Silibinin efficacy in a rat model of pulmonary arterial hypertension using monocrotaline and chronic hypoxia.

BACKGROUND: C-X-C chemokine receptor type 4 (CXCR4) may be involved in the development of pulmonary arterial hypertension (PAH). CXCR4 inhibitor AMD3100 was described to have a positive effect on the prevention of pulmonary arterial muscularization in PAH models. Silibinin is a traditional medicine that has an antagonistic effect on CXCR4. We investigated the effect of silibinin using rat models of PAH. METHODS: PAH was induced by a single subcutaneous injection of monocrotaline. The rats were maintained in a chronic hypoxic condition (10% O2) with or without silibinin. To evaluate the efficacy of silibinin on PAH, right ventricular systolic pressure (RVSP), Fulton index (weight ratio of right ventricle to the left ventricle and septum), percent medial wall thickness (% MT), and vascular occlusion score (VOS) were measured and calculated. Immunohistochemical analysis was performed targeting CXCR4 and c-Kit. Reverse transcription-quantitative polymerase chain reaction was performed for the stem cell markers CXCR4, stromal cell derived factor-1 (SDF-1), c-Kit, and stem cell factor (SCF), and the inflammatory markers monocyte chemoattractant protein 1 (MCP1), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNFα). Statistical analyses were performed using t-test and one-way analysis of variance with Bonferroni's post hoc test. RESULTS: Silibinin treatment for 1 week reduced RVSP and Fulton index. Treatment for 2 weeks reduced RVSP, Fulton index, % MT, and VOS, as well as downregulating the expression of CXCR4, SDF-1, and TNFα in pulmonary arteries. In contrast, treatment for 3 weeks failed to ameliorate PAH. The time-course study demonstrated that RVSP, Fulton index, % MT, and VOS gradually increased over time, with a decrease in the expression of CXCR4 and TNFα occurring after 2 weeks of PAH development. After 3 weeks, SDF-1, c-Kit, and SCF began to decrease and, after 5 weeks, MCP1 and IL-6 gradually accumulated. CONCLUSIONS: The CXCR4 inhibitor silibinin can ameliorate PAH, possibly through the suppression of the CXCR4/SDF-1 axis, until the point where PAH becomes a severe and irreversible condition. Silibinin results in reduced pulmonary arterial pressure and delays pulmonary arteriolar occlusion and pulmonary vascular remodeling.

Animal Disease Models and Patient-iPS-Cell-Derived In Vitro Disease Models for Cardiovascular Biology-How Close to Disease?

Currently, zebrafish, rodents, canines, and pigs are the primary disease models used in cardiovascular research. In general, larger animals have more physiological similarities to humans, making better disease models. However, they can have restricted or limited use because they are difficult to handle and maintain. Moreover, animal welfare laws regulate the use of experimental animals. Different species have different mechanisms of disease onset. Organs in each animal species have different characteristics depending on their evolutionary history and living environment. For example, mice have higher heart rates than humans. Nonetheless, preclinical studies have used animals to evaluate the safety and efficacy of human drugs because no other complementary method exists. Hence, we need to evaluate the similarities and differences in disease mechanisms between humans and experimental animals. The translation of animal data to humans contributes to eliminating the gap between these two. In vitro disease models have been used as another alternative for human disease models since the discovery of induced pluripotent stem cells (iPSCs). Human cardiomyocytes have been generated from patient-derived iPSCs, which are genetically identical to the derived patients. Researchers have attempted to develop in vivo mimicking 3D culture systems. In this review, we explore the possible uses of animal disease models, iPSC-derived in vitro disease models, humanized animals, and the recent challenges of machine learning. The combination of these methods will make disease models more similar to human disease.

Stem Cell Studies in Cardiovascular Biology and Medicine: A Possible Key Role of Macrophages.

