Moreover, these lines have been successfully targeted using Cas9/CRISPR technology, and the mutant cells have been shown to remain germ line competent. Therefore, these new NRG ESC lines combined with genome editing nucleases bring a powerful genetic tool that facilitates the generation of new NOD‐based mouse models with the aim to improve the existing xenograft models.     

Critical roles of hMAGEA2 in induced pluripotent stem cell pluripotency,proliferation, and differentiation

Induced pluripotent stem (iPS) cells are important for clinical application and stem cell research. Although human melanoma‐associated antigen A2 (hMAGEA2) expression is known to affect differentiation in embryonic stem cells, its specific role in iPS cells remains unclear. To evaluate the function of hMAGEA2 and its characteristics in iPS cells, we produced hMAGEA2‐overexpressing iPS cells from hMAGEA2‐overexpressing transgenic mice. Although the iPS cells with overexpressed hMAGEA2 did not differ in morphology, their pluripotency, and self‐renewal related genes (Nanog, Oct3/4, Sox2, and Stat3), expression level was significantly upregulated. Moreover, hMAGEA2 contributed to the promotion of cell cycle progression, thereby accelerating cell proliferation. Through embryoid body formation in vitro and teratoma formation in vivo, we demonstrated that hMAGEA2 critically decreases the differentiation ability of iPS cells. These data indicate that hMAGEA2 intensifies the self‐renewal, pluripotency, and degree of proliferation of iPS cells, while significantly repressing their differentiation efficiency. Therefore, our findings prove that hMAGEA2 plays key roles in iPS cells.     

Stem cells in asexual reproduction of Enchytraeus japonensis (Oligochaeta, Annelid): proliferation and migration of neoblasts

Enchytraeus japonensis is a small oligochaete that reproduces mainly asexually by fragmentation (autotomy) and regeneration. As sexual reproduction can also be induced, it is a good animal model for the study of both somatic and germline stem cells. To clarify the features of stem cells in regeneration, we investigated the proliferation and lineage of stem cells in E. japonensis. Neoblasts, which have the morphological characteristics of undifferentiated cells, were found to firmly adhere to the posterior surface of septa in each trunk segment. Also, smaller neoblast‐like cells, which are designated as N‐cells in this study, were located dorsal to the neoblasts on the septa. By conducting 5‐bromo‐2′‐deoxyuridine (BrdU)‐labeling‐experiments, we have shown that neoblasts are slow‐cycling (or quiescent) in intact growing worms, but proliferate rapidly in response to fragmentation. N‐cells proliferate more actively than do neoblasts in intact worms. The results of pulse‐chase experiments indicated that neoblast and N‐cell lineage mesodermal cells that incorporated BrdU early in regeneration migrated toward the autotomized site to form the mesodermal region of the blastema, while the epidermal and intestinal cells also contributed to the blastema locally near the autotomized site. We have also shown that neoblasts have stem cell characteristics by expressing Ej‐vlg2 and by the activity of telomerase during regeneration. Telomerase activity was high in the early stage of regeneration and correlated with the proliferation activity in the neoblast lineage of mesodermal stem cells. Taken together, our results indicate that neoblasts are mesodermal stem cells involved in the regeneration of E. japonensis.     

HIF1α‐mediated AIMP3 suppression delays stem cell aging via the induction of autophagy

