Myocardial protection by nanomaterials formulated with CHIR99021 and FGF1.

The mortality of patients suffering from acute myocardial infarction is linearly related to the infarct size. As regeneration of cardiomyocytes from cardiac progenitor cells is minimal in the mammalian adult heart, we have explored a new therapeutic approach, which leverages the capacity of nanomaterials to release chemicals over time to promote myocardial protection and infarct size reduction. Initial screening identified 2 chemicals, FGF1 and CHIR99021 (a Wnt1 agonist/GSK-3ß antagonist), which synergistically enhance cardiomyocyte cell cycle in vitro. Poly-lactic-co-glycolic acid nanoparticles (NPs) formulated with CHIR99021 and FGF1 (CHIR + FGF1-NPs) provided an effective slow-release system for up to 4 weeks. Intramyocardial injection of CHIR + FGF1-NPs enabled myocardial protection via reducing infarct size by 20%-30% in mouse or pig models of postinfarction left ventricular (LV) remodeling. This LV structural improvement was accompanied by preservation of cardiac contractile function. Further investigation revealed that CHIR + FGF1-NPs resulted in a reduction of cardiomyocyte apoptosis and increase of angiogenesis. Thus, using a combination of chemicals and an NP-based prolonged-release system that works synergistically, this study demonstrates a potentially novel therapy for LV infarct size reduction in hearts with acute myocardial infarction.

N-cadherin overexpression enhances the reparative potency of human-induced pluripotent stem cell-derived cardiac myocytes in infarcted mouse hearts.

AIMS: In regenerative medicine, cellular cardiomyoplasty is one of the promising options for treating myocardial infarction (MI); however, the efficacy of such treatment has shown to be limited due to poor survival and/or functional integration of implanted cells. Within the heart, the adhesion between cardiac myocytes (CMs) is mediated by N-cadherin (CDH2) and is critical for the heart to function as an electromechanical syncytium. In this study, we have investigated whether the reparative potency of human-induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) can be enhanced through CDH2 overexpression. METHODS AND RESULTS: CDH2-hiPSC-CMs and control wild-type (WT)-hiPSC-CMs were cultured in myogenic differentiation medium for 28 days. Using a mouse MI model, the cell survival/engraftment rate, infarct size, and cardiac functions were evaluated post-MI, at Day 7 or Day 28. In vitro, conduction velocities were significantly greater in CDH2-hiPSC-CMs than in WT-hiPSC-CMs. While, in vivo, measurements of cardiac functions: left ventricular (LV) ejection fraction, reduction in infarct size, and the cell engraftment rate were significantly higher in CDH2-hiPSC-CMs treated MI group than in WT-hiPSC-CMs treated MI group. Mechanistically, paracrine activation of ERK signal transduction pathway by CDH2-hiPSC-CMs, significantly induced neo-vasculogenesis, resulting in a higher survival of implanted cells. CONCLUSION: Collectively, these data suggest that CDH2 overexpression enhances not only the survival/engraftment of cultured CDH2-hiPSC-CMs, but also the functional integration of these cells, consequently, the augmentation of the reparative properties of implanted CDH2-hiPSC-CMs in the failing hearts.

Cardiomyocytes from CCND2-overexpressing human induced-pluripotent stem cells repopulate the myocardial scar in mice: A 6-month study.

