Bone marrow-derived mesenchymal stem cells inhibit vascular smooth muscle cell proliferation and neointimal hyperplasia after arterial injury in rats

We investigated whether mesenchymal stem cell (MSC)-based treatment could inhibit neointimal hyperplasia in a rat model of carotid arterial injury and explored potential mechanisms underlying the positive effects of MSC therapy on vascular remodeling/repair. Sprague-Dawley rats underwent balloon injury to their right carotid arteries. After 2 days, we administered cultured MSCs from bone marrow of GFP-transgenic rats (0.8 × 10⁶ cells, n = 10) or vehicle (controls, n = 10) to adventitial sites of the injured arteries. As an additional control, some rats received a higher dose of MSCs by systemic infusion (3 × 10⁶ cells, tail vein; n = 4). Local vascular MSC administration significantly prevented neointimal hyperplasia (intima/media ratio) and reduced the percentage of Ki67 + proliferating cells in arterial walls by 14 days after treatment, despite little evidence of long-term MSC engraftment. Notably, systemic MSC infusion did not alter neointimal formation. By immunohistochemistry, compared with neointimal cells of controls, cells in MSC-treated arteries expressed reduced levels of embryonic myosin heavy chain and RM-4, an inflammatory cell marker. In the presence of platelet-derived growth factor (PDGF-BB), conditioned medium from MSCs increased p27 protein levels and significantly attenuated VSMC proliferation in culture. Furthermore, MSC-conditioned medium suppressed the expression of inflammatory cytokines and RM-4 in PDGF-BB-treated VSMCs. Thus, perivascular administration of MSCs may improve restenosis after vascular injury through paracrine effects that modulate VSMC inflammatory phenotype.     

Repairing chronic myocardial infarction with autologous mesenchymal stem cells engineered tissue in rat promotes angiogenesis and limits ventricular remodeling

Background

Tissue engineering scaffold constitutes a new strategy of myocardial repair. Here, we studied the contribution of a patch using autologous mesenchymal stem cells (MSCs) seeded on collagen-1 scaffold on the cardiac reconstruction in rat model of chronic myocardial infarction (MI).

Methods

Patches were cultured with controlled MSCs (growth, phenotype and potentiality). Twenty coronary ligated rats with tomoscingraphy (SPECT)-authenticated transmural chronic MI were referred into a control group (n = 10) and a treated group (n = 10) which beneficiated an epicardial MSC-patch engraftment. Contribution of MSC-patch was tested 1-mo after using non-invasive SPECT cardiac imaging, invasive hemodynamic assessment and immunohistochemistry.

Results

3D-collagen environment affected the cell growth but not the cell phenotype and potentiality. MSC-patch integrates well the epicardial side of chronic MI scar. In treated rats, one-month SPECT data have documented an improvement of perfusion in MI segments compared to control (64 ± 4% vs 49 ± 3% p = 0.02) and a reduced infarction. Contractile parameter dp/dtmax and dp/dtmin were improved (p & 0.01). Histology showed an increase of ventricular wall thickness (1.75 ± 0.24 vs 1.35 ± 0.32 mm, p &0.05) and immunochemistry of the repaired tissue displayed enhanced angiogenesis and myofibroblast-like tissue.

Conclusion

3D-MSC-collagen epicardial patch engraftment contributes to reverse remodeling of chronic MI.     

Regulation of alternative macrophage activation by MSCs derived hypoxic conditioned medium,via the TGF-β1/Smad3 pathway

Macrophages are re-educated and polarized in response to myocardial infarction (MI). The M2 anti-inflammatory phenotype is a known dominator of late stage MI. Mesenchymal stem cells (MSCs) represent a promising tool for cell therapy, particularly heart related diseases. In general, MSCs induce alteration of the macrophage subtype from M1 to M2, both in vitro and in vivo. We conjectured that hypoxic conditions can promote secretome productivity of MSCs. Hypoxia induces TGF-β1 expression, and TGF-β1 mediates M2 macrophage polarization for anti-inflammation and angiogenesis in infarcted areas. We hypothesized that macrophages undergo advanced M2 polarization after exposure to MSCs in hypoxia. Treatment of MSCs derived hypoxic conditioned medium (hypo-CM) promoted M2 phenotype and neovascularization through the TGF-β1/Smad3 pathway. In addition, hypo-CM derived from MSCs improved restoration of ischemic heart, such as attenuating cell apoptosis and fibrosis, and ameliorating microvessel density. Based on our results, we propose a new therapeutic method for effective MI treatment using regulation of macrophage polarization.      