Stem cells are used in cardiovascular biology and biomedicine, and research in this field is expanding. Two types of stem cells have been used in research: induced pluripotent and somatic stem cells. Stem cell research in cardiovascular medicine has developed rapidly following the discovery of different types of stem cells. Induced pluripotent stem cells (iPSCs) possess potent differentiation ability, unlike somatic stem cells, and have been postulated for a long time. However, differentiating into adult-type mature and functional cardiac myocytes (CMs) remains difficult. Bone marrow stem/stromal cells (BMSCs), adipose-derived stem cells (ASCs), and cardiac stem cells (CSCs) are somatic stem cells used for cardiac regeneration. Among somatic stem cells, bone marrow stem/stromal cells (BMSCs) were the first to be discovered and are relatively well-characterized. BMSCs were once thought to have differentiation ability in infarcted areas of the heart, but it has been identified that paracrine cytokines and micro-RNAs derived from BMSCs contributed to that effect. Moreover, vesicles and exosomes from these cells have similar effects and are effective in cardiac repair. The molecular signature of exosomes can also be used for diagnostics because exosomes have the characteristics of their origin cells. Cardiac stem cells (CSCs) differentiate into cardiomyocytes, smooth muscle cells, and endothelial cells, and supply cardiomyocytes during myocardial infarction by differentiating into newly formed cardiomyocytes. Stem cell niches and inflammatory cells play important roles in stem cell regulation and the recovery of damaged tissues. In particular, chemokines can contribute to the communication between inflammatory cells and stem cells. In this review, we present the current status of this exciting and promising research field.

Myogenic differentiation in atrium-derived adult cardiac pluripotent cells and the transcriptional regulation of GATA4 and myogenin on ANP promoter.

We established cardiac pluripotent stem-like cells from the left atrium (LA-PCs) of adult rat hearts. These cells could differentiate not only into beating myocytes but also into cells of other lineages, including adipocytes and endothelial cells in the methylcellulose-based medium containing interleukin-3 (IL-3), interleukin-6 (IL-6), and stem cell factor (SCF). In particular, IL-3 and SCF contributed to the differentiation into cardiac troponin I-positive cells. Notably, small population of LA-PCs coexpressed GATA4 and myogenin, which are markers specific to cardiomyocytes and skeletal myocytes, respectively, and could differentiate into both cardiac and skeletal myocytes. Therefore, we investigated the involvement of these two tissue-specific transcription factors in the cardiac transcriptional activity. Coexpression of GATA4 and myogenin synergistically activated GATA4-specific promoter of the atrial natriuretic peptide gene. This combinatorial function was shown to be dependant on the GATA site, but independent of the E-box. The results of chromatin immunoprecipitation and electrophoretic mobility shift assays suggested that myogenin bound to GATA4 on the GATA elements and the C-terminal Zn-finger domain of GATA4 and the N-terminal region of myogenin were required for this synergistic activation of transcription. Taken together, these two transcription factors could be involved in the myogenesis of LA-PCs.

Induced Pluripotent Stem Cell-Derived Cardiomyocytes with SCN5A R1623Q Mutation Associated with Severe Long QT Syndrome in Fetuses and Neonates Recapitulates Pathophysiological Phenotypes.

The SCN5A R1623Q mutation is one of the most common genetic variants associated with severe congenital long QT syndrome 3 (LQT3) in fetal and neonatal patients. To investigate the properties of the R1623Q mutation, we established an induced pluripotent stem cell (iPSC) cardiomyocyte (CM) model from a patient with LQTS harboring a heterozygous R1623Q mutation. The properties and pharmacological responses of iPSC-CMs were characterized using a multi-electrode array system. The biophysical characteristic analysis revealed that R1623Q increased open probability and persistent currents of sodium channel, indicating a gain-of-function mutation. In the pharmacological study, mexiletine shortened FPDcF in R1623Q-iPSC-CMs, which exhibited prolonged field potential duration corrected by Fridericia's formula (FPDcF, analogous to QTcF). Meanwhile, E4031, a specific inhibitor of human ether-a-go-go-related gene (hERG) channel, significantly increased the frequency of arrhythmia-like early after depolarization (EAD) events. These characteristics partly reflect the patient phenotypes. To further analyze the effect of neonatal isoform, which is predominantly expressed in the fetal period, on the R1623Q mutant properties, we transfected adult form and neonatal isoform SCN5A of control and R1623Q mutant SCN5A genes to 293T cells. Whole-cell automated patch-clamp recordings revealed that R1623Q increased persistent Na+ currents, indicating a gain-of-function mutation. Our findings demonstrate the utility of LQT3-associated R1623Q mutation-harboring iPSC-CMs for assessing pharmacological responses to therapeutic drugs and improving treatment efficacy. Furthermore, developmental switching of neonatal/adult Nav1.5 isoforms may be involved in the pathological mechanisms underlying severe long QT syndrome in fetuses and neonates.

