Morphology Of Stem Cells Research Articles

Mesenchymal stem cells isolated from both distal femurs of patients with unilateral trauma or osteoarthritis of the knee exhibit similar in-vitro ability of bone formation

BackgroundBone fractures are a common cause of hospital admission. Currently, treatment consists of conservative regimens or operation. However, regenerative medicine introduces a possible new addition to established treatments. Evidence suggests that application of autologous mesenchymal stem cells can enhance bone regeneration, by differentiating into osteoblasts.This study investigates whether mesenchymal stem cells, isolated from bone marrow in sites of trauma or osteoarthritis, exhibit reduced proliferation and osteogenic differentiation in-vitro, compared to stem cells isolated from non-traumatic and non-osteoarthritic sites. If these pathologies are detrimental to the quality, clinicians should prioritize bone marrow from unafflicted sites. Methods17 patients were enrolled. 7 had recent unilateral trauma to the knee, requiring arthroscopy. 10 had x-ray verified unilateral osteoarthritis of the knee and were scheduled for arthroplasty. Stem cells were isolated from bone marrow aspirated perioperatively from both distal femurs. In-vitro osteogenic activity was assessed through alkaline phosphatase measurement, RNA-expression and alizarin red staining. Proliferation was measured using a growth curve. Results29 out of 34 primary cultures were successful, forming colonies with characteristic stem cell-morphology. There was no difference in mononuclear cell yield of aspirates or stem cell-yield from primary culture between non-osteoarthritic and arthritic knees or non-traumatic and traumatic knees. There was no significant difference in in-vitro osteogenic capability or proliferation. ConclusionOur findings suggest that stem cells from sites afflicted by osteoarthritis or trauma can be utilized for bone regeneration with identical results as MSCs isolated from non-traumatic and non-osteoarthritic sites. However, clinical studies are needed to confirm this assumption.

GMP-compatible and xeno-free cultivation of mesenchymal progenitors derived from human-induced pluripotent stem cells

BackgroundHuman mesenchymal stem cells are a strong candidate for cell therapies owing to their regenerative potential, paracrine regulatory effects, and immunomodulatory activity. Yet, their scarcity, limited expansion potential, and age-associated functional decline restrict the ability to consistently manufacture large numbers of safe and therapeutically effective mesenchymal stem cells for routine clinical applications. To overcome these limitations and advance stem cell treatments using mesenchymal stem cells, researchers have recently derived mesenchymal progenitors from human-induced pluripotent stem cells. Human-induced pluripotent stem cell-derived progenitors resemble adult mesenchymal stem cells in morphology, global gene expression, surface antigen profile, and multi-differentiation potential, but unlike adult mesenchymal stem cells, it can be produced in large numbers for every patient. For therapeutic applications, however, human-induced pluripotent stem cell-derived progenitors must be produced without animal-derived components (xeno-free) and in accordance with Good Manufacturing Practice guidelines.MethodsIn the present study we investigate the effects of expanding mesodermal progenitor cells derived from two human-induced pluripotent stem cell lines in xeno-free medium supplemented with human platelet lysates and in a commercial high-performance Good Manufacturing Practice-compatible medium (Unison Medium).ResultsThe results show that long-term culture in xeno-free and Good Manufacturing Practice-compatible media somewhat affects the morphology, expansion potential, gene expression, and cytokine profile of human-induced pluripotent stem cell-derived progenitors but supports cell viability and maintenance of a mesenchymal phenotype equally well as medium supplemented with fetal bovine serum.ConclusionsThe findings support the potential to manufacture large numbers of clinical-grade human-induced pluripotent stem cell-derived mesenchymal progenitors for applications in personalized regenerative medicine.Graphical abstract

MicroRNA changes of bone marrow-derived mesenchymal stem cells differentiated into neuronal-like cells by Schwann cell-conditioned medium.