Regulation of transplanted mesenchymal stem cells by the lung progenitor niche in rats with chronic obstructive pulmonary disease

Background

Stem cell transplantation is a promising method for the treatment of chronic obstructive pulmonary disease (COPD), and mesenchymal stem cells (MSCs) have clinical potential for lung repair/regeneration. However, the rates of engraftment and differentiation are generally low following MSC therapy for lung injury. In previous studies, we constructed a pulmonary surfactant-associated protein A (SPA) suicide gene system, rAAV-SPA-TK, which induced apoptosis in alveolar epithelial type II (AT II) cells and vacated the AT II cell niche. We hypothesized that this system would increase the rates of MSC engraftment and repair in COPD rats.

Methods

The MSC engraftment rate and morphometric changes in lung tissue in vivo were investigated by in situ hybridization, hematoxylin and eosin staining, Masson’s trichrome staining, immunohistochemistry, and real-time PCR. The expression of hypoxia inducible factor (HIF-1α) and stromal cell-derived factor-1 (SDF-1), and relationship between HIF-1α and SDF-1 in a hypoxic cell model were analyzed by real-time PCR, western blotting, and enzyme-linked immunosorbent assay.

Results

rAAV-SPA-TK transfection increased the recruitment of MSCs but induced pulmonary fibrosis in COPD rats. HIF-1α and SDF-1 expression were enhanced after rAAV-SPA-TK transfection. Hypoxia increased the expression of HIF-1α and SDF-1 in the hypoxic cell model, and SDF-1 expression was augmented by HIF-1α under hypoxic conditions.

Conclusions

Vacant AT II cell niches increase the homing and recruitment of MSCs to the lung in COPD rats. MSCs play an important role in lung repair and promote collagen fiber deposition after induction of secondary damage in AT II cells by rAAV-SPA-TK, which involves HIF-1α and SDF-1 signaling.     

Sonic Hedgehog Gene Delivery to the Rodent Heart Promotes Angiogenesis via iNOS/Netrin-1/PKC Pathway

Background

We hypothesized that genetic modification of mesenchymal stem cells (MSCs) with Sonic Hedgehog (Shh) transgene, a morphogen during embryonic development and embryonic and adult stem cell growth, improved their survival and angiogenic potential in the ischemic heart via iNOS/netrin/PKC pathway.

Methods/Principal Findings

MSCs from young Fisher-344 rat bone marrow were purified and transfected with pCMV Shh plasmid (^ShhMSCs). Immunofluorescence, RT-PCR and Western blotting showed higher expression of Shh in ^ShhMSCs which also led to increased expression of angiogenic and pro-survival growth factors in ^ShhMSCs. Significantly improved migration and tube formation was seen in ^ShhMSCs as compared to empty vector transfected MSCs (^EmpMSCs). Significant upregulation of netrin-1 and iNOS was observed in ^ShhMSCs in PI3K independent but PKC dependent manner. For in vivo studies, acute myocardial infarction model was developed in Fisher-344 rats. The animals were grouped to receive 70 µl basal DMEM without cells (group-1) or containing 1×10^{6 Emp}MSCs (group-2) and ^ShhMSCs (group-3). Group-4 received recombinant netrin-1 protein injection into the infarcted heart. FISH and sry-quantification revealed improved survival of ^ShhMSCs post engraftment. Histological studies combined with fluorescent microspheres showed increased density of functionally competent blood vessels in group-3 and group-4. Echocardiography showed significantly preserved heart function indices post engraftment with ^ShhMSCs in group-3 animals.

Conclusions/Significance

Reprogramming of stem cells with Shh maximizes their survival and angiogenic potential in the heart via iNOS/netrin-1/PKC signaling.     