BACKGROUND: Cardiomyocytes that have been differentiated from CCND2-overexpressing human induced-pluripotent stem cells (hiPSC-CCND2OE CMs) can proliferate when transplanted into mouse hearts after myocardial infarction (MI). However, it is unknown whether remuscularization can replace the thin LV scar and if the large muscle graft can electrophysiologically synchronize to the recipient myocardium. Our objectives are to evaluate the structural and functional potential of hiPSC-CCND2OE CMs in replacing the LV thin scar. METHODS: NOD/SCID mice were treated with hiPSC-CCND2OE CMs (i.e., the CCND2OE group), hiPSC-CCND2WT CMs (the CCND2WT group), or an equal volume of PBS immediately after experimentally-induced myocardial infarction. The treatments were administered to one site in the infarcted zone (IZ), two sites in the border zone (BZ), and a fourth group of animals underwent Sham surgery. RESULTS: Six months later, engrafted cells occupied >50% of the scarred region in CCND2OE animals, and exceeded the number of engrafted cells in CCND2WT animals by ~8-fold. Engrafted cells were also more common in the IZ than in the BZ for both cell-treatment groups. Measurements of cardiac function, infarct size, wall thickness, and cardiomyocyte hypertrophy were significantly improved in CCND2OE animals compared to animals from the CCND2WT or PBS-treatment groups. Measurements in the CCND2WT and PBS groups were similar, and markers for cell cycle activation and proliferation were significantly higher in hiPSC-CCND2OE CMs than in hiPSC-CCND2WT CMs. Optical mapping of action potential propagation indicated that the engrafted hiPSC-CCND2OE CMs were electrically coupled to each other and to the cells of the native myocardium. No evidence of tumor formation was observed in any animals. CONCLUSIONS: Six months after the transplantation, CCND2-overexpressing hiPSC-CMs proliferated and replaced >50% of the myocardial scar tissue. The large graft hiPSC-CCND2OE CMs also electrically integrated with the host myocardium, which was accompanied by a significant improvement in LV function.

Functional Tissue Engineering: A Prevascularized Cardiac Muscle Construct for Validating Human Mesenchymal Stem Cells Engraftment Potential In Vitro.

The influence of somatic stem cells in the stimulation of mammalian cardiac muscle regeneration is still in its early stages, and so far, it has been difficult to determine the efficacy of the procedures that have been employed. The outstanding question remains whether stem cells derived from the bone marrow or some other location within or outside of the heart can populate a region of myocardial damage and transform into tissue-specific differentiated progenies, and also exhibit functional synchronization. Consequently, this necessitates the development of an appropriate in vitro three-dimensional (3D) model of cardiomyogenesis and prompts the development of a 3D cardiac muscle construct for tissue engineering purposes, especially using the somatic stem cell, human mesenchymal stem cells (hMSCs). To this end, we have created an in vitro 3D functional prevascularized cardiac muscle construct using embryonic cardiac myocytes (eCMs) and hMSCs. First, to generate the prevascularized scaffold, human cardiac microvascular endothelial cells (hCMVECs) and hMSCs were cocultured onto a 3D collagen cell carrier (CCC) for 7 days under vasculogenic culture conditions; hCMVECs/hMSCs underwent maturation, differentiation, and morphogenesis characteristic of microvessels, and formed dense vascular networks. Next, the eCMs and hMSCs were cocultured onto this generated prevascularized CCCs for further 7 or 14 days in myogenic culture conditions. Finally, the vascular and cardiac phenotypic inductions were characterized at the morphological, immunological, biochemical, molecular, and functional levels. Expression and functional analyses of the differentiated progenies revealed neo-cardiomyogenesis and neo-vasculogenesis. In this milieu, for instance, not only were hMSCs able to couple electromechanically with developing eCMs but were also able to contribute to the developing vasculature as mural cells, respectively. Hence, our unique 3D coculture system provides us a reproducible and quintessential in vitro 3D model of cardiomyogenesis and a functioning prevascularized 3D cardiac graft that can be utilized for personalized medicine.

A Novel Human Tissue-Engineered 3-D Functional Vascularized Cardiac Muscle Construct.

