Ethanol-based garlic extract prevents malignant evolution of non-invasive breast tumor cells induced by moderate hypoxia.

BACKGROUND: In breast cancer, low oxygen availability is associated with a more aggressive phenotype and with malignant evolution of non-invasive cells. Natural compounds have long attracted attention in cancer treatment, and in recent years garlic (Allium sativum) organosulfur derivatives have been shown to negatively affect growth and invasion of tumor cells. METHODS: Homemade ethanol-based garlic extract (GE) was administered to MCF7 and MCF10DCIS breast tumor cell lines grown under moderate hypoxia. Cell cycle, epithelial-to-mesenchymal transition and cancer stem cell markers were evaluated. RESULTS: We revealed that, in the non-invasive MCF10DCIS cells but not in the post-EMT MCF7 cells, low oxygen availability induced the decrease of E-cadherin and the increase of vimentin and motility, that were prevented by GE administration. In both cell lines, treatment with GE counteracted the up-modulation of CD133 positive cells induced by hypoxia. CONCLUSIONS: Overall, our data firstly revealed anti-cancer properties of garlic in non-invasive breast cancer cells. In particular, they demonstrated a protective role of this natural product against the hypoxia-induced increase of molecules that play crucial roles in tumor evolution, suggesting that garlic derivatives can be considered in new approaches for preventing progression of breast tumors from non-invasive to infiltrating lesions.

Development of CD133 Targeting Multi-Drug Polymer Micellar Nanoparticles for Glioblastoma - In Vitro Evaluation in Glioblastoma Stem Cells.

PURPOSE: Glioblastoma (GBM) is a malignant brain tumor with a poor long-term prognosis due to recurrence from highly resistant GBM cancer stem cells (CSCs), for which the current standard of treatment with temozolomide (TMZ) alone will unlikely produce a viable cure. In addition, CSCs regenerate rapidly and overexpress methyl transferase which overrides the DNA-alkylating mechanism of TMZ, leading to resistance. The objective of this research was to apply the concepts of nanotechnology to develop a multi-drug therapy, TMZ and idasanutlin (RG7388, a potent mouse double minute 2 (MDM2) antagonist), loaded in functionalized nanoparticles (NPs) that target the GBM CSC subpopulation, reduce the cell viability and provide possibility of in vivo preclinical imaging. METHODS: Polymer-micellar NPs composed of poly(styrene-b-ethylene oxide) (PS-b-PEO) and poly(lactic-co-glycolic) acid (PLGA) were developed by a double emulsion technique loading TMZ and/or RG7388. The NPs were covalently bound to a 15-nucleotide base-pair CD133 aptamer to target the CD133 antigen expressed on the surfaces of GBM CSCs. For diagnostic functionality, the NPs were labelled with radiotracer Zirconium-89 (89Zr). RESULTS: NPs maintained size range less than 100 nm, a low negative charge and exhibited the ability to target and kill the CSC subpopulation when TMZ and RG7388 were used in combination. The targeting function of CD133 aptamer promoted killing in GBM CSCs providing impetus for further development of targeted nanosystems for localized therapy in future in vivo models. CONCLUSIONS: This work has provided a potential clinical application for targeting GBM CSCs with simultaneous diagnostic imaging.

470 nm LED Irradiation Inhibits the Invasiveness of CD133-positive Human Colorectal Cancer Stem Cells by Suppressing the Cyclooxygenase-2/prostaglandin E2 Pathway.