TGF-beta superfamily regulates a switch that mediates differentiation either into adipocytes or myocytes in left atrium derived pluripotent cells (LA-PCS).

Many stem cell studies have focused on the subject of cell fate and the signal molecules that modulate the regulatory switches for a given differentiation pathway. Genome-wide screens for cell fate determination signals require a cell source that differentiates purely into a single cell type. From adult rat left atrium, we established LA-PCs that differentiates into cardiac/skeletal myocytes or adipocytes with almost 100% purity. In this study, we compared gene expression profiles of undifferentiated LA-PCs with those of differentiated cells [adipocytes (Adi) or cardiac/skeletal myocytes (Myo)] to identify the signals that set the regulatory switch for adipocyte or myocyte differentiation. Microarray analysis verified the feasibility of genome-wide screening by this method. Using a pathway analysis screen, we found that members of the TGF-beta superfamily signal transduction pathways modulate the adipocyte/myocyte differentiation switch. Further analysis determined that recombinant TGF-beta inhibits adipogenesis and induces myogenesis simultaneously in a dose-dependent manner. Moreover, noggin induces differentiation into fully developed beating cardiac myocytes in vitro. These results provided new insight into the molecules that modulate the differentiation switch and validated a screening method for their identification.

Silibinin Upregulates CXCR4 Expression in Cultured Bone Marrow Cells (BMCs) Especially in Pulmonary Arterial Hypertension Rat Model.

Previously we reported that silibinin ameliorated pulmonary arterial hypertension (PAH) in rat PAH models, possibly through the suppression of the CXCR4/SDF-1, until the point where PAH became a severe and irreversible condition. To further investigate how silibinin ameliorates PAH, we first attempted to clarify its effect on bone marrow cells (BMCs), since the CXCR4/SDF-1 axis is known to regulate stem cell migration and attachment in BM niches. Rat PAH models were established through a combination of a single subcutaneous injection of monocrotaline (MCT) and chronic hypoxic conditions (10% O2). BMCs were harvested and cultured, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and flow cytometry (FCM) were performed to investigate whether silibinin affected CXCR4 expression. Silibinin upregulated the gene expression of stem cell related markers CXCR4, SDF-1, SCF, and c-Kit, inflammatory markers IL-6 and TNFα, mesenchymal stem cell (MSC)-related markers CD44 and CD29, and the granulocyte/monocyte-macrophage marker CD14 in cultured BM in PAH rats, but not in normal rats, except CXCR4. FCM showed that silibinin increased the CXCR4-positive cell population in a granulocyte fraction of cultured BMCs. However, immunohistochemical (IHC) staining showed no significant change in CXCR4 expression in the BM of the tibias. These results suggest that silibinin increases the expression of CXCR4 in BM, and the increased CXCR4-positive cells could be granulocytes/monocyte-macrophages.

Involvement of CXCR4 in Normal and Abnormal Development.

CXC motif chemokine receptor type 4 (CXCR4) is associated with normal and abnormal development, including oncogenesis. The ligand of CXCR4 is stromal cell-derived factor (SDF), also known as CXC motif ligand (CXCL) 12. Through the SDF-1/CXCR4 axis, both homing and migration of hematopoietic (stem) cells are regulated through niches in the bone marrow. Outside of the bone marrow, however, SDF-1 can recruit CXCR4-positive cells from the bone marrow. SDF/CXCR4 has been implicated in the maintenance and/or differentiation of stemness, and tissue-derived stem cells can be associated with SDF-1 and CXCR4 activity. CXCR4 plays a role in multiple pathways involved in carcinogenesis and other pathologies. Here, we summarize reports detailing the functions of CXCR4. We address the molecular signature of CXCR4 and how this molecule and cells expressing it are involved in either normal (maintaining stemness or inducing differentiation) or abnormal (developing cancer and other pathologies) events. As a constituent of stem cells, the SDF-1/CXCR4 axis influences downstream signal transduction and the cell microenvironment.