Organ tissue engineering, including cardiovascular tissues, has been an area of intense investigation. The major challenge to these approaches has been the inability to vascularize and perfuse the in vitro engineered tissue constructs. Attempts to provide oxygen and nutrients to the cells contained in the biomaterial constructs have had varying degrees of success. The aim of this current study is to develop a three-dimensional (3-D) model of vascularized cardiac tissue to examine the concurrent temporal and spatial regulation of cardiomyogenesis in the context of postnatal de novo vasculogenesis during stem cell cardiac regeneration. In order to achieve the above aim, we have developed an in vitro 3-D functional vascularized cardiac muscle construct using human induced pluripotent stem cell-derived embryonic cardiac myocytes (hiPSC-ECMs) and human mesenchymal stem cells (hMSCs). First, to generate the prevascularized scaffold, human cardiac microvascular endothelial cells (hCMVECs) and hMSCs were co-cultured onto a 3-D collagen cell carrier (CCC) for 7 days under vasculogenic culture conditions. In this milieu, hCMVECs/hMSCs underwent maturation, differentiation, and morphogenesis characteristic of microvessels, and formed extensive plexuses of vascular networks. Next, the hiPSC-ECMs and hMSCs were co-cultured onto this generated prevascularized CCCs for further 7 or 14 days in myogenic culture conditions. Finally, the vascular and cardiac phenotypic inductions were analyzed at the morphological, immunological, biochemical, molecular, and functional levels. Expression and functional analyses of the differentiated cells revealed neo-angiogenesis and neo-cardiomyogenesis. Thus, our unique 3-D co-culture system provided us the apt in vitro functional vascularized 3-D cardiac patch that can be utilized for cellular cardiomyoplasty.

Nitric Oxide Modulates Postnatal Bone Marrow-Derived Mesenchymal Stem Cell Migration.

Nitric oxide (NO) is a small free-radical gas molecule, which is highly diffusible and can activate a wide range of downstream effectors, with rapid and widespread cellular effects. NO is a versatile signaling mediator with a plethora of cellular functions. For example, NO has been shown to regulate actin, the microfilament, dependent cellular functions, and also acts as a putative stem cell differentiation-inducing agent. In this study, using a wound-healing model of cellular migration, we have explored the effect of exogenous NO on the kinetics of movement and morphological changes in postnatal bone marrow-derived mesenchymal stem cells (MSCs). Cellular migration kinetics and morphological changes of the migrating MSCs were measured in the presence of an NO donor (S-Nitroso-N-Acetyl-D,L-Penicillamine, SNAP), especially, to track the dynamics of single-cell responses. Two experimental conditions were assessed, in which SNAP (200 µM) was applied to the MSCs. In the first experimental group (SN-1), SNAP was applied immediately following wound formation, and migration kinetics were determined for 24 h. In the second experimental group (SN-2), MSCs were pretreated for 7 days with SNAP prior to wound formation and the determination of migration kinetics. The generated displacement curves were further analyzed by non-linear regression analysis. The migration displacement of the controls and NO treated MSCs (SN-1 and SN-2) was best described by a two parameter exponential functions expressing difference constant coefficients. Additionally, changes in the fractal dimension (D) of migrating MSCs were correlated with their displacement kinetics for all the three groups. Overall, these data suggest that NO may evidently function as a stop migration signal by disordering the cytoskeletal elements required for cell movement and proliferation of MSCs.

Modulation of the migration and differentiation potential of adult bone marrow stromal stem cells by nitric oxide.

Nitric oxide (NO) is a diffusible free radical, which serves as a pluripotent intracellular messenger in numerous cell systems. NO has been demonstrated to regulate actin dependent cellular functions and functions as a putative inductive agent in directing stem cells differentiation. In this study, we investigated the effect of exogenous NO on the kinetics of movement and morphological changes in adult bone marrow stromal cells (BMSCs) in a wound healing model of cellular migration. Cellular migration and morphological changes were determined by measurement of changes in the area and fractal dimension of BMSCs monolayer as a function of time in the presence of an NO donor (S-Nitroso-N-Acetyl-D,L-Penicillamine, SNAP) compared to untreated BMSCs. Response of the BMSCs' actin cytoskeleton and desmin to NO was assessed by determining changes in their integrated optical density (IOD) and fractal dimension at 24 h and 7 days. NO suppressed BMSCs' migration accompanied by a reduction in cell size, with maintenance of their stellate to polygonal morphology. In response to NO, the actin cytoskeleton expressed an increase in randomness but maintained a constant amount of F-actin relative to the cell size. The presence of NO also induced an increase in randomly organized cytoplasmic desmin. These data suggest that NO has an apparent inductive effect on adult BMSCs and is capable of initiating phenotypic change at the gross cellular, cytoskeletal and molecular levels. It is apparent, however, that additional factors or conditions are required to further drive the differentiation of adult BMSCs into specific phenotypes, such as cardiomyocytes.