BACKGROUND/AIM: Recurrence and metastasis of cancer caused by cancer stem cells (CSCs) is a challenge to overcome. Low level laser therapy is a new treatment strategy to suppress their invasiveness. We have assessed the inhibitory effects of 470 nm blue LED on the invasiveness of them to determine the molecular mechanisms of anti-invasiveness. MATERIALS AND METHODS: The effects of blue LEDs on their viability, proliferation and invasion were analyzed using MTT and transwell methods. In addition, the anti-invasiveness effect of blue LED on them was evaluated by zymography, semi-quantitative RT-PCR and western blot analysis. RESULTS: Irradiation with blue LED at 3 J/cm2 resulted in inhibition of their viability, proliferation and invasiveness. Their matrix metalloproteinase 2 (MMP-2) and MMP-9 activities were reduced by blue LED irradiation. Semi-quantitative RT-PCR also showed similar results. In addition, western blotting analyses showed that cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) synthesis were significantly inhibited by LED irradiation in CD133+ colorectal CSCs. CONCLUSION: Down-regulation of the COX-2/PGE2 signaling pathway by blue LED irradiation led to reduce expression of MMP-2 and MMP-9, inhibiting the invasiveness of CD133+ colorectal CSC.

Characterization of Adult Canine Kidney Epithelial Stem Cells That Give Rise to Dome-Forming Tubular Cells.

Dome formation can occur in cultured tubular epithelial cells originating from various tissues, including the mammary gland and the kidney. The isolation and characterization of normal kidney epithelial stem cells that give rise to dome-forming tubular cells have never been reported. We attempted to isolate and characterize canine kidney epithelial stem cells using a simple cell culture method that we have previously used to isolate other adult human stem cells. Dome-forming kidney epithelial cells were derived from dissociated adult canine kidney tissues that were cultured in a modified keratinocyte serum-free medium supplemented with N-acetyl-l-cysteine, l-ascorbic acid 2-phosphate, nicotinamide, and fetal bovine serum. These cells exhibited high self-renewal capacity in long-term culture (growth for >13 months and 30 cumulative population doublings) and exhibited characteristics of stem cells, including (1) deficiency in gap junctional intercellular communication, (2) anchorage-independent growth, (3) expression of stem cell markers octamer-binding transcription factor 4 and SRY (sex determining region Y)-box 2, (4) expression of cell surface markers CD24 and CD133, and (5) multipotent differentiation into osteoblasts, adipocytes, chondrocytes, and dome-forming tubular cells. Most of these characteristics are shared by the well-known canine renal tubule-derived immortalized Madin-Darby Canine Kidney cell line. Furthermore, the putative canine kidney stem cells developed in this study formed budding tubule-like organoids on Matrigel and required high cell density (>4,000 cells/cm2) for sustained growth and confluency for dome formation. The signal transducer and activator of transcription-3 (STAT3) phosphorylation inhibitor, AG490, inhibited colony-forming efficiency and dome formation, whereas lipopolysaccharide, an activator of STAT3, increased colony-forming efficiency in a dose-dependent manner. These results are consistent with the hypothesis that high cell density induces STAT3 expression, which promotes both stem cell self-renewal and differentiation into tubular cells. Our novel cell culture method should be useful for the future development of normal human kidney stem cells for clinical applications and for studying mechanisms of nephrotoxicity.

Lusianthridin targeting of lung cancer stem cells via Src-STAT3 suppression.

BACKGROUND: Cancer stem cells (CSCs) are well-recognized as a majority cause of treatment failure and can give rise to relapse. The discovery of compounds attenuating CSCs' properties is crucial for enabling advances in novel therapeutics to limit recurrence. CSCs' features in lung cancer are regulated through a reduction in Src-STAT3-c-Myc, which drives cancer progression, drug resistance, and metastasis. METHODS: The effect of lusianthridin suppresses CSC-like phenotypes was determined by 3D culture and anchorage independent growth. The expression of CSC markers and associated proteins were determined by Western blot analyses. Protein ubiquitination and degradation were assessed using immunoprecipitation. RESULTS: Herein, we report that lusianthridin, a pure compound from Dendrobium venustum, dramatically suppressed CSCs in lung cancer cells as verified by several CSC phenotype assessments and CSC markers. The CSC phenotypes in lusianthridin-treated cells were suppressed through downregulation of Src-STAT3-c-Myc pathways. Ectopic Src introduced by the transfection augmented CSC phenotypes in lung cancer cells through STAT3 (increased active p-STAT3Tyr705) and c-Myc signals, while the ShRNA-Src transfection or Src inhibitor dasatinib exhibited opposite results. Treatment of the Src-overexpressing cells with lusianthridin resulted in the reversal of active STAT3 (p-STAT3Tyr705) and c-Myc as well as the CSC marker CD133. Importantly, we confirmed the CSC-targeted activity of lusianthridin in CSC-rich primary lung cancer cells. The compound dramatically inhibited the formation of tumor spheres of primary lung cancer cells. Finally, we demonstrated that after CSC-attenuation by lusianthridin, the lung cancer cells exhibited significantly higher susceptibility to chemotherapeutic drugs. Such a sensitizing effect caused by pro-survival suppression and pro-apoptotic induction together with the abolishment of stemness indicated by the decrease in CSC markers CD133, ABCG2, and ALDH1A1. CONCLUSION: These findings revealed a novel pharmacological action and the underlying mechanism of lusianthridin in negatively regulating CSC-like phenotypes and sensitizing resistant cancer cells to cemetery.