A perivascular origin for mesenchymal stem cells in multiple human organs

Mesenchymal stem cells (MSCs), the archetypal multipotent progenitor cells derived in cultures of developed organs, are of unknown identity and native distribution. We have prospectively identified perivascular cells, principally pericytes, in multiple human organs including skeletal muscle, pancreas, adipose tissue, and placenta, on CD146, NG2, and PDGF-Rbeta expression and absence of hematopoietic, endothelial, and myogenic cell markers. Perivascular cells purified from skeletal muscle or nonmuscle tissues were myogenic in culture and in vivo. Irrespective of their tissue origin, long-term cultured perivascular cells retained myogenicity; exhibited at the clonal level osteogenic, chondrogenic, and adipogenic potentials; expressed MSC markers; and migrated in a culture model of chemotaxis. Expression of MSC markers was also detected at the surface of native, noncultured perivascular cells. Thus, blood vessel walls harbor a reserve of progenitor cells that may be integral to the origin of the elusive MSCs and other related adult stem cells.     

Human CD34+/CD90+ ASCs Are Capable of Growing as Sphere Clusters,Producing High Levels of VEGF and Forming Capillaries

Background

Human adult adipose tissue is an abundant source of mesenchymal stem cells (MSCs). Moreover, it is an easily accessible site producing a considerable amount of stem cells.

Methodology/Principal Findings

In this study, we have selected and characterized stem cells within the stromal vascular fraction (SVF) of human adult adipose tissue with the aim of understanding their differentiation capabilities and performance. We have found, within the SVF, different cell populations expressing MSC markers – including CD34, CD90, CD29, CD44, CD105, and CD117 – and endothelial-progenitor-cell markers – including CD34, CD90, CD44, and CD54. Interestingly, CD34⁺/CD90⁺ cells formed sphere clusters, when placed in non-adherent growth conditions. Moreover, they showed a high proliferative capability, a telomerase activity that was significantly higher than that found in differentiated cells, and contained a fraction of cells displaying the phenotype of a side population. When cultured in adipogenic medium, CD34⁺/CD90⁺ quickly differentiated into adipocytes. In addition, they differentiated into endothelial cells (CD31⁺/VEGF⁺/Flk-1⁺) and, when placed in methylcellulose, were capable of forming capillary-like structures producing a high level of VEGF, as substantiated with ELISA tests.

Conclusions/Significance

Our results demonstrate, for the first time, that CD34⁺/CD90⁺ cells of human adipose tissue are capable of forming sphere clusters, when grown in free-floating conditions, and differentiate in endothelial cells that form capillary-like structures in methylcellulose. These cells might be suitable for tissue reconstruction in regenerative medicine, especially when patients need treatments for vascular disease.     

Defined serum-free media for in vitro expansion of adipose-derived mesenchymal stem cells

BackgroundThere is a growing interest in mesenchymal stem cells (MSCs) because they are regarded as good candidates for cell therapy. Adipose tissue represents an easily accessible source to derive mesenchymal stem cells (Ad-MSCs) non-invasively in large numbers. The aim of this study was to evaluate a defined serum-free medium for in vitro expansion of MSCs as a prerequisite for their clinical use.MethodsAdipose tissue was isolated from healthy donors. Cells were isolated and expanded for five passages in serum-free medium (Mesencult-XF) and Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (DMEM-FBS). MSC morphology, marker expression, viability, population doubling time and differentiation potential toward osteogenic and adipogenic lineages were evaluated. Bone marrow MSCs were included as controls.ResultsAd-MSCs cultured in Mesencult-XF had shorter population doubling time (33.3 ± 13.7 h) compared with those cultured in DMEM-FBS (54.3 ± 41.0 h, P < 0.05). Ad-MSCs cultured in Mesencult-XF displayed a stable morphology and surface marker expression and a higher differentiation potential in comparison to Ad-MSCs cultured in DMEM-FBS.ConclusionsThe defined serum-free and xeno-free Mesencult-XF media appear to be a good choice for Ad-MSCs, but it is not as good in supporting culture of bone marrow MSCs when the cells are to be used for clinical purposes.     