Senescence in stem cells, which occurs as a consequence of chronic responses to the environment, defines the capacity of stem cells for proliferation and differentiation as well as their potential for tissue regeneration and homeostasis maintenance. Although stem cells reside under low oxygen pressure and the availability of oxygen is known to be a crucial determinant in their fate, the key modulators in stem cell aging and the underlying mechanism have yet to be unraveled. Human placenta‐derived mesenchymal stem cells (hpMSCs) were cultured under hypoxia (3% O₂) or normoxia (21% O₂) to investigate the key factors that regulate stem cell senescence under hypoxic conditions. RNA sequencing results suggested that the expression of aminoacyl‐tRNA synthetase‐interacting multifunctional protein 3 (AIMP3, EEF1E1), an aging inducer, in the hpMSCs was dramatically repressed under hypoxia with concurrent suppression of the aging marker p16^INK4a. The hpMSCs that overexpressed AIMP3 under hypoxic conditions displayed significantly decreased proliferation and fewer stem cell characteristics, whereas the downregulation of AIMP3 ameliorated the age‐related senescence of MSCs. Consistent with the results of the hpMSCs, MSCs isolated from the adipose tissue of AIMP3‐overexpressing mice exhibited decreased stem cell functions. Interestingly, AIMP3‐induced senescence is negatively regulated by hypoxia‐inducible factor 1α (HIF1α) and positively regulated by Notch3. Furthermore, we showed that AIMP3 enhanced mitochondrial respiration and suppressed autophagic activity, indicating that the AIMP3‐associated modulation of metabolism and autophagy is a key mechanism in the senescence of stem cells and further suggesting a novel target for interventions against aging.     

Coupling of the radiosensitivity of melanocyte stem cells to their dormancy during the hair cycle

Current studies have revealed that stem cells are more radiosensitive than mature cells. As somatic stem cells are mostly kept in a quiescent state, this conflicts with Bergonié and Tribondeau's law that actively mitotic cells are the most radiosensitive. In this study, we focused on hair graying to understand the stress‐resistance of melanocyte stem cells (McSCs). We used Dct‐H2B‐GFP transgenic mice which enables the stable visualization of McSCs and an anti‐Kit monoclonal antibody which selectively eradicates amplifying McSCs. The results demonstrate that quiescent McSCs are rather radiosensitive, but the coexistence of non‐quiescent McSCs provides the stem cell pool with radioresistance. The irradiated quiescent McSCs prematurely differentiate in the niche upon their activation without sufficiently renewing themselves for cyclic hair pigmentation. These data indicate that tissue radiosensitivity is largely dependent on the state of somatic stem cells under their local microenvironment.     

Effect of TET1 regulating MGMT on chemotherapy resistance of oral squamous cell carcinoma stem cells

The study was to evaluate the effect of ten‐eleven translocation 1 (TET1) regulating o6‐methylguanine‐DNA methyltransferase (MGMT) in chemotherapy resistance of oral squamous cell carcinoma (OSCC) stem cells. OSCC stem cells were divided into the blank, negative control (NC), TET1‐siRNA, TET1‐siRNA + MGMT‐OE, and MGMT‐OE groups. Methylation‐specific polymerase chain reaction (MSP), qRT‐PCR and Western blotting were conducted to detect the methylation status of MGMT, expressions of TET1, MGMT, ABCG2, and Oct‐4. Cell proliferation, cisplatin chemosensitivity, and cell cycle and apoptosis, were detected using CCK8 and flow cytometry. A chromatin immunoprecipitation (ChIP) assay was employed for detecting the link between TET1 and MGMT gene promoters. In comparison to the NC group, the TET1‐siRNA group exhibited increased levels of MGMT methylation, the number of apoptotic cells and cisplatin chemosensitivity consisting of varying concentrations, however, decreased levels of mRNA and protein expressions of TET1 as well as MGMT, cell viability, the number of cells in the S phase, and protein expressions of ABCG2 and Oct‐4 were all have diminished amounts. The TET1‐siRNA + MGMT‐OE and MGMT‐OE groups had higher MGMT mRNA and protein expression, as well as increased protein expressions of ABCG2 and Oct‐4, greater cell activity, higher number of cells in the S phase, decreased apoptotic rates in cells and decreased cisplatin chemosensitivity with different concentrations. Our study provided evidence that low‐expression of TET1 in OSCC stem cells may stimulate MGMT promoter methylation, while inhibiting MGMT mRNA expression, this ultimately strengthens the sensitivity of OSCC stem cells in regards to chemotherapeutics.     