Effect of Shogaol on the Expression of Intestinal Stem Cell Markers in Experimentally Induced Colitis in BALB/c Mice.

AIM: This study is aimed at investigating the effect of Shogaol, a phenolic constituent of ginger, on dextran sodium sulfate- (DSS-) induced ulcerative colitis (UC) in mice in comparison with 6-thioguanine (6-TG), an immune-suppressant chemotherapeutic medicine used for treatment of ulcerative colitis. MATERIAL & METHODS: Thirty-six adult, male and female BALB/c mice were randomly divided into six groups: group 1 (control negative) not exposed to DSS and did not receive any treatment, group 2 (control positive) exposed to DSS but did not receive any treatment, group 3 exposed to DSS and treated by 0.1 mg/kg of 6-thioguanine, and groups 4, 5, and 6 exposed to DSS and treated by 10, 20, and 40 mg/kg b.w. Shogaol, respectively. At day 56, the mice were checked for their disease activity index (DAI) and they were sacrificed. The colons of the mice were examined for length measurement, histological index score, and the expression of CD133 and CD34 stem cell markers. RESULTS: Shogaol showed a better curative effect than did 6-TG in repairing the colonic mucosal damages in DSS-exposed mice as indicated by the levels of CD133 and CD34 expression in the colonic crypts and by the DAI score, colon length measurements, & histological index score which were significantly reduced in mice treated by Shogaol, particularly the 20 and 40 mg/kg BW doses. CONCLUSION: The results of this study indicated that oral treatment with the ginger-derived substance Shogaol could be better than the conventional immunosuppressive chemotherapeutic remedy 6-TG in treatment of DSS-induced UC.

Nucleus-targeted nano delivery system eradicates cancer stem cells by combined thermotherapy and hypoxia-activated chemotherapy.

Many efforts have focused on the cancer stem cell (CSC) targeting nano delivery system, however, the anticancer therapy efficacy is relative low due to the highly drug-resistance and drug efflux. Nucleus-targeted drug delivery is a promising strategy for reverse the drug resistance and drug efflux of CSCs, but in vivo nucleus-targeted drug delivery has been challenging. Herein, we designed a mesoporous silica nanoparticle (MSN)-based nucleus-targeted system, which could directly target the CSCs and further enter the nucleus by the surface modification of anti-CD133 and thermal-triggered exposure of TAT peptides under an alternating magnetic field (AMF). The nucleus-targeted drug release ultimately leads to an exhaustive apoptosis of the CSCs through combined thermotherapy and hypoxia-activated chemotherapy. In vivo, the nucleus-targeted nano delivery system efficiently inhibits the tumor growth without notable side effects during the course of treatment. Molecular mechanism study illustrates that the system effectively eliminates the CSCs by blocking the hypoxia signaling pathway. This designed nucleus-targeted nano delivery system is expected to provide new insights for developing efficient platforms for CSC-targeted cancer therapy.

α-Ketoglutarate Promotes Pancreatic Progenitor-Like Cell Proliferation.