The mechanical coupling of adult marrow stromal stem cells during cardiac regeneration assessed in a 2-D co-culture model.

Postnatal cardiomyocytes undergo terminal differentiation and a restricted number of human cardiomyocytes retain the ability to divide and regenerate in response to ischemic injury. However, whether these neo-cardiomyocytes are derived from endogenous population of resident cardiac stem cells or from the exogenous double assurance population of resident bone marrow-derived stem cells that populate the damaged myocardium is unresolved and under intense investigation. The vital challenge is to ameliorate and/or regenerate the damaged myocardium. This can be achieved by stimulating proliferation of native quiescent cardiomyocytes and/or cardiac stem cell, or by recruiting exogenous autologous or allogeneic cells such as fetal or embryonic cardiomyocyte progenitors or bone marrow-derived stromal stem cells. The prerequisites are that these neo-cardiomyocytes must have the ability to integrate well within the native myocardium and must exhibit functional synchronization. Adult bone marrow stromal cells (BMSCs) have been shown to differentiate into cardiomyocyte-like cells both in vitro and in vivo. As a result, BMSCs may potentially play an essential role in cardiac repair and regeneration, but this concept requires further validation. In this report, we have provided compelling evidence that functioning cardiac tissue can be generated by the interaction of multipotent BMSCs with embryonic cardiac myocytes (ECMs) in two-dimensional (2-D) co-cultures. The differentiating BMSCs were induced to undergo cardiomyogenic differentiation pathway and were able to express unequivocal electromechanical coupling and functional synchronization with ECMs. Our 2-D co-culture system provides a useful in vitro model to elucidate various molecular mechanisms underpinning the integration and orderly maturation and differentiation of BMSCs into neo-cardiomyocytes during myocardial repair and regeneration.

A 3-D cardiac muscle construct for exploring adult marrow stem cell based myocardial regeneration.

Adult bone marrow stromal cells (BMSCs) are capable of differentiating into cardiomyocyte-like cells in vitro and contribute to myocardial regeneration in vivo. Consequently, BMSCs may potentially play a vital role in cardiac repair and regeneration. However, this concept has been limited by inadequate and inconsistent differentiation of BMSCs into cardiomyocytes along with poor survival and integration of neo-cardiomyocytes after implantation into ischemic myocardium. In order to overcome these barriers and to explore adult stem cell based myocardial regeneration, we have developed an in vitro model of three-dimensional (3-D) cardiac muscle using rat ventricular embryonic cardiomyocytes (ECMs) and BMSCs. When ECMs and BMSCs were seeded sequentially onto a 3-D tubular scaffold engineered from topographically aligned type I collagen-fibers and cultured in basal medium for 7, 14, 21, or 28 days, the maturation and co-differentiation into a cardiomyocyte lineage was observed. Phenotypic induction was characterized at morphological, immunological, biochemical and molecular levels. The observed expression of transcripts coding for cardiomyocyte phenotypic markers and the immunolocalization of cardiomyogenic lineage-associated proteins revealed typical expression patterns of neo-cardiomyogenesis. At the biochemical level differentiating cells exhibited appropriate metabolic activity and at the ultrastructural level myofibrillar and sarcomeric organization were indicative of an immature phenotype. Our 3-D co-culture system sustains the ECMs in vitro continuum of differentiation process and simultaneously induces the maturation and differentiation of BMSCs into cardiomyocyte-like cells. Thus, this novel 3-D co-culture system provides a useful in vitro model to investigate the functional role and interplay of developing ECMs and BMSCs during cardiomyogenic differentiation.

