Establishment and molecular characterization of breast cancer mesenchymal stem cell line derived from human non-metastasis breast cancer tumor

Breast cancer remains a leading cause of morbidity and mortality in women mainly because of the propensity of primary breast tumors to metastasize. It is composed of heterogeneous cell populations with different biological properties. Breast cancer-initiating cells have been recently identified in breast carcinoma as CD44+/CD24-/low cells, which display stem cell like properties. In the present study, we have isolated breast cancer stem cells from non-metastasis tumor tissue, which is presently at passage 18 and designated as human Breast Cancer Mesenchymal Stem Cells (hBCMSCs) line. These cells showed spindle shaped morphology and formed mammos-pheres as well as pluripotency clones indicating their stem cell nature. Molecular marker study confirmed mesenchymal nature as well as pluripotency, plasticity and oncogenicity of these cells. The hBCMSCs cell line may likely contain a heterogeneous population of malignant cells. Interestingly, we also found that these cells exhibit BRCA 2 mutation, which was found in Indian population. Overall, this study revealed that hBCMSCs cell line may represent a suitable in vitro model to study the mechanism of breast cancer which further leads to an identification of molecular targets for future breast cancer targeted therapy.

Characterization of a mesenchymal stem cell line that differentiates to bone and provides niches supporting mouse and human hematopoietic stem cells

Identification of mouse cell lines with properties of primary multipotential mesenchymal stromal cells (MSC) is required to facilitate the use of mouse models for evaluation of mechanisms in bone formation, hematopoiesis and cellular therapies for regenerative medicine. Primary murine MSC vary between strains, are difficult to grow in vitro and have inconsistent properties. The main aim of the study was to establish OMA-AD cells as an appropriate model system to conduct studies on MSC, bone formation and hematopoiesis. OMA-AD cells were isolated by differential trypsinization of C57BL/6J mouse bone marrow (BM) cells. The cells were then repassaged, cloned and characterized. OMA-AD cells were immortal and non-tumorigenic, differentiated readily to all mesenchymal cell types including bone, supported mouse and human hematopoiesis and were immunosuppressive. Our results demonstrated that OMA-AD cells possessed the properties of primary MSC. In addition, these cells grew readily and consistently, thereby facilitating future studies of bone formation, hematopoiesis and mesenchymal cells for regenerative medicine.

Human dental pulp stem cells differentiate into neural precursors but not into mature functional neurons

Large numbers of neuronal cells are needed for regenerative medicine to treat patients suffering from central nervous system diseases and deficits such as Parkinson’s disease and spinal cord injury. One suggestion has been the utilization of human dental pulp stem cells (hDPSCs) for production of neuronal cells which would offer a patient-specific cell source for these treatments. Neuronal differentiation of hDPSCs has been described previously. Here, we tested the differentiation of DPSCs into neuronal cells with previously reported protocol and characterized the cells according to their morphology, gene and protein expressions and most importantly according to their spontaneous electrical functionality with microelectrode array platform (MEA). Our results showed that even though hDPSC-derived neural progenitor stage cells could be produced, these cells did not mature further into functional neuronal cells. Thus, utilization of DPSCs as a cell source for producing grafts to treat neurological deficits requires more efforts before being optimal.

Mesenchymal Stromal Cells Derived from Human Embryonic Stem Cells, Fetal Limb and Bone Marrow Share a Common Phenotype but Are Transcriptionally and Biologically Different

Mesenchymal stromal cells (MSCs) can be obtained from several sources and the significant differences in their properties make it crucial to investigate the differentiation potential of MSCs from different sources to determine the optimal source of MSCs. We investigated if this biological heterogeneity in MSCs from different sources results in different mechanisms for their differentiation. In this study, we compared the gene expression patterns of phenotypically defined MSCs derived from three ontogenically different sources: Embryonic stem cells (hES-MSCs), Fetal limb (Flb-MSCs) and Bone Marrow (BM-MSCs). Differentially expressed genes between differentiated cells and undifferentiated controls were compared across the three MSC sources. We found minimal overlap (5% - 16%) in differentially expressed gene sets among the three sources. Flb-MSCs were similar to BM-MSCs based on differential gene expression patterns. Pathway analysis of the differentially expressed genes using Ingenuity Pathway Analysis (IPA) revealed a large variation in the canonical pathways leading to MSC differentiation. The similar canonical pathways among the three sources were lineage specific. The Flb-MSCs showed maximum overlap of canonical pathways with the BM-MSCs, indicating that the Flb-MSCs are an intermediate source between the less specialised hES-MSC source and the more specialised BM-MSC source. The source specific pathways prove that MSCs from the three ontogenically different sources use different biological pathways to obtain similar differentiation outcomes. Thus our study advocates the understanding of biological pathways to obtain optimal sources of MSCs for various clinical applications.