A major source of ß cell generation is pancreatic progenitor-like cell differentiation. Multiple studies have confirmed that stem cell metabolism plays important roles in self-renewal and proliferation. In the absence of glucose, glutamine provides the energy for cell division and growth. Furthermore, α-ketoglutarate (αKG), a precursor for glutamine synthesis, is sufficient for enabling glutamine-independent cell proliferation. We have demonstrated that αKG contributes to the large-scale proliferation of pancreatic progenitor-like cells that can provide an ample amount of clinically relevant ß cells. We compared the mRNA expression of a subset of genes, the abundance of ATP, reactive oxide species, mitochondrial number, and the colony-forming frequency between mouse pancreatic CD133⁺ and CD133- cells. We employed Real-Time PCR, immunostaining and passage assays to investigate self-renewal and proliferation of pancreatic progenitor-like cells in a 3D culture system in the presence and absence of αKG. The energy metabolism of CD133⁺ cells was more prone to oxidative phosphorylation. However, in the 3D culture system, when αKG was supplemented to the culture medium, the proliferation of the pancreatic progenitor-like cells was significantly elevated. We confirmed that the presence of αKG correlated with the up-regulation of Ten-Eleven Translocation (Tet). αKG can promote the proliferation of pancreatic progenitor-like cells via the up-regulation of Tet.

Wogonin suppresses stem cell-like traits of CD133 positive osteosarcoma cell via inhibiting matrix metallopeptidase-9 expression.

BACKGROUND: Several efforts have been deployed to cure osteosarcoma, a high-grade malignant bone tumour in children and adolescents. However, some challenges such as drug resistance, relapse, and tumour metastasis remain owing to the existence of cancer stem cells (CSC). There is an urgent need to develop cost-effective and safe therapies. METHODS: Wogonin, an extract from the root of Scutellaria baicalensis, has long been considered as a promising natural and safe compound for anti-tumourigenesis, particularly to inhibit tumour invasion and metastasis. Hoechst 33,342 staining, wound healing assay, sphere formation assay, western blotting, and gelatin zymography assays were performed in CD133 positive osteosarcoma cell. RESULTS: In this study, we examined the effect of Wogonin on the mobility of human osteosarcoma CSC. Wogonin induces apoptosis of human osteosarcoma CSC, inhibits its mobility in vitro via downregulation of MMP-9 expression, and represses its renewal ability. CONCLUSIONS: We demonstrated that Wogonin decreases the renewal capacity of CSC. By inhibiting the formation of and reducing the size of spheres, Wogonin at a concentration of 40-80 µM effectively minimizes potential risk from CSC. Taken together, we have demonstrated a new approach for developing a potential therapy for osteosarcoma.

Suppression of cancer stem-like phenotypes in NCI-H460 lung cancer cells by vanillin through an Akt-dependent pathway.

Cancer stem cells (CSCs) have been reported as a major cause of cancer metastasis and the failure of cancer treatment. Cumulative studies have indicated that protein kinase B (Akt) and its downstream signaling pathway, including CSC markers, play a critical role in the aggressive behavior of this cancer. In this study, we investigated whether vanillin, a major component in Vanilla planifolia seed, could suppress cancer stemness phenotypes and related proteins in the human non-small cell lung cancer NCI­H460 cell line. A non-toxic concentration of vanillin suppressed spheroid and colony formation, two hallmarks of the cancer stemness phenotype, in vitro in NCI­H460 cells. Western blot analysis revealed that the CSC markers CD133 and ALDH1A1 and the associated transcription factors, Oct4 and Nanog, were extensively downregulated by vanillin. Vanillin also attenuated the expression and activity of Akt, a transcription regulator upstream of CSCs, an action that was confirmed by treatment with the Akt inhibitor perifosine. Furthermore, the ubiquitination of Akt was elevated in response to vanillin treatment prior to proteasomal degradation. This finding indicates that vanillin can inhibit cancer stem cell-like behavior in NCI­H460 cells through the induction of Akt-proteasomal degradation and reduction of downstream CSC transcription factors. This inhibitory effect of vanillin may be an alternative approach in the treatment against lung cancer metastasis and its resistance to chemotherapy.