Regulation of osteogenic differentiation of rat bone marrow stromal cells on 2D nanorod substrates.

Bone marrow stromal cells (BMSCs) possess multi-lineage differentiation potential and can be induced to undergo differentiation into various cell types with the correct combination of chemical and environmental factors. Although, they have shown great prospects in therapeutic and medical applications, less is known about their behavior on nanosurfaces mimicking the extra cellular matrix (ECM). In this report we have employed 2D substrates coated with tobacco mosaic virus (TMV) nanorods to study the differentiation process of BMSCs into osteoblast like cells. TMV is a rod-shaped plant virus with an average length of 300 nm and diameter of 18 nm. The osteogenic differentiation of BMSCs on TMV was studied over time points of 7, 14 and 21 days. We examined the temporal gene expression changes during these time points by real-time quantitative PCR (RT-qPCR) analysis. As expected, osteo-specific genes (osteocalcin, osteopontin and osteonectin) were upregulated and showed a maximum change in expression on TMV at 14 days which was 7 days earlier than on tissue culture plastic (TCP). Based on the genes expression profile generated by RT-qPCR experiments, we proposed that the early interaction of cells with TMV triggers on signaling pathways which regulate speedy expression of osteocalcin in turn, resulting in early mineralization of the cells. To further investigate these regulating factors we studied global changes in gene expression (DNA microarray analyses) during osteogenic differentiation on the nanosubstrate. Multitudes of genes were affected by culturing cells on nanorod substrate, which corroborated our initial PCR findings. Microarray analysis further revealed additional targets influenced by the presence of nanorods on the surface, of which, the expression of bone morphogenetic protein 2 (BMP2) was of particular interests. Further investigation into the temporal change of BMP2, revealed that it acts as a major promoter in signaling the early regulation of osteocalcin on TMV coated substrates.

A three-dimensional model of vasculogenesis.

Postnatal bone marrow contains various subpopulations of resident and circulating stem cells (HSCs, BMSCs/MSCs) and progenitor cells (MAPCs, EPCs) that are capable of differentiating into one or more of the cellular components of the vascular bed in vitro as well as contribute to postnatal neo-vascularization in vivo. When rat BMSCs were seeded onto a three-dimensional (3-D) tubular scaffold engineered from topographically aligned type I collagen fibers and cultured either in vasculogenic or non-vasculogenic media for 7, 14, 21 or 28 days, the maturation and co-differentiation into endothelial and/or smooth muscle cell lineages were observed. Phenotypic induction of these substrate-grown cells was assayed at transcript level by real-time PCR and at protein level by confocal microscopy. In the present study, the observed upregulation of transcripts coding for vascular phenotypic markers is reminiscent of an in vivo expression pattern. Immunolocalization of vasculogenic lineage-associated markers revealed typical expression patterns of vascular endothelial and smooth muscle cells. These endothelial cells exhibited high metabolism of acetylated low-density lipoprotein. In addition to the induced monolayers of endothelial cells, the presence of numerous microvascular capillary-like structures was observed throughout the construct. At the level of scanning electron microscopy, smooth-walled cylindrical tube-like structures with smooth muscle cells and/or pericytes attached to its surface were elucidated. Our 3-D culture system not only induces the maturation and differentiation of BMSCs into vascular cell lineages but also supports microvessel morphogenesis. Thus, this unique in vitro model provides an excellent platform to study the temporal and spatial regulation of postnatal de novo vasculogenesis, as well as attack the lingering limit in developing engineered tissues, that is perfusion.

Material properties and bone marrow stromal cells response to in situ crosslinkable RGD-functionlized lactide-co-glycolide scaffolds.