Conditional TGF‐β1 treatment increases stem cell‐like cell population in myoblasts

The limitation in successfully acquiring large populations of stem cell has impeded their application. A new method based on the dedifferentiation of adult somatic cells to generate induced multipotent stem cells would allow us to obtain a large amount of autologous stem cells for regenerative medicine. The current work was proposed to induce a sub‐population of cells with characteristics of muscle stem cells from myoblasts through conditional treatment of transforming growth factor (TGF)‐β₁. Our results show that a lower concentration of TGF‐β₁ is able to promote C2C12 myoblasts to express stem cell markers as well as to repress myogenic proteins, which involves a mechanism of dedifferentiation. Moreover, TGF‐β₁ treatment promoted the proliferation‐arrested C2C12 myoblasts to re‐enter the S‐phase. We also investigated the multi‐differentiation potentials of the dedifferentiated cells. TGF‐β₁ pre‐treated C2C12 myoblasts were implanted into mice to repair dystrophic skeletal muscle or injured bone. In addition to the C2C12 myoblasts, similar effects of TGF‐β₁ were also observed in the primary myoblasts of mice. Our results suggest that TGF‐β₁ is effective as a molecular trigger for the dedifferentiation of skeletal muscle myoblasts and could be used to generate a large pool of progenitor cells that collectively behave as multipotent stem cell‐like cells for regenerative medicine applications.     

Bone marrow and adipose mesenchymal stem cells attenuate cardiac fibrosis induced by methotrexate in rats

Mesenchymal stem cells (MSCs) are an ideal adult stem cell with capacity for self‐renewal and differentiation with an extensive tissue distribution. The present study evaluates the therapeutic effects of bone marrow mesenchymal stem cells (BM‐MSCs) or adipose‐derived mesenchymal stem cells (AD‐MSCs) against the development of methotrexate (MTX)‐induced cardiac fibrosis versus dexamethasone (DEX). Rats were allocated into five groups; group 1, received normal saline orally; group 2, received MTX (14 mg/kg/week for 2 weeks); groups 3 and 4, treated once with 2 × 10 ⁶ cells of MTX + BM‐MSCs and MTX + AD‐MSCs, respectively; and group 5, MTX + DEX (0.5 mg/kg, for 7 days, P.O.). MTX induced cardiac fibrosis as marked changes in oxidative biomarkers and elevation of triglyceride, cholesterol, aspartate aminotransferase, gamma‐glutamyl transferase, creatine kinase, and caspase‐3, as well as deposited collagen. These injurious effects were antagonized after treatment with MSCs. So, MSCs possessed antioxidant, antiapoptotic, as well antifibrotic effects, which will perhaps initiate them as notable prospective for the treatment of cardiac fibrosis.     

Modulation of tumorigenesis and oestrogen receptor‐α expression by cell culture conditions in a stem cell‐derived breast epithelial cell line

Background information. The common phenotypes of cancer and stem cells suggest that cancers arise from stem cells. Oestrogen is one of the few most important determinants of breast cancer, as shown by several lines of convincing evidence. We have previously reported a human breast epithelial cell type (Type 1 HBEC) with stem cell characteristics and ERα (oestrogen receptor α) expression. A tumorigenic cell line, M13SV1R2, was developed from this cell type after SV40 (simian virus 40) large T‐antigen transfection and X‐ray irradiation. The cell line, however, was not responsive to oestrogen for cell growth or tumour development. In the present study, we tested the hypothesis that deprivation of growth factors and hormones may change the tumorigenicity and oestrogen response of this cell line. Results. The M13SV1R2 cells lost their tumorigenicity after culturing in a growth factor/hormone‐deprived medium for >10 passages (referred to as R2d cells) concomitant with the expression of two tumour suppressor genes, namely those coding for maspin and α6 integrin. However, these cells acquired oestrogen responsiveness in cell growth and tumour development. By immunocytochemistry, Western blotting and flow cytometry analysis, oestrogen treatment of R2d cells was found to induce many important effects related to breast carcinogenesis, namely: (i) the emergence of a subpopulation of cells expressing CD44^+/high/CD24^?/low breast tumour stem cell markers; (ii) the induction of EMT (epithelial‐to‐mesenchymal transition); (iii) the acquisition of metastatic ability; and (iv) the expression of COX‐2 (cyclo‐oxygenase‐2) through a CD44‐mediated mechanism. Conclusion. An oestrogen‐responsive cell line with ERα and CD44⁺/CD24^?/low expression can be derived from breast epithelial stem cells. The tumorigenicity and oestrogen response of these cells could depend on the cell culture conditions. The findings of this study have implications in regard to the origins of (1) ERα‐positive breast cancers, (2) CD44⁺/CD24^?/low breast tumour stem cells and (3) the metastatic ability of breast cancer.     