Bone marrow-derived mesenchymal stem cells differentiate into neurons under the induction of Schwann cells. However, key microRNAs and related pathways for differentiation remain unclear. This study screened and identified differentially expressed microRNAs in bone marrow-derived mesenchymal stem cells induced by Schwann cell-conditioned medium, and explored targets and related pathways involved in their differentiation into neuronal-like cells. Primary bone marrow-derived mesenchymal stem cells were isolated from femoral and tibial bones, while primary Schwann cells were isolated from bilateral saphenous nerves. Bone marrow-derived mesenchymal stem cells were cultured in unconditioned (control group) and Schwann cell-conditioned medium (bone marrow-derived mesenchymal stem cell + Schwann cell group). Neuronal differentiation of bone marrow-derived mesenchymal stem cells induced by Schwann cell-conditioned medium was observed by time-lapse imaging. Upon induction, the morphology of bone marrow-derived mesenchymal stem cells changed into a neural shape with neurites. Results of quantitative reverse transcription-polymerase chain reaction revealed that nestin mRNA expression was upregulated from 1 to 3 days and downregulated from 3 to 7 days in the bone marrow-derived mesenchymal stem cell + Schwann cell group. Compared with the control group, microtubule-associated protein 2 mRNA expression gradually increased from 1 to 7 days in the bone marrow-derived mesenchymal stem cell + Schwann cell group. After 7 days of induction, microRNA analysis identified 83 significantly differentially expressed microRNAs between the two groups. Gene Ontology analysis indicated enrichment of microRNA target genes for neuronal projection development, regulation of axonogenesis, and positive regulation of cell proliferation. Kyoto Encyclopedia of Genes and Genomes pathway analysis demonstrated that Hippo, Wnt, transforming growth factor-beta, and Hedgehog signaling pathways were potentially associated with neural differentiation of bone marrow-derived mesenchymal stem cells. This study, which carried out successful microRNA analysis of neuronal-like cells differentiated from bone marrow-derived mesenchymal stem cells by Schwann cell induction, revealed key microRNAs and pathways involved in neural differentiation of bone marrow-derived mesenchymal stem cells. All protocols were approved by the Animal Ethics Committee of Institute of Radiation Medicine, Chinese Academy of Medical Sciences on March 12, 2017 (approval number: DWLI-20170311).

Quantitative Investigation of the Process Parameters of Electrohydrodynamic Direct-Writing and Their Effects on Fiber Morphology and Cell Adhesion

Electrohydrodynamic direct-writing (EHD-DW), a near-field electrospinning process, is considered as a promising technique to generate nanofibers in a non-contact, continuous, and controllable manner. However, the effect of process parameters on EHD-DW fibers' structures and the effect of fibers' structures on their properties have not yet been fully investigated. In this work, polycaprolactone (PCL) solution was used to fabricate EHD-DW fibers for investigating the process-structure-property correlations of EHD-DW. Three working modes of the electrospinning process and their corresponding regions in the process parameter space were studied. The surface roughness of EHD-DW fibers fabricated at different process parameters was measured. The impact of the surface roughness on the number and morphology of human mesenchymal stem cells (hMSCs) attached to the EHD-DW fibers was investigated. The results suggest that the surface roughness, which can be controlled by the process parameters of EHD-DW, can significantly affect the attachment of hMSCs. The cell density upon seeding can be largely improved by tuning the process parameters to achieve a favorable surface roughness. This work revealed the process-structure-property correlations of EHD-DW technology. Guidelines for controlling the morphology and cellular adhesion of EHD-DW fibers are provided based on the results of this study.

The Effect of Scaffold Modulus on the Morphology and Remodeling of Fetal Mesenchymal Stem Cells.

Hydrogel materials have been successfully used as matrices to explore the role of biophysical and biochemical stimuli in directing stem cell behavior. Here, we present our findings on the role of modulus in guiding bone marrow fetal mesenchymal stem cell (BMfMSC) fate determination using semi-synthetic hydrogels made from PEG-fibrinogen (PF). The BMfMSCs were cultivated in the PF for up to 2 weeks to study the influence of matrix modulus (i.e