In situ crosslinkable biomaterials with degradation profiles that can be tailored to a particular application are indispensable for treating irregularly shaped defects and for fabrication of shape-selective scaffolds. The objective of this work was to synthesize ultra low molecular weight functionalized PLA and PLGA macromers that can be grafted with bioactive peptides and crosslinked in situ to fabricate biodegradable functional scaffolds. In situ crosslinkable lactide-co-glycolide macromer (cMLGA; "c" for crosslinkable, "M" for macromer, and "LGA" for lactide-co-glycolide) was synthesized by anionic polymerization of lactide and glycolide monomers followed by condensation polymerization with fumaryl chloride. The cMLA (100% L-lactide) and cMLGA macromers formed porous crosslinked scaffolds with NVP as the crosslinker. The mass loss of the crosslinked cMLA and cMLGA was linear with incubation time in vitro (zero-order degradation) and the degradation rate depended on the ratio of lactide to glycolide. cMLGA scaffold with 1:1 lactide to glycolide ratio completely degraded after 4 weeks while the cMLA lost less than 40% of its initial mass after 35 weeks. When cMLA scaffold was functionalized with acrylated integrin-binding Ac-GRGD amino acid sequence, bone marrow stromal (BMS) cells attached and spread on the cMLA scaffold and exhibited focal-point cell adhesion. The mRNA expression levels of collagen-1alpha, osteonectin, and osteopontin for BMS cells seeded in the scaffolds with 1 and 5% Ac-GRGD were upregulated compared with those without Ac-GRGD. cMLGA is attractive as in situ crosslinkable macromer for fabrication of functional scaffolds with degradation characteristics that can be tailored to a particular application.

The promotion of osteoblastic differentiation of rat bone marrow stromal cells by a polyvalent plant mosaic virus.

To investigate the role that the micro/nano-environment plays on the differentiation pathway of bone marrow stromal cells (BMSCs) into osteoblasts, we employed a 2D substrate coated with turnip yellow mosaic virus (TYMV) particles. TYMV is a non-enveloped icosahedral plant virus which has an average diameter 28 nm and the protein cage structure consists of 180 identical subunits. The temporal effect of TYMV coated substrate on the adhesion and differentiation capacity of the BMSCs was monitored for selected time periods of 7, 14 and 21 days. We examined the gene expression profile of BMSCs cultured in primary media (undifferentiated cells) and cells induced to osteoblast lineage by real time PCR analysis. To further corroborate our findings, we investigated the expression of osteogenic markers using immunohistochemistry and cytochemical staining. As expected, the genes involved in the process of osteogenic differentiation were activated more during the growth of cells under osteogenic media. In addition, we found that the BMSCs induced to undergo osteogenic differentiation on TYMV coated substrates formed fully mineralized nodules comprising of osteoblast-like cells around day 14. Comparing the gene expression pattern of BMSCs induced to osteogenic differentiation under standard culture conditions with the cells induced on TYMV substrates, we found significant differences in the temporal expression and level of expression of several key genes. Our findings indicate that TYMV, as a biogenic nanoparticle, can be employed as a model to modulate the nano-environment of the substrates in order to gain an insight into the role that the micro/nano-environment has in regulating adhesion, growth and differentiation of BMSCs towards osteogenic lineage, which will be vital for designing compatible biomaterials for tissue engineering purposes.

A three-dimensional tubular scaffold that modulates the osteogenic and vasculogenic differentiation of rat bone marrow stromal cells.