Tumor suppressors Sav/scrib and oncogene ras regulate stem‐cell transformation in adult Drosophila malpighian tubules

An increasing body of evidence suggests that tumors might originate from a few transformed cells that share many properties with normal stem cells. However, it remains unclear how normal stem cells are transformed into cancer stem cells (CSCs). Here, we demonstrated that mutations causing the loss of tumor suppressor Salvador (Sav) or Scribble (Scrib) or activation of the oncogene Ras transform normal stem cells into CSCs through a multistep process in the adult Drosophila Malpighian Tubules (MTs). In wild‐type MTs, each stem cell generates one self‐renewing and one differentiating daughter cell. However, in flies with loss‐of‐function sav or scrib or gain‐of‐function Ras mutations, both daughter cells grew and behaved like stem cells, leading to the formation of tumors in MTs. Ras functioned downstream of Sav and Scrib in regulating the stem‐cell transformation. The Ras‐transformed stem cells exhibited many of the hallmarks of cancer, such as increased proliferation, reduced cell death, and failure to differentiate. We further demonstrated that several signal transduction pathways (including MEK/MAPK, RhoA, PKA, and TOR) mediate Ras' function in the stem‐cell transformation. Therefore, we have identified a molecular mechanism that regulates stem‐cell transformation, and this finding may lead to strategies for preventing tumor formation in certain organs. J. Cell. Physiol. 224: 766–774, 2010. © 2010 Wiley‐Liss, Inc.     

Chronic ethanol exposure of human pancreatic normal ductal epithelial cells induces cancer stem cell phenotype through SATB2

The incidence of pancreatic cancer is on the rise. Risk factors for pancreatic cancer include alcohol toxicity and metabolic conditions such as obesity, hypertension, dyslipidaemia, insulin resistance and type 2 diabetes. However, the molecular mechanism by which chronic alcohol consumption contributes to pancreatic cancer is not well understood. The purpose of the study was to demonstrate the effects of long‐term chronic ethanol exposure on the transformation of human pancreatic normal ductal epithelial (HPNE) cells. Our data showed that ethanol‐transformed HPNE cells were more progressively transformed exhibiting spheroids and colonies, and anchorage‐independent growth. These transformed cells contained high levels of reactive oxygen species and induced SATB2 expression. Furthermore, during ethanol‐induced cellular transformation, cells gained the phenotypes of cancer stem cells (CSCs) by expressing pluripotency maintaining factors (Oct4, Sox2, cMyc and KLF4) and stem cell markers (CD24, CD44 and CD133). Ethanol‐induced SATB2 can bind to the promoters of KLF4, Oct4, cMyc, Sox2, Bcl‐2 and XIAP genes. Suppression of SATB2 expression in ethanol‐transformed HPNE cells inhibited cell proliferation, colony formation and markers of CSCs and pluripotency. These data suggest that chronic alcohol consumption may contribute toward the development of pancreatic cancer by converting HPNE cells to cancer stem‐like cells.     

Combined pre‐conditioning with salidroside and hypoxia improves proliferation,migration and stress tolerance of adipose‐derived stem cells