High expression of CXCR4 and stem cell markers in a monocrotaline and chronic hypoxia-induced rat model of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a severe and fatal clinical syndrome. C-X-C chemokine receptor type 4 (CXCR4) is known to serve a key role in recruiting mesenchymal stem cells (MSCs) from the bone marrow. In the present study, a rat model of PAH induced by 5 weeks of chronic hypoxia and treatment with a single injection of monocrotaline (60 mg/kg) was used to investigate the involvement of CXCR4 in PAH. Successful establishment of the PAH model was confirmed by significant differences between the PAH and control groups in right ventricular systolic pressure, Fulton index, wall thickness, vascular occlusion score determined by immunohistochemical staining and the expression of inflammatory markers measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The expression of CXCR4 and other stem cell markers were compared in the PAH and control groups. RT-qPCR showed that the expression of CXCR4, SCF, c-Kit, and CD29, which are expressed in MSCs, was significantly higher in the PAH group. Immunohistochemical staining also showed that the numbers of CXCR4-, c-Kit- and CD90-positive cells were significantly higher in the PAH group. These results suggest that CXCR4 is involved in the pathogenesis of PAH and that stem cells may serve an important role in pulmonary vascular remodeling.

Therapeutic efficacy of valproic acid in a combined monocrotaline and chronic hypoxia rat model of severe pulmonary hypertension.

BACKGROUND: Pulmonary hypertension (PH) is a serious disease with poor prognosis. Reports show that cells in remodeled pulmonary arteries of PH patients have similar characteristics to cancer cells, such as exuberant inflammation, increased proliferation, and decreased apoptosis. An ideal strategy for developing PH therapies is to directly target pulmonary vascular remodeling. High levels of histone deacetylase (HDAC) expression and activity are found in certain cancers, and research has shown the potential of HDAC inhibitors in repressing tumor growth via anti-inflammatory and anti-proliferative effects. To date, little is known about the effectiveness of HDAC inhibitors against pulmonary vascular remodeling in severe PH. OBJECTIVE: To investigate whether class I HDAC inhibitors suppress or reverse the development of severe PH in rats. METHODS: Male Sprague-Dawley rats were injected with a single, subcutaneous dose of monocrotaline (60 mg/kg), and were exposed to chronic hypoxia to induce severe PH. Valproic acid, a class I HDAC inhibitor, was administered to rats daily via gastric gavage (300 mg/kg) in a PH prevention study (during the first 3 weeks) or a PH reversal study (from 3 to 5 weeks). At the end of experiment, hemodynamic indices were measured, ventricular hypertrophy indices were calculated and vascular remodeling phenotypes were analyzed. RESULTS: After 3 weeks exposure to a combined stimulation of monocrotaline and chronic hypoxia, rats exhibited a reduced body weight, elevated right ventricular systolic pressure, an increased Fulton index, right ventricle weight ratio, medial wall thickness and muscularized peripheral pulmonary arteries. These parameters for PH evaluation were exacerbated from 3 to 5 weeks. Daily administration of valproic acid therapy prevented and partially reversed the development of severe PH in rats, and decreased inflammation and proliferation in remodeled pulmonary arteries. CONCLUSION: These data show that class I HDAC inhibitors may be effective for treating severe PH.

Cardiomyocyte regeneration.

The heart was initially believed to be a terminally differentiated organ; once the cardiomyocytes died, no recovery could be made to replace the dead cells. However, around a decade ago, the concept of cardiac stem cells (CSCs) in adult hearts was proposed. CSCs differentiate into cardiomyocytes, keeping the heart functioning. Studies have proved the existence of stem cells in the heart. These somatic stem cells have been studied for use in cardiac regeneration. Moreover, recently, induced pluripotent stem cells (iPSCs) were invented, and methodologies have now been developed to induce stable cardiomyocyte differentiation and purification of mature cardiomyocytes. A reprogramming method has also been applied to direct reprogramming using cardiac fibroblasts into cardiomyocytes. Here, we address cardiomyocyte differentiation of CSCs and iPSCs. Furthermore, we describe the potential of CSCs in regenerative biology and regenerative medicine.

3D-culture system for heart regeneration and cardiac medicine.

3D cultures have gained attention in the field of regenerative medicine for their usefulness as in vitro model of solid tissues. Bottom-up technology to generate artificial tissues or organs is prospective and an attractive approach that will expand as the field of regenerative medicine becomes more translational. We have characterized c-kit positive cardiac stem cells after long-term cultures and established a 3D-nanoculture system using collagen scaffolds. By combining informatics-based studies, including proteomic analyses and microarrays, we sought to generate methods that modeled cardiac regeneration which can ultimately be used to build artificial hearts. Here, we describe the use of biodegradable beads or 3D cultures to study cardiac regeneration. We summarize recent work that demonstrates that, by using a combination of molecular analyses with 3D cultures, it is possible to evaluate concise mechanisms of solid tissue stem cell biology.