Bone marrow stromal cells (BMSCs) or mesenchymal stem cells (MSCs) are a heterogeneous population of cells that are multipotent. When rat BMSCs were seeded onto a 3-dimensional (3-D) tubular scaffold engineered from aligned type I collagen strands and cultured in osteogenic medium, they simultaneously matured and differentiated into osteoblastic and vascular cell lineages. In addition, these osteoblasts produced mineralized matricellular deposits. BMSCs were seeded at a density of 2 x 10(6) cells/15 mm tube and cultured in basal or osteogenic medium for 3, 6, and 9 days. These cells were subsequently processed for real-time reverse-transcriptase polymerase chain reaction (RT-qPCR), immunohistochemical, cytochemical, and biochemical analyses. Immunolocalization of lineage-specific proteins was visualized using confocal microscopy. In the present study, the expression pattern of key osteogenic markers significantly differed in response to basal and osteogenic media. Alkaline phosphatase activity and calcium content increased significantly over the observed period of time in osteogenic medium. The observed up-regulation of transcripts coding for osteoblastic phenotypic markers is reminiscent of in vivo expression patterns. Abundant sheets of Pecam (CD31) -, Flk-1 (vascular endothelial growth factor receptor-2) -, CD34-, tomato lectin-, and alpha-smooth muscle actin-positive cells were observed in these tube cultures. Moreover, nascent capillary-like vessels were also seen amid the osteoblasts in osteogenic cultures. Our 3-D culture system augmented the maturation and differentiation of BMSCs into osteoblasts. Thus, our in vitro model provides an excellent opportunity to study the concurrent temporal and spatial regulation of osteogenesis and vasculogenesis during bone development.

The influence of proepicardial cells on the osteogenic potential of marrow stromal cells in a three-dimensional tubular scaffold.

It is well established that the process of neovascularization or neoangiogenesis is coupled to the development and maturation of bone. Bone marrow stromal cells (BMSCs) or mesenchymal stem cells (MSCs) comprise a heterogeneous population of cells that can be differentiated in vitro into both mesenchymal and non-mesenchymal cell lineages. When both rat BMSCs and quail proepicardia (PEs) were seeded onto a three-dimensional (3-D) tubular scaffold engineered from aligned collagen type I strands and co-cultured in osteogenic media, the maturation and co-differentiation into osteoblastic and vascular cell lineages were observed. In addition, these cells produced abundant mineralized extracellular matrix materials and vessel-like structures. BMSCs were seeded at a density of 2 x 10(6)cells/15 mm tube and cultured in basal media for 3 days. Subsequently, on day 3, PEs were seeded onto the same tubes and the co-culture was continued for another 3, 6 or 9 days either in basal or in osteogenic media. Differentiated cells were subjected to immunohistochemical, cytochemical and biochemical analyses. Phenotypic induction was analyzed at mRNA level by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Immunolocalization of key osteogenic and vasculogenic lineage specific markers were examined using confocal scanning laser microscopy. In osteogenic tube cultures, both early and late osteogenic markers were observed and were reminiscent of in vivo expression pattern. Alkaline phosphatase activity and calcium content significantly increased over the observed period of time in osteogenic medium. Abundant interlacing fascicles of QCPN, QH1, isolectin and alpha-smooth muscle actin (alpha-SMA) positive cells were observed in these tube cultures. These cells formed extensive arborizations of nascent capillary-like structures and were seen amidst the developing osteoblasts in osteogenic cultures. The 3-D culture system not only generated de novo vessel-like structures but also augmented the maturation and differentiation of BMSCs into osteoblasts. Thus, this novel co-culture system provides a useful in vitro model to investigate the functional role and effects of neovascularization in the proliferation, differentiation and maturation of BMSC derived osteoblasts.

Age related changes in Fas (CD95) and Fas ligand gene expression and cytokine profiles in healthy Indians.