Oxidative stress after ischaemia impairs the function of transplanted stem cells. Increasing evidence has suggested that either salidroside (SAL) or hypoxia regulates growth of stem cells. However, the role of SAL in regulating function of hypoxia‐pre–conditioned stem cells remains elusive. Thus, this study aimed to determine the effect of SAL and hypoxia pre‐conditionings on the proliferation, migration and tolerance against oxidative stress in rat adipose‐derived stem cells (rASCs). rASCs treated with SAL under normoxia (20% O₂) or hypoxia (5% O₂) were analysed for the cell viability, proliferation, migration and resistance against H₂O₂‐induced oxidative stress. In addition, the activation of Akt, Erk1/2, LC3, NF‐κB and apoptosis‐associated pathways was assayed by Western blot. The results showed that SAL and hypoxia treatments synergistically enhanced the viability (fold) and proliferation of rASCs under non‐stressed conditions in association with increased autophagic flux and activation of Akt, Erk1/2 and LC3. H₂O₂‐induced oxidative stress, cytotoxicity, apoptosis, autophagic cell death and NF‐κB activation were inhibited by SAL or hypoxia, and further attenuated by the combined SAL and hypoxia pre‐treatment. The SAL and hypoxia pre‐treatment also enhanced the proliferation and migration of rASCs under oxidative stress in association with Akt and Erk1/2 activation; however, the combined pre‐treatment exhibited a more profound enhancement in the migration than proliferation. Our data suggest that SAL combined with hypoxia pre‐conditioning may enhance the therapeutic capacity of ASCs in post‐ischaemic repair.     

Musashi 1‐positive cells derived from mouse embryonic stem cells can differentiate into neural and intestinal epithelial‐like cells in vivo

Msi1 (Musashi 1) is regarded as a marker for neural and intestinal epithelial stem cells. However, it is still unclear whether Msi1‐positive cells derived from mouse embryonic stem cells have the ability to differentiate into neural or intestinal epithelial cells. A pMsi1–GFP (green fluorescent protein) reporter plasmid was constructed in order to sort Msi1‐positive cells out of the differentiated cell population. The GFP‐positive cells (i.e. Msi1‐positive cells) were sorted by FACS and were hypodermically engrafted into the backs of NOD/SCID (non‐obese diabetic/severe combined immunodeficient) mice. The presence of neural and intestinal epithelial cells in the grafts was detected. Msi1 was highly expressed in the GFP‐positive cells, but not in the GFP‐negative cells. The markers for neural cells (Nestin and Tubulin β III) and intestinal epithelial cells [FABP2 (fatty acid binding protein 2), Lyz (lysozyme) and ChA (chromogranin A)] were more highly expressed in the grafts from Msi1‐positive cells than those from Msi1‐negative cells (P<0.05). The grafts from the Msi1‐negative cells contained more mesodermal‐like tissues than those from the Msi1‐positive cells. The pMsi1–GFP vector can be used to sort Msi1‐positive cells from a cell population derived from mouse embryonic stem cells. The Msi1‐positive cells can differentiate into neural and intestinal epithelial‐like cells in vivo.     

Mouse 3T3 fibroblasts under the influence of fibroblasts isolated from stroma of human basal cell carcinoma acquire properties of multipotent stem cells

Background information. Multipotent mesenchymal stem cells can participate in the formation of a microenvironment stimulating the aggressive behaviour of cancer cells. Moreover, cells exhibiting pluripotent ESC (embryonic stem cell) markers (Nanog and Oct4) have been observed in many tumours. Here, we investigate the role of cancer‐associated fibroblasts in the formation of stem cell supporting properties of tumour stroma. We test the influence of fibroblasts isolated from basal cell carcinoma on mouse 3T3 fibroblasts, focusing on the expression of stem cell markers and plasticity in vitro by means of microarrays, qRT‐PCR (quantitative real‐time PCR) and immunohistochemistry. Results. We demonstrate the biological activity of the cancer stromal fibroblasts by influencing the 3T3 fibroblasts to express markers such as Oct4, Nanog and Sox2 and to show differentiation potential similar to mesenchymal stem cells. The role of growth factors such as IGF2 (insulin‐like growth factor 2), FGF7 (fibroblast growth factor 7), LEP (leptin), NGF (nerve growth factor) and TGFβ (transforming growth factor β), produced by the stromal fibroblasts, is established to participate in their bioactivity. Uninduced 3T3 do not express the stem cell markers and show minimal differentiation potential. Conclusions. Our observations indicate the pro‐stem cell activity of cancer‐associated fibroblasts and underline the role of epithelial—mesenchymal interaction in tumour biology.