Cell shape and cardiosphere differentiation: a revelation by proteomic profiling.

Stem cells (embryonic stem cells, somatic stem cells such as neural stem cells, and cardiac stem cells) and cancer cells are known to aggregate and form spheroid structures. This behavior is common in undifferentiated cells and may be necessary for adapting to certain conditions such as low-oxygen levels or to maintain undifferentiated status in microenvironments including stem cell niches. In order to decipher the meaning of this spheroid structure, we established a cardiosphere clone (CSC-21E) derived from the rat heart which can switch its morphology between spheroid and nonspheroid. Two forms, floating cardiospheres and dish-attached flat cells, could be switched reversibly by changing the cell culture condition. We performed differential proteome analysis studies and obtained protein profiles distinct between spherical forms and flat cells. From protein profiling analysis, we found upregulation of glycolytic enzymes in spheroids with some stress proteins switched in expression levels between these two forms. Evidence has been accumulating that certain chaperone/stress proteins are upregulated in concert with cellular changes including proliferation and differentiation. We would like to discuss the possible mechanism of how these aggregates affect cell differentiation and/or other cellular functions.

ACVRL1 gene variant in a patient with vein of Galen aneurysmal malformation.

Although mutations in the RASA1 gene in vein of Galen aneurysmal malformation (VGAM) and an endoglin gene mutation in a VGAM patient with a family history of hereditary hemorrhagic telangiectasia (HHT) have been identified, most VGAM cases have no mutation in these genes. We sought to detect mutations in other genes related to HHT. We screened for mutations in RASA1 and three genes (endoglin, activin receptor-like kinase 1 (ACVRL1), encoding ALK1, and SMAD4) related to HHT in four VGAM patients. One variant (c.652 C>T p.R218W) in ACVRL1 was identified. Immunoblotting revealed that the ALK1-R218W protein could not promote SMAD1/5/8 phosphorylation by BMP9 stimulation. On the other hand, wild-type ALK1 could enhance the phosphorylation as expected. Furthermore, the transcriptional activation of ALK1-R218W was less efficient than that of wild-type ALK1. We identified 1 variant in ACVRL1 in a VGAM patient. These findings suggest that the ACVRL1 variant-R218W may be associated with the pathogenesis of VGAM.

Stem cells for cardiac regeneration and possible roles of the transforming growth factor-ß superfamily.

Abstract Heart failure is a leading cause of death worldwide. Studies of stem cell biology are essential for developing efficient treatments. Recently, we established and characterized c-kit-positive cardiac stem cells from the adult rat heart. Using a MethoCult culture system with a methyl-cellulose-based medium, stem-like left-atrium-derived pluripotent cells could be regulated to differentiate into skeletal/cardiac myocytes or adipocytes with almost 100% purity. Microarray and pathway analyses of these cells showed that transforming growth factor-ß1 (TGF-ß1) and noggin were significantly involved in the differentiation switch. Furthermore, TGF-ß1 may act as a regulator for this switch because it simultaneously inhibits adipogenesis and activates myogenesis in a dose-dependent manner. However, the effect of TGF-ß varies with developmental stage, dosage, and timing of treatment. In the present review, the findings of recent studies, in particular the use of c-kit-positive cardiac stem cells, are discussed. The effects of the TGF-ß superfamily on differentiation, especially on adipogenesis and/or myogenesis, have important implications for future regenerative medicine.

Prospective in vitro models of channelopathies and cardiomyopathies.

An in vitro heart disease model is a promising model used for identifying the genes responsible for the disease, evaluating the effects of drugs, and regenerative medicine. We were interested in disease models using a patient-induced pluripotent stem (iPS) cell-derived cardiomyocytes because of their similarity to a patient's tissues. However, as these studies have just begun, we would like to review the literature in this and other related fields and discuss the path for future models of molecular biology that can help to diagnose and cure diseases, and its involvement in regenerative medicine. The heterogeneity of iPS cells and/or differentiated cardiomyocytes has been recognized as a problem. An in vitro heart disease model should be evaluated using molecular biological analyses, such as mRNA and micro-RNA expression profiles and proteomic analysis.