Actein ameliorates hepatobiliary cancer through stemness and p53 signaling regulation.

Actein is isolated from the rthizomes of Cimicifuga foetida, which is a triterpene glycoside, displaying suppressive effects on breast cancer cells proliferation. However, the effects of actein treatment on liver injury, tending to cancer, have little to be known. Thus, the study is conducted to explore the role of actein in early liver cancer. Diethylnitrosamine (DEN) was used to induce liver cancer in mice followed by actein treatment at different concentrations. DEN caused steatohepatitis supported by fibrosis and inflammation, which were ameliorated for actein administration. Liver histology of mice with DEN treatment displayed hepatobiliary cysts, reversed by actein. Cell proliferation markers of Cyclin Ds and p53, as well as cancer stem cell markers of CD133 were highly increased in liver tissue samples from DEN-induced mice, and actein showed inhibitory role in these signals expression. Actein-reduced up-regulation of Hif-1α and VEGFR1 in DEN-stimulated liver tissue of mice was seen. Taken together, DEN promoted liver cancer progression, which was ameliorated by actein, supplying a potential therapeutic strategy for liver cancer in future.

Regeneration of rat corpora cavernosa tissue by transplantation of CD133+ cells derived from human bone marrow and placement of biodegradable gel sponge sheet.

The objective is to develop an easier technique for regenerating corpora cavernosa tissue through transplantation of human bone marrow-derived CD133 + cells into a rat corpora cavernosa defect model. We excised 2 mm × 2 mm squares of the right corpora cavernosa of twenty-three 8-week-old male nude rats. Alginate gel sponge sheets supplemented with 1 × 10 4 CD133 + cells were then placed over the excised area of nine rats. Functional and histological evaluations were carried out 8 weeks later. The mean intracavernous pressure/mean arterial pressure ratio for the nine rats (0.34258 ± 0.0831) was significantly higher than that for eight rats with only the excision (0.0580 ± 0.0831, P = 0.0238) and similar to that for five rats for which the penis was exposed, and there was no excision (0.37228 ± 0.1051, P = 0.8266). Immunohistochemical analysis revealed that the nine fully treated rats had venous sinus-like structures and quantitative reverse transcription polymerase chain reaction analysis of extracts from their alginate gel sponge sheets revealed that the amounts of mRNA encoding the nerve growth factor (NGF), and vascular endothelial growth factor (VEGF) were significantly higher than those for rats treated with alginate gel sheets without cell supplementation (NGF: P = 0.0309; VEGF: P < 0.0001). These findings show that transplantation of CD133 + cells accelerates functional and histological recovery in the corpora cavernosa defect model.

Dual drug-loaded biofunctionalized amphiphilic chitosan nanoparticles: Enhanced synergy between cisplatin and demethoxycurcumin against multidrug-resistant stem-like lung cancer cells.

Lung cancer kills more humans than any other cancer and multidrug resistance (MDR) in cancer stem-like cells (CSC) is emerging as a reason for failed treatments. One concept that addresses this root cause of treatment failure is the utilization of nanoparticles to simultaneously deliver dual drugs to cancer cells with synergistic performance, easy to envision - hard to achieve. (1) It is challenging to simultaneously load drugs of highly different physicochemical properties into one nanoparticle, (2) release kinetics may differ between drugs and (3) general requirements for biomedical nanoparticles apply. Here self-assembled nanoparticles of amphiphilic carboxymethyl-hexanoyl chitosan (CHC) were shown to present nano-microenvironments enabling simultaneous loading of hydrophilic and hydrophobic drugs. This was expanded into a dual-drug nano-delivery system to treat lung CSC. CHC nanoparticles were loaded/chemically modified with the anticancer drug cisplatin and the MDR-suppressing Chinese herbal extract demethoxycurcumin, followed by biofunctionalization with CD133 antibody for enhanced uptake by lung CSC, all in a feasible one-pot preparation. The nanoparticles were characterized with regard to chemistry, size, zeta potential and drug loading/release. Biofunctionalized and non-functionalized nanoparticles were investigated for uptake by lung CSC. Subsequently the cytotoxicity of single and dual drugs, free in solution or in nanoparticles, was evaluated against lung CSC at different doses. From the dose response at different concentrations the degree of synergy was determined through Chou-Talalay's Plot. The biofunctionalized nanoparticles promoted synergistic effects between the drugs and were highly effective against MDR lung CSC. The efficacy and feasible one-pot preparation suggests preclinical studies using relevant disease models to be justified.