Comparative Characterization of Cells from the Various Compartments of the Human Umbilical Cord Shows that the Wharton’s Jelly Compartment Provides the Best Source of Clinically Utilizable Mesenchymal Stem Cells

The human umbilical cord (UC) is an attractive source of mesenchymal stem cells (MSCs) with unique advantages over other MSC sources. They have been isolated from different compartments of the UC but there has been no rigorous comparison to identify the compartment with the best clinical utility. We compared the histology, fresh and cultured cell numbers, morphology, proliferation, viability, stemness characteristics and differentiation potential of cells from the amnion (AM), subamnion (SA), perivascular (PV), Wharton’s jelly (WJ) and mixed cord (MC) of five UCs. The WJ occupied the largest area in the UC from which 4.61 ± 0.57 x 10⁶ /cm fresh cells could be isolated without culture compared to AM, SA, PV and MC that required culture. The WJ and PV had significantly lesser CD40+ non-stem cell contaminants (26-27%) compared to SA, AM and MC (51-70%). Cells from all compartments were proliferative, expressed the typical MSC-CD, HLA, and ESC markers, telomerase, had normal karyotypes and differentiated into adipocyte, chondrocyte and osteocyte lineages. The cells from WJ showed significantly greater CD24+ and CD108+ numbers and fluorescence intensities that discriminate between MSCs and non-stem cell mesenchymal cells, were negative for the fibroblast-specific and activating-proteins (FSP, FAP) and showed greater osteogenic and chondrogenic differentiation potential compared to AM, SA, PV and MC. Cells from the WJ offer the best clinical utility as (i) they have less non-stem cell contaminants (ii) can be generated in large numbers with minimal culture avoiding changes in phenotype, (iii) their derivation is quick and easy to standardize, (iv) they are rich in stemness characteristics and (v) have high differentiation potential. Our results show that when isolating MSCs from the UC, the WJ should be the preferred compartment, and a standardized method of derivation must be used so as to make meaningful comparisons of data between research groups.     

Wharton’s jelly‐derived stromal cells and their cell therapy applications in allogeneic haematopoietic stem cell transplantation

For decades, mesenchymal stromal cells (MSCs) have been of great interest in the fields of regenerative medicine, tissue engineering and immunomodulation. Their tremendous potential makes it desirable to cryopreserve and bank MSCs to increase their accessibility and availability. Postnatally derived MSCs seem to be of particular interest because they are harvested after delivery without ethical controversy, they have the capacity to expand at a higher rate than adult‐derived MSCs, in which expansion decreases with ageing, and they have demonstrated immunological and haematological supportive properties similar to those of adult‐derived MSCs. In this review, we focus on MSCs obtained from Wharton''s jelly (the mucous connective tissue of the umbilical cord between the amniotic epithelium and the umbilical vessels). Wharton''s jelly MSCs (WJ‐MSCs) are a good candidate for cellular therapy in haematology, with accumulating data supporting their potential to sustain haematopoietic stem cell engraftment and to modulate alloreactivity such as Graft Versus Host Disease (GVHD). We first present an overview of their in‐vitro properties and the results of preclinical murine models confirming the suitability of WJ‐MSCs for cellular therapy in haematology. Next, we focus on clinical trials and discuss tolerance, efficacy and infusion protocols reported in haematology for GVHD and engraftment.     

Increased strength in the Col-Tgel induces apoptosis in the human dental pulp stem cells: 3D culturing of human dental pulp stem cells at different strengths of collagen

Human dental pulp stem cells (HDPSCs) have great potential to be used in regenerative medicine. To use these stem cells effectively for this purpose, they should be grown in a 3D cell culture that mimics their natural niches instead of a 2D conventional cell culture. The aim of this study was to grow the HDPSCs in the 3D cell culture created by Transglutaminase-crosslinked collagen hydrogels (Col-Tgel) in two different strengths to find a suitable 3D cell culture environment for these stem cells. Two stiffness of the 3D Col-Tgel were used to grow the HDPSCs: soft and medium matrix with strength of 0.9–1.5 kPa and 14–20 kPa, respectively. HDPSCs express markers similar to MSCs, therefore seven such markers were analyzed in the HDPSCs during their growth in the 2D and in the 3D soft and medium Col-Tgel. The CD105 and CD90 markers were significantly (p < 0.05) downregulated in HDPSCs cultured in both 3D cell culture conditions compared with HDPSCs in 2D cell culture. Furthermore, CD34 marker, a negative marker, expressed by a few cells in HDPSCs culture was upregulated (p < 0.05) in HDPSCs cultured in medium 3D Col-Tgel, indicating cells that expressing the marker grow better in medium 3D Col-Tgel. The apoptosis results revealed that HDPSCs in medium 3D Col-Tgel had the least number of live cells and a significantly (p < 0.05) higher early apoptosis rate compared to HDPSCs in 2D and 3D Col-Tgel medium. MTT analysis also showed a significant difference among the three cell culture conditions. We conclude that HDPSCs cultured on 3D soft Col-Tgel showed better proliferation than cells cultured in 3D medium gel. These results demonstrate that the ideal environment to grow HDPSCs in 3D is the soft Col-Tgel not medium Col-Tgel.     