Adult cardiac-derived stem cells: differentiation and survival regulators.

At present, heart failure is one of the most concerning diseases worldwide. To develop efficient treatments, it is necessary to gain a better understanding of the biological characteristics of stem cells in the heart. We recently established and characterized c-kit-positive cardiac stem cells obtained from adult rats. Moreover, we established left atrium-derived pluripotent cells that can differentiate either into skeletal/cardiac myocytes or adipocytes in a methylcellulose-based Methocult medium with almost 100% purity. Microarray and signaling pathway analyses showed that transforming growth factor (TGF)-ß is a key molecule in the regulation of the differentiation switch. Indeed, TGF-ß1 simultaneously inhibits adipogenesis and activates myogenesis in a dose-dependent manner. However, the effect of TGF-ß varies with the developmental stage, dosage, and timing of the treatment.

Proteomic comparison of spherical aggregates and adherent cells of cardiac stem cells.

BACKGROUND: Previously published papers showed that cardiac stem cells (CSCs) form (cardio)sphere. However, recent studies questioned the significance of the sphere-formation as one of the characteristics of CSCs. We isolated c-kit-positive cardiac stem cells, cultured as bulk (CSC-BC) and characterized them previously. Among them, CSC-BC21 formed an extraordinary number of spheres. Using a clone derived from this bulk culture, we investigated the effect of sphere-formation on differentiation and performed proteomics analysis comparing two statuses, cardiosphere and dish substrate attachment. METHODS: We performed sphere-forming assay to compare the sphere-forming ability among CSC-BCs. The cloned cells from CSC-BC21, which had distinct sphere-forming ability, were cultured in a differentiation medium (DM) to induce cardiac myocyte differentiation. We performed RT-PCR analysis to investigate if cardiosphere-formation affects cardiac myocyte gene expression level. Furthermore, proteome analysis was performed to compare floating cardiosphere (flCS) and dish-attached cardiosphere-derived cells (daCS). RESULTS: One of the cloned cells, CSC-21E expressed higher troponin I message than CSC-BC21. Moreover, the message level of troponin I was enhanced when they had experienced cardiosphere prior to the treatment of myocyte differentiation medium. The change from flCS to daCS accompanied up-regulation of chaperones and down regulation of glycolytic and other metabolic enzymes. Calreticulin and Hsp 90 were among the up-regulated chaperons. Calreticulin is known to be an essential component of cardiogenesis. CONCLUSION: These results suggest that the switch from aggregated sphere to the cell attachment, is important for advancing the cardiac cell differentiation.

Micro and nano-scale in vitro 3D culture system for cardiac stem cells.

Despite the success to prevent or limit cardiovascular diseases, the restoration of the function of a damaged heart remains a formidable challenge. Cardiac stem cells (CSCs), with the capacity to differentiate into cardiomyocytes, hold great potential as a source of cells for regenerative medicine. A major challenge facing the clinical application of differentiated CSCs, however, is theability to generate sufficient numbers of cells with the desired phenotype. We previously established cell lines of CSCs using a c-kit antibody from adult rat hearts for use in regenerative medicine. C-kit -positive cardiac cells are well recognized as CSCs and have the potential to differentiate into cardiomyocytes. Here, before implant these cells in vivo, we first developed three-dimensional culture system (3D) using micro- and nano-scaled material. Sheets of poly(glycolic acid) (PGA) were fabricated by electrospinning. Composites of collagen-PGA were prepared that contained 0, 1.5, 3 or 6 mg of electrospun PGA nanofibers. The nanofibers were added as a sheet that formed a layer within the collagen sponge. The sponges were freeze-dried and then dehydrothermally crosslinked. A scanning electron microscopy (SEM)-based analysis of the surface of the sponges demonstrated a uniform collagenous structure regardless of the amount of PGA nanofibres included. The PGA nanofibers significantly enhanced the compressive strength of the collagen sponge. More CSCs attached to the collagen sponge incorporating 6 mg of PGA nanofibers than the sponge without PGA nanofibers. The attachment and proliferation of CSCs in the 3D culture was enhanced by incubation in a bioreactor perfusion system compared with 3D static and two-dimensional (2D; i.e. tissue culture plates) culture systems. The use of micro- and nano-scale materials in the fabrication of composites together with a 3D culture system is a very promising way to promote the culture of stem cells. (c) 2009 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.