Ageing in human and animal models show changes in many aspects of protective immunity, particularly lymphopoenia and progressive decline in immune functions leading to increased frequency of infection and neoplasia. However, the exact mechanism of these defects is still unclear. In this study, elderly subjects showed a decline in CD3+ and CD4+ T-cell subsets as well as serum IL-2 levels. Serum IL-6 was significantly raised while expression of its signaling receptor gp130 was significantly impaired in elderly as compared to the younger ones. Additionally, all the elderly individuals showed constitutive expression of Fas and FasL mRNA; however, none of the younger individuals expressed mRNA transcripts constitutively although induced expression was seen in both the groups. Similarly, frequency of Fas and FasL expressing CD4+ and CD8+ T-cell subsets were significantly (p < 0.001) higher in elderly subjects as compared to the younger ones. Elderly individuals also showed a significantly (p < 0.001) higher frequency of activation induced cell death (AICD). Since interaction of Fas with its cognate ligand (FasL) activates death inducing caspases leading to apoptosis, and gp130 induces anti-apoptotic signal through STAT-3 pathway, these results suggest that the decline in protective immune functions in aged individuals may be related to Fas and FasL mediated apoptosis of peripheral T-cell subsets.

Novel germline mutations in the BRCA1 and BRCA2 genes in Indian breast and breast-ovarian cancer families.

The two major hereditary breast/ovarian cancer predisposition tumor suppressor genes, BRCA1 and BRCA2 that perform apparently generic cellular functions nonetheless cause tissue-specific syndromes in the human population when they are altered, or mutated in the germline. However, little is known about the contribution of BRCA1 and BRCA2 mutations to breast and/or ovarian cancers in the Indian population. We have screened for mutations the entire BRCA1 and BRCA2 coding sequences, and intron-exon boundaries, as well as their flanking intronic regions in sixteen breast or breast and ovarian cancer families of Indian origin. We have also analyzed 20 female patients with sporadic breast cancer regardless of age and family history, and 69 unrelated normal individuals as control. Thus a total of 154 samples were screened for BRCA1 and BRCA2 mutations using a combination of polymerase chain reaction-mediated site directed mutagenesis (PSM), polymerase chain reaction-single stranded conformation polymorphism assay (PCR-SSCP) and direct DNA sequencing of PCR products (DS). Twenty-one sequence variants including fifteen point mutations were identified. Five deleterious pathogenic, protein truncating frameshift and non-sense mutations were detected in exon 2 (c.187_188delAG); and exon 11 (c.3672G>T) [p.Glu1185X] of BRCA1 and in exon 11 (c.5227dupT, c.5242dupT, c.6180dupA) of BRCA2 (putative mutations - four novel) as well as fourteen amino acid substitutions were identified. Twelve BRCA1 and BRCA2 missense variants were identified as unique and novel. In the cohort of 20 sporadic female patients no mutations were found.

BRCA1 germline mutations in Indian familial breast cancer.

Germline mutation analysis of BRCA1 gene has demonstrated significant allelic heterogeneity. These differences represent historical influences of migration, population structure and geographic or cultural isolation. To date, there have been no reports of Indian families with mutations in BRCA1. We have screened for mutations in selected coding exons of BRCA1 and their flanking intron regions in three breast or breast and ovarian cancer families with family history of three or more cases of breast cancer under age 45 and/or ovarian cancer at any age. We have also analyzed 10 female patients with sporadic breast cancer regardless of age and family history, as well as 50 unrelated normal individuals as controls. Thus a total of 90 samples were analyzed for BRCA1 mutations using polymerase chain reaction-mediated site directed mutagenesis (PSM) and single stranded conformation polymorphism (SSCP) analysis for various selected exons followed by sequencing of variant bands. Eight point mutations were identified. Two deleterious pathogenic, protein truncating non-sense mutations were detected in exon 11 (E1250X) and exon 20 (E1754X) and six novel and unique amino acid substitutions (F1734S, D1739Y, V1741G, Q1747H, P1749A, R1753K). One complex missense mutation of exon 20 [V1741G; P1749A] was seen in two out of three families and another complex combination of missense and non-sense mutations of the same exon [V1741G; E1754X] was observed in only one family. These complex mutations exist only in breast cancer families but not in control populations of women. Three splice site variants (IVS20+3A>C, IVS20+4A>T, IVS20+5A>T) and two intronic variants (IVS20+21_22insG, IVS20+21T>G) were also detected. In the group of 10 sporadic female patients no mutations were found.