Simultaneous impact of atorvastatin and mesenchymal stem cells for glioblastoma multiform suppression in rat glioblastoma multiform model

Glioblastoma multiform (GBM) is known as an aggressive glial neoplasm. Recently incorporation of mesenchymal stem cells with anti-tumor drugs have been used due to lack of immunological responses and their easy accessibility. In this study, we have investigated the anti-proliferative and apoptotic activity of atorvastatin (Ator) in combination of mesenchymal stem cells (MSCs) on GBM cells in vitro and in vivo. The MSCs isolated from rats and characterized for their multi-potency features. The anti-proliferative and migration inhibition of Ator and MSCs were evaluated by MTT and scratch migration assays. The annexin/PI percentage and cell cycle arrest of treated C6 cells were evaluated until 72 h incubation. The animal model was established via injection of C6 cells in the brain of rats and subsequent injection of Ator each 3 days and single injection of MSCs until 12 days. The growth rate, migrational phenotype and cell cycle progression of C6 cells decreased and inhibited by the interplay of different factors in the presence of Ator and MSCs. The effect of Ator and MSCs on animal models displayed a significant reduction in tumor size and weight. Furthermore, histopathology evaluation proved low hypercellularity and mitosis index as well as mild invasive tumor cells for perivascular cuffing without pseudopalisading necrosis and small delicate vessels in Ator?+?MSCs condition. In summary, Ator and MSCs delivery to GBM model provides an effective strategy for targeted therapy of brain tumor.

     

Ascorbic acid induces in vitro proliferation of human subcutaneous adipose tissue derived mesenchymal stem cells with upregulation of embryonic stem cell pluripotency markers Oct4 and SOX 2

Mesenchymal stem cells (MSCs) have immense therapeutic potential because of their ability to self-renew and differentiate into various connective tissue lineages. The in vitro proliferation and expansion of these cells is necessary for their use in stem cell therapy. Recently our group has developed and characterized mesenchymal stem cells from subcutaneous and visceral adipose tissue. We observed that these cells show a slower growth rate at higher passages and therefore decided to develop a supplemented medium, which will induce proliferation. Choi et al. have recently shown that the use of ascorbic acid enhances the proliferation of bone marrow derived MSCs. We therefore studied the effect of ascorbic acid on the proliferation of MSCs and characterized their phenotypes using stem cell specific molecular markers. It was observed that the use of 250 μM ascorbic acid promoted the significant growth of MSCs without loss of phenotype and differentiation potential. There was no considerable change in gene expression of cell surface markers CD105, CD13, Nanog, leukemia inhibitory factor (LIF) and Keratin 18. Moreover, the MSCs maintained in the medium supplemented with ascorbic acid for a period of 4 weeks showed increase in pluripotency markers Oct4 and SOX 2. Also cells in the experimental group retained the typical spindle shaped morphology. Thus, this study emphasizes the development of suitable growth medium for expansion of MSCs and maintenance of their undifferentiated state for further therapeutic use.     

The characteristics of endothelial progenitor cells derived from mononuclear cells of rat bone marrow in different culture conditions

Endothelial progenitor cells (EPCs) derived from bone marrow are known to be heterogeneous. In this study, we tried to find favorable conditions that induce the differentiation of mononuclear cells (MNCs) from bone marrow into EPCs. The differentiation capacity of MNCs from rat bone marrow was investigated in different conditions, such as different media, different induction times and different culture surfaces. The cell morphology and endothelial biomarkers associated with differentiated MNCs were studied. Our results indicated that MNCs cultured in EGM-2MV (Endothelial cell basal medium-2, plus SingleQuots of growth supplements) developed a bursiform shape, a late EPC-like morphology, while MNCs cultured in complete medium (CM, M199 with 10% FBS, 20 ng/mL VEGF and 10 ng/mL bFGF) showed a spindle shape, an early EPC-like morphology. Cells of both morphologies were able to incorporate DiI-ac-LDL and bind lectin in vitro. MNCs cultured in EGM-2MV exhibited a higher proliferation rate and higher eNOS expression than MNCs cultured in CM. MNCs cultured in EGM-2MV had the ability to form tubes on Matrigel. Flow cytometry results indicated that CD133 expression was highest at day 12 and that the greatest number of cells positive for both FLK-1 and CD133 appeared at day 20 from cells cultured in dishes without fibronectin coating. In addition, the expression levels of CD133, CD31 and FLK-1/CD133 were not significantly different between cells of different shapes. Our experiments suggest that MNCs from bone marrow can be differentiated into late EP-like cells in EGM-2MV, which have the ability to rapidly proliferate. These MNCs can also be differentiated into early EP-like cells in CM. Additionally, fibronectin may not be necessary for the differentiation of EPCs to mature ECs after three generations. Differentiated MNCs from bone marrow in EGM-2MV have the characteristics of EPCs, although the expression levels of EPC markers were lower than previously reported.     