Analysis of proteomic profiling of mouse embryonic stem cells derived from fertilized, parthenogenetic and androgenetic blastocysts

Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of preimplantation embryos. ESCs exhibit true pluripotency, i.e., the ability to differentiate into cells of all three germ layers in the developing embryo. We used 2-DE MALDI-TOF/TOF to identify differentially expressed proteins among three types of mouse embryonic stem cells (ESCs) derived from ferti-lized, parthenogenetic, and androgenetic (fESC, pESC and aESC, respectively) blastocysts. We detected more than 800 proteins on silver- stained gels of whole protein extracts from each type of ESC. Of these, 52 differentially expressed proteins were identified by the MALDI–TOF/TOF analyzer, including 32 (fESCs vs. pESCs), 28 (fESCs vs. aESCs) and 17 (pESCs vs. aESCs) prominent proteins with significantly higher or lower expression relative to the comparison cells. Among the 32 proteins from fESCs, 12 were significantly increased in expression and 20 were reduced in expression in fESCs com-pared with pESCs. Similarly, 10 of 28 and 8 of 17 proteins were more highly expressed in fESCs and pESCs compared with aESCs, respectively. In contrast, 18 of 28 and 9 of 17 proteins were reduced in expression in fESCs and pESCs compared with aESCs, respectively. Of the eight protein candidates in fESCs, four were in-creased and four were decreased in expression relative to both pESCs and aESCs in the 2-DE analysis. Differential expression of these pro-teins were confirmed by mRNA expression analysis using real time RT-PCR. For three pro-teins, ANXA5, CLIC1 and SRM, Western blot analysis corroborated the expression patterns indicated by the 2-DE results. ANXA5 and CLIC1 were increased in expression and SRM was de-creased in expression in fESCs compared with both pESCs and aESCs. The differentially ex-pressed proteins identified in the present study warrant further investigation towards the goal of their potential application in ESC therapy.

Enrichment of CD9⁺spermatogonial stem cells from goat (<i>Capra aegagrus hircus</i>) testis using magnetic microbeads

The well documented source for adult multipotent stem cells is spermatogonial stem cells (SSCs) of mammalian testis. It is foundation of spermatogenesis in the testis throughout adult life by balancing self-renewal and differentiation. SSCs isolation from mammalian testis is difficult because of their scarcity and the lack of well characterized cell surface markers. Thus, the isolation of SSCs is of great interest for exploration of spermatogonial physiology and therapeutic approaches for fertility preservation. CD9 is a surface marker expressed in mouse and rat male germline stem cells. In this study, CD9 positive SSCs were successfully isolated from the goat testis using enzymatic digestion followed by three step purification: Differential plating, Percoll discontinuous density gradient followed by Magnetic activated cell sorting (MACS). Percoll discontinuous density gradient showed significant differences in the percentage of CD9+ SSCs across individual fraction. The fraction 36% and 40% gave the highest percentage of CD9+ SSCs i.e. 82% ± 1.2 and 9.2% ± 1.3 respectively. Magnetic activated cell sorting of CD9+ cells in the magnetic fraction of goat testes was in the range of 15% - 18% which is upto threefolds. CD9+ SSCs were further recovered with appreciable efficiency after immunomagnetic isolation by using various bead: cells ratio in which 4:1 ratio gave the highest yield of 69.06 × 105 with 18% of CD9+ SSCs. Magnetic activated cell sorting using anti-CD9 antibodies provides an efficient and fast approach as compared to conventional approaches such as differential plating and percoll discontinuous density gradient for enrichment strategy for spermatogonial stem cells from goat testes for undertaking research on basic and applied reproductive biology.

Human embryonic stem cell lines with ccr5-del32 allele conferring resistance to HIV

A 32bp deletion in the chemokine receptor 5 (CCR5) gene (CMKBR5) was shown to be linked to HIV resistance. Bone marrow transplantation from the homozygous CCR5-del32 donor to a CDC Stage 2 HIV-positive recipient was demonstrated to confer a HIV resistance, resulting in discontinuation of antiretroviral therapy. In search for an unlimited source of CCR5-del32 cells for transplantation purposes, we tested 137 human embryonic stem cell (hESC) lines from the Reproductive Genetics Institute’s hESC lines collection, and report here the finding of 12 hESC lines with the CCR5-del32 allele, one of which represents a unique partenogenetic ESC line containing two copies of this deletion and may be studied for utility in stem cell transplantation treatment of HIV.

Initial Experience with Tricortical Iliac Crest Bone Graft and Human Amniotic Allograft in Evans Calcaneal Osteotomy

Background: Adult acquired flatfoot deformity is generally mediated with an Evans procedure where a wedge of bone is placed into the calcaneus to better align the foot and decrease the deformity. The purpose of this study was to assess the efficacy and safety of human amniotic allograft applied to allogeneic tri-cortical grafts in Evans calcaneal osteotomy. Methods: The medical records of patients who had Evans calcaneal osteotomy with implantation of tri-cortical iliac crest bone graft with human anmiotic allograft for surgical management of adult acquired flatfoot deformity with 2 years follow-up data were reviewed. Results: A total of 63 patients (mean age: 33.3 yr, range: 18 - 66 yr) were enrolled with adult acquired flatfoot deformity. Median time to weight-bearing was 6 weeks. Time to wearing normal shoes was 10 weeks, and time to radiographic healing was 16 weeks. Conclusions: The use of human amniotic allograft did not diminish the long term outcome of procedure or the short term benchmarks for healing after surgery. There were no nonunion, wound dehiscence, infection, or allergic or immune reaction reported. This retrospective study demonstrated that tri-cortical iliac crest bone graft and HAA could be safely used in Evans calcaneal osteotomy with favorable results.

Human Amniotic Allograft in Use on Talar Dome Lesions: A Prospective Report of 37 Patients

One of the most challenging joint conditions facing ankle surgeons today is the treatment of Osteochondritis Dissecans (OCD) of the talar dome. The use of human amniotic allograft (HAA) in various surgical procedures, has been proven to facilitate bone growth and both soft tissue and cartilage healing. The authors of this paper propose the addition of HAA to the surgical repair of talar dome lesions to improve postoperative results, specifically pain reduction. For the study, 37 patients were identified having an OCD lesion of the talus measuring no larger than 2 cm2. All patients were treated surgically with an arthroscopic micro-fracture repair along with the addition of HHA. Modified ACFAS ankle scores were taken pre-operatively and at 3 months, 12 months, and at 24 months postoperatively. Visual analog scores were also taken preoperative and 24 months postoperatively. The size of the talar lesions documented with pre-operative MRI’s was compared with intra-operative measurements for each patient. Additional surgical repairs, comorbidities and any complications were also recorded and evaluated. All patients were treated with micro-fracture with HAA. Postoperative ACFAS scores for 3 months, 12 months and 24 months were significantly improved (p < 0.0001) compared with average preoperative scores. Additionally, VAS scores were also significantly improved when comparing the average pre-operative (4.9) and post-operative (1.1) pain scores (p < 0.0001). The size of the lesions documented by pre-operative MRI correlated to intra-operative measurements. There were no identified complications. The addition of HAA to arthroscopic micro-fracture repair of talar dome lesions measuring less than 2 cm2?has shown to significantly improve both post-operative VAS scores, when compared to preoperative scores. This improvement in ACFAS and VAS scores speaks to the potential use of HAA in the treatment of OCD.

Long-term phenotypic characterization of human bone marrow and adipose tissue derived mesenchymal stromal cells

We present methods to characterize mesenchymal stromal cells (MSC) over long time periods in vitro. The methods entail passaging cells multiple times and performing differentiation studies with the cells at each passage. Using an array of surface markers and flow cytometric quantification, the data can be correlated to traditional measures of differentiation such as PCR and staining. Using these methods to quantify the amount of differentiation, we concluded that many common MSC markers do not specifically define MSCs with true stem cell properties. Additionally, adipose-derived as opposed to bone marrow-derived MSCs show long-term CD34+ labeling. The methods described can be used to help identify stem cell markers and to characterize the state of stem cells in vitro. Compiling these data from multiple laboratories would be helpful to determine source, extraction and culture methods needed to obtain high yields of useful stem cells.

The xenotropic microRNA gene information for stem cell researches and clinical applications

MicorRNA (miRNA) is a small noncoding RNA and a miRNA is the gene. The identification of the human miRNA gene and its application have been performed and then it has been proceeding to explain about functioning of miRNAs in miRNA-messenger RNA targeting, profiling of miRNAs for diseases, transduction of the miRNA gene expression, production of human-induced pluripotent stem (iPS) cells by miRNA, embryonic stem (ES) cells and cancer development upon miRNA. The RNA information supplied by the miRNA gene, and the RNA gene information could expand to intracellular, intercellular, intraorgan, interorgan, intraspecies and interspecies. Therefore, the implantation of ES and iPS cells from donors would deliver xenotropic miRNAs to the acceptor. The therapeutic efficacy for treatment of iPS-derived cell implantation is the most important for clinical development of the stem cell researches but the xenotropic miRNA gene assessment with iPS-derived cells should substantially be completed for a safe and an exact application of the stem cell researches.

Repeated application of autologous bone marrow-derived stem cell therapy in patients with severe Buerger’s disease

Stem cell therapy (SCT) is a promising and prospective approach in the treatment of patients with severe peripheral arterial disorders, primarily with Buerger’s disease. However, very little is known about the duration of the effect of SCT, and to our best knowledge no data are available on the efficacy and safety of repeated SCT in patients with Buerger’s disease. Here we report on two patients with severe Buerger’s disease, who received repeated autologous bone marrow-derived stem cell therapy. Our results show that a second SCT, applied to a previously treated leg 30 or 36 months after the first treatment was efficient in both cases. After twelve months, the clinical state of the repeatedly treated lower limb improved spectacularly and non-healing ulcers healed more rapidly than after the first SCT. No severe adverse events were detected. Thus repeated SCT offers a safe and efficient treatment option for relapsing patients at the advanced stage of Buerger’s disease.

Increased osteogenesis with hydroxyapatite constructs combined with serially-passaged bone marrow-derived mesenchymal stem cells

We have previously reported on both the osteogenic potential of hydroxyapatite (HA) combined with bone marrow-derived mesenchymal stem cells (BMSCs) and a method involving osteogenic matrix cell sheet transplantation of BMSCs. In the present study, we assessed the osteogenic potential of serially-passaged BMSCs, both in vitro and in vivo. We also assessed whether an additional cell-loading technique can regain the osteogenic potential of the constructs combined with serially-passaged BMSCs. The present study revealed that passage (P) 1 cells cultured in osteogenic-induced medium showed strong positive staining for alkaline phosphatase (ALP) and Alizarin Red S, whereas P3 cells showed faint staining for ALP, with no Alizarin Red S staining. Staining of P1, P2 and P3 cells were progressively weaker, indicating that the osteogenic potential of the serially-passaged rat BMSCs is lost after P3 in vitro. The in vivo study showed that little bone formation was observed in the HA constructs seeded with P3 cells, 4 weeks after subcutaneous implantation. However, P3 cell/HA constructs which had increased cell-loading showed abundant bone formation within the pores of the HA construct. ALP and osteocalcin mRNA expression in these constructs was significantly higher than that of constructs with regular cell-seeding. The present study indicates that the osteogenic potential of the constructs with serially-passaged BMSCs is increased by additional cell-loading. This method can be applied to cases requiring hard tissue reconstruction, where BMSCs require serial expansion of cells.

Establishment and molecular characterization of human dermal mesenchymal-like stem cells derived from human scalp biopsy of androgenetic alopecia patient

Development of Dermal cell line has great scope in the field of skin related diseases and regenerative medicine. Alopecia leads to a skin disorder causing balding and its mechanism is not yet understood. In the present study, we have developed and characterized a heterogeneous population of human dermal mesenchymal-like stem cell line from scalp biopsy of androgenetic alopecia patient with a view to isolate cells from the bulge region of the hair follicle. Our study showed that the dermal cells isolated from dermis of skin showed epithelial-like cells expressing CD34 and Keratin 18, which are characteristic of bulge hair follicle cells. These cells also expressed mesenchymal phenotypes and pluripotency markers such as Oct4, Nanog and SOX2. These cells were designated as “Human Dermal Mesenchymal-like Stem Cells (hDMSCs)”. To confirm their epithelial phenotypes, we have grown these cells at low serum concentration and it was observed that 3% serum concentration provided optimum conditions for their growth and maintenance of characteristics. The hDMSCs cells are presently at passage 10. This study reports the establishment of human dermal mesenchymal-like cell line from the dermis of Alopecia patient, which may be used as an in vitro model system to study the mechanism of Alopecia and other related skin disorders.

An addition of medium-dose ATG to conditioning regimens favours the long-term survival of patients with allogeneic hematopoietic stem cell transplantation

Long-term survival of 116 leukemia/MDS patients received allo-SCT conditioned by a regimen with ATG-F or without ATG-F was analysed, together with the impact of ATG-F on the long-term survival, GVHD and disease relapse. Seventy patients received an ATG-F containing conditioning regimen FBCA, and 46 patients received a non-ATG-F FBC regimen. The FBCA regimen was associated with a 5-year survival of 65.4% in the complete HLA-matched group and 39.3% in the HLA-mismatched group. The difference between the two groups was significant (P = 0.012). For the FBC conditioning regimen, the 5-year overall survival of HLA-matched patients and the HLA-mismatched patients was 34.2% and 24.2% respectively (P = 0.216). The incidence of cGVHD was 32.9% and 83.6% in the FBCA and FBC condition regimen group respectively. Only 2.9% of the cases showed extensive cGVHD in the FBCA group while it was 69.4% in the FBC group (P = 0.00). Multivariate analysis indicated that relapse was related to the disease status and HLA typing, but unrelated to the conditioning regimens whether or not ATG-F was used (HR 0.54, P = 0.109). We conclude that the addition of ATG-F to conditioning regimen favours the longterm survival of allo-SCT.

Plasmid vector based generation of transgenic mesenchymal stem cells with stable expression of reporter gene in caprine

The production of cells capable of expressing gene(s) of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and a novel method of stem cell therapy in the various diseases. Achieving high levels of transgene expression for the longer period of time, without adversely affecting cell viability and differentiation capacity of the cells, is crucial. In the present study, we investigated the efficiency of plasmid vector for the production of transgenic cMSCs and examined any functional change of cells after transfection. To do so first we have collected bone marrows from the adult goats and cultured them for isolation of mesenchymal stem cells (cBM-MSCs). These cells were characterized using MSC specific markers including differentiation into osteocytes and adipocytes. Transfection with plasmid vector did not adversely affect cBM-MSCs morphology, viability or differentiation potential, and transgene expression levels were unaffected beyond passage 12th. The results indicated that we have been able to generate transgenic caprine MSC (tcBM-MSC) and transfection of cBM-MSCs using plasmid vector resulted in very high and stable transfection efficiency. This finding may have considerable significance in improving the efficacy of MSC-based therapies and their tracking in animal model.

Defining umbilical cord blood stem cells

Umbilical cord blood is the blood found in the vessels of the umbilical cord and placenta. It has been shown that this blood contains at least three populations of stem cells, each with unique features and properties. Due to the absence of standardized criteria for characterizing and naming cord blood stem cells, different terms and acronyms have been introduced to describe certain cell populations. Besides the confusion caused by the introduction of these different names, some of the terms used by different groups are inaccurate and misleading when considering the molecular and cellular properties of such cells. Hence, in this review we provide simple and direct descriptions of different populations of stem cells in umbilical cord blood in an attempt to clarify the confusion caused by the existence of multiple names given to certain cord blood stem cells. We also discuss the potential use of umbilical cord blood stem cells as a therapeutic tool for several diseases and disorders in light of ongoing clinical trials.

Expression of the polycomb group gene <i>Bmi</i>1 does not affect the prognosis of pediatric acute lymphoblastic leukemia

The Polycomb group protein Bmi1 is a constituent of the Polycomb repressive complex 1, and it is an important molecule for the regulation of the self-renewal of hematopoietic stem cells. In the field of clinical hematology, there are reports that the level of Bmi1 expression in blast cells is related to the prognosis of acute myeloid leukemia, chronic myeloid leukemia, and myelodysplastic syndrome. We investigated whether the level of Bmi1 expression in leukemic cells is related to the prognosis and the characteristics of childhood acute lymphoblastic leukemia. In all the leukemic blast cells, Bmi1 gene expression was lower value than that in normal B cells. There were no correlations between the level of Bmi1 gene expression in leukemic blast cells and other parameters, including prognosis. Here, we report that the level of Bmi1 expression in blast cells is not related to the prognosis of pediatric acute lymphoblastic leukemia.

NSC-induced D-neurons are decreased in striatum of schizophrenia: Possible cause of mesolimbic dopamine hyperactivity

Neural stem cell (NSC) hypofunction is an etiological hypothesis of schizophrenia. Although dopamine (DA) dysfunction is also a widely accepted hypothesis, molecular background of mesolimbic DA hyperactivity has not yet been well known. Here, the author proposes “D-cell hypothesis”, accounting for molecular basis of mesolimbic DA hyperactivity of schizophrenia, by NSC hypofunction and decrease of putative NSC-induced D-cells. The “D-cell” is defined as “non-monoaminergic aromatic L-amino acid decarboxylase (AADC)-containing cell”. D-cells produce trace amines, and also take up amine precursors and convert them to amines by decarboxylation. The author reported “dopa-decarboxylating neurons specific to the human striatum”, that is, “D-neurons” in the human striatum, and decrease of striatal D-neurons in patients with schizophrenia. Trace amine-associated receptor, type 1 (TAAR1), a subtype of trace amine receptors, having a quite number of ligands such as tyramine, β-phenylethylamine (PEA) and methamphetamine, has modulating functions on monoamine neurons. It has been known that reduced binding of ligands to TAAR1 receptors on DA terminal of DA neurons of the midbrain ventral tegmental area (VTA) increased firing frequency of VTA DA neurons. In brains of schizophrenia, NSC hypofunction in the subventricular zone of lateral ventricle may cause decrease of D-neurons in the striatum and nucleus accumbens, and may result in decrease of trace amine signals. Decrease of trace amine signals to TAAR1 on VTA DA neurons may increase firing frequency of VTA DA neurons, and may finally cause mesolimbic DA hyperactivity. Increased stimulation to DA D2 receptors of NSCs might suppress NSC proliferation, and may induce additional mesolimbic DA hyperactivity as well as D-cell decrease. This novel theory, “D-cell hypothesis”, possibly explains mesolimbic DA hyperactivity in pathogenesis of schizophrenia.

Pretreatment with antioxidants prevent bone injury by improving bone marrow microenvironment for stem cells

Irradiation induces bone injury by generating free radicals that adversely affect the microenvironment for Mesenchymal stem cells (MSCs) and damages bone marrow blood vessels. We wished to investigate the efficacy of antioxidant administration in protecting stem cell microenvironments and promoting bone marrow vasculature recovery after radiation treatment. The antioxidant ascorbic acid was administered 3 times at a dosage: 150 mg/kg/day to experimenttal groups 3 days before targeted radiation by a unique Small Animal Radiation Research Platform (SARRP). Histological staining indicated that antioxidant treated mice had less severe bone marrow damage 1 week after irradiation with substantial marrow cellular recovery 4 weeks later. Flow cytometry analysis showed that antioxidant administration was correlated with a rebound in MSC quantity in bone marrow. Anti-oxidant treatment was also observed to allow for better vasculature retention and recovery through angiographic imaging. Our data suggests that pre-treatment with ascorbic acid serves to improve bone marrow microenvironments for bone marrow stem cells after radiation treatment.