Isolation, Culture and Identification of Choriocarcinoma Stem-Like Cells from the Human Choriocarcinoma Cell-Line JEG-3.

BACKGROUND/AIMS: Cancer stem cells (CSCs) exhibit enhanced proliferative capacity and resistance to chemotherapy; however, choriocarcinoma CSCs have not yet been reported. In this study the human choriocarcinoma cell line JEG-3 was cultured in serum free media, and the characteristics of suspension and parental adherent JEG-3 cells were compared. METHODS: Cell proliferation, colony-formation, soft agar clonogenicity, and transwell invasion assays were performed in vitro, and tumor xenografts in BALB/c nude mice were used to evaluate stem cell properties. RESULTS: In serum-supplemented medium (SSM), JEG-3 cells were 4.51 ± 1.71% CD44+, 7.67 ± 2.67% CD133+, and 13.85 ± 2.95% ABCG2+. In serum-free medium (SFM), the expression of these markers increased to 53.08 ± 3.15%, 47.40 ± 2.67%, and 78.70 ± 7.16%, respectively. Moreover, suspension JEG-3 cells exhibited enhanced colony-formation capability as well as invasive and proliferative ability in vitro, alongside enhanced tumorigenic properties in vivo. Suspension JEG-3 cells also exhibited resistance to the chemotherapeutic drugs methotrexate, fluorouracil and etoposide. When seeded in serum supplemented medium, suspension JEG-3 cells readopted an adherent phenotype and continued to differentiate with no significant difference in the morphology between suspension and parent cells. CONCLUSION: In this study, choriocarcinoma stem-like cells (CSLCs) were isolated from the human choriocarcinoma JEG-3 cell line by SFM culture and characterized.

LGR5 and CD133 as prognostic and predictive markers for fluoropyrimidine-based adjuvant chemotherapy in colorectal cancer.

BACKGROUND: Expression of leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5) gene is associated with a metastatic phenotype and poor prognosis in colorectal cancer (CRC). CD133 expression is a putative cancer stem cell marker and a proposed prognostic marker in CRC, whereas the predictive value of CD133 expression for effect of adjuvant chemotherapy in CRC is unclear. MATERIAL AND METHODS: For the study of LGR5 mRNA and CD133 expression, tissue microarrays from 409 primary CRC stage II and III tumors, where patients had been randomized to adjuvant chemotherapy or surgery only, were available. LGR5 mRNA and CD133 expression were assessed by in situ hybridization (ISH) and immunohistochemistry (IHC), respectively. LGR5 mRNA and CD133 expression as prognostic and predictive markers were evaluated by univariate and multivariate analyses. RESULTS: For all CRC patients, positive LGR5 mRNA and CD133 expression were associated with classic adenocarcinoma histology type (p = 0.001 and p = 0.014, respectively). Positive LGR5 mRNA expression was also associated with smaller tumor diameter for CRC stage II (p = 0.005), but not for CRC stage III (p = 0.054). For CRC stage II, lack of LGR5 mRNA expression was associated with longer time to recurrence (TTR) in Kaplan-Meier (p = 0.045) and in multivariate Cox analysis (HR 0.27, 95% CI 0.08-0.95, p = 0.041). For colon cancer stage III patients, lack of CD133 expression was associated with better effect of adjuvant chemotherapy (p = 0.016) in Kaplan-Meier univariate analysis, but the interaction between CD133 and adjuvant chemotherapy was not statistically significant in multivariate analysis (HR 0.59, 95% CI 0.18-1.89, p = 0.374). CONCLUSION: LGR5 mRNA expression is a prognostic factor for CRC stage II patients, whereas the value of CD133 expression as prognostic and predictive biomarker is inconclusive.

Autologous cell therapy with CD133+ bone marrow-derived stem cells for refractory Asherman's syndrome and endometrial atrophy: a pilot cohort study.

STUDY QUESTION: Could cell therapy using autologous peripheral blood CD133+ bone marrow-derived stem cells (BMDSCs) offer a safe and efficient therapeutic approach for patients with refractory Asherman's syndrome (AS) and/or endometrial atrophy (EA) and a wish to conceive? SUMMARY ANSWER: In the first 3 months, autologous cell therapy, using CD133+ BMDSCs in conjunction with hormonal replacement therapy, increased the volume and duration of menses as well as the thickness and angiogenesis processes of the endometrium while decreasing intrauterine adhesion scores. WHAT IS KNOWN ALREADY: AS is characterized by the presence of intrauterine adhesions and EA prevents the endometrium from growing thicker than 5 mm, resulting in menstruation disorders and infertility. Many therapies have been attempted for these conditions, but none have proved effective. STUDY DESIGN, SIZE, DURATION: This was a prospective, experimental, non-controlled study. There were 18 patients aged 30-45 years with refractory AS or EA were recruited, and 16 of these completed the study. Medical history, physical examination, endometrial thickness, intrauterine adhesion score and neoangiogenesis were assessed before and 3 and 6 months after cell therapy. PARTICIPANTS/MATERIALS, SETTING, METHODS: After the initial hysteroscopic diagnosis, BMDSC mobilization was performed by granulocyte-CSF injection, then CD133+ cells were isolated through peripheral blood aphaeresis to obtain a mean of 124.39 million cells (range 42-236), which were immediately delivered into the spiral arterioles by catheterization. Subsequently, endometrial treatment after stem cell therapy was assessed in terms of restoration of menses, endometrial thickness (by vaginal ultrasound), adhesion score (by hysteroscopy), neoangiogenesis and ongoing pregnancy rate. The study was conducted at Hospital Clínico Universitario of Valencia and IVI Valencia (Spain). MAIN RESULTS AND THE ROLE OF CHANCE: All 11 AS patients exhibited an improved uterine cavity 2 months after stem cell therapy. Endometrial thickness increased from an average of 4.3 mm (range 2.7-5) to 6.7 mm (range 3.1-12) ( ITALIC! P = 0.004). Similarly, four of the five EA patients experienced an improved endometrial cavity, and endometrial thickness increased from 4.2 mm (range 2.7-5) to 5.7 mm (range 5-12) ( ITALIC! P = 0.03). The beneficial effects of the cell therapy increased the mature vessel density and the duration and intensity of menses in the first 3 months, with a return to the initial levels 6 months after the treatment. Three patients became pregnant spontaneously, resulting in one baby boy born, one ongoing pregnancy and a miscarriage. Furthermore, seven pregnancies were obtained after fourteen embryo transfers, resulting in three biochemical pregnancies, one miscarriage, one ectopic pregnancy, one baby born and one ongoing pregnancy. LIMITATIONS, REASONS FOR CAUTION: Limitations of this pilot study include the small sample size and the lack of control group. WIDER IMPLICATIONS OF THE FINDINGS: This novel autologous cell therapy is a promising therapeutic option for patients with these incurable pathologies and a wish to conceive. STUDY FUNDING/COMPETING INTERESTS: This study was funded by the Spanish Ministry of Science and Innovation (SAF 2012-31017, Principal Investigator C.S.), Spanish Ministry of Health (EC11-299, Principal Investigator C.S.) and Regional Valencian Ministry of Education (PROMETEOII/2013/018, Principal Investigator C.S.). Four authors (X.S., I.C., A.P. and C.S.) are co-inventors of the patent resulting from this work (Application number: 62/013,121). S.C., C.A., F.R., J.F., J.P. and J.R. have no conflict of interest in relation to this work. TRIAL REGISTRATION NUMBER: This study was registered with ClinicalTrials.gov (NCT02144987).