Neural stem cells transplanted into intact brains as neurospheres form solid grafts composed of neurons, astrocytes and oligodendrocyte precursors

Neural stem cells (NSCs) are tissue-specific stem cells with self-renewal potential that can give rise to neurons and glia in vivo and in vitro. The aim of this study was to transplant NSCs as whole neurospheres into intact brain and assess the fate and phenotype of their progeny generated in vivo. We isolated NSCs from E14 foetal rat forebrains and cultured them in basic fibroblast and epidermal growth factor-supplemented serum-free medium in the form of neurospheres in vitro. Neurospheres were transplanted into the intact brains of 2 Wistar rats and after a period of 3 weeks, grafted brains were examined immunohistochemically. Neurospheres formed solid grafts that were found in the lateral ventricle and in the velum interpositum under the hippocampus. The majority of cells in the transplanted tissue were identified as beta-III-tubulin(+), NeuN(+), PanNF(+) and synaptophysin(+) neurons and were accumulated throughout the graft centre. GFAP(+) astrocytes were scattered throughout the entire graft and astrocyte processes delimited the outer and perivascular surfaces. A great number of NG2(+) oligodendrocyte precursors was detected. Nestin(+) endothelial cells were found to line capillaries growing in the transplant. These data indicate that nestin(+) NSCs prevailing in neurospheres differentiate following transplantation into nestin(-) neuronal and glial cells which confirms the multipotency of NSCs. Three weeks posttransplantation neuronal and astrocyte cells reached terminal differentiation (formation of synaptic vesicles and superficial and perivascular limiting membranes) while elements of oligodendroglial cell lineage remained immature. Grafting stem cells as non-dissociated neurospheres provide cells with favourable conditions which facilitate cell survival, proliferation and differentiation. However, in the intact brain, grafted neurosphere cells were not found to integrate with the brain parenchyma and formed a compact structure demarcated from its surroundings.     

The effects of adipose-derived stem cells on CD133-expressing bladder cancer cells

Mesenchymal stem cells (MSCs) hold great promise as therapeutic agents in regenerative medicine. They are also considered as a preferred cell source for urinary tract reconstruction. However, as MSCs exhibit affinity to tumor microenvironment, possible activation of tumor-initiating cells remains a major concern in the application of stem cell-based therapies for patients with a bladder cancer history. To analyze the influence of adipose-derived stem cells (ASCs) on bladder cancer cells with stem cell-like properties, we isolated CD133-positive bladder cancer cells and cultured them in conditioned medium from ASCs (ASC-CM). Our results showed that parental 5637 and HB-CLS-1 cells showed induced clonogenic potential when cultured in ASC-CM. Soluble mediators secreted by ASCs increased proliferation and viability of unsorted cells as well as CD133+ and CD133− subpopulations. Furthermore, incubation with ASC-CM modulated activation of intracellular signaling pathways. Soluble mediators secreted by ASCs increased phosphorylation of AKT1/2/3 (1.4-fold, P < 0.05), ERK1/2 (1.6-fold, P < 0.02), and p70 S6K (1.4-fold) in CD133+ cells isolated from 5637 cell line. In turn, decreased phosphorylation of those three proteins involved in PI3K/Akt and MAPK signaling was observed in CD133+ cells isolated from HB-CLS-1 cell line. Our results revealed that bladder cancer stem-like cells are responsive to signals from ASCs. Paracrine factors secreted by locally-delivered ASCs may, therefore, contribute to the modulation of signaling pathways involved in cancer progression, metastasis, and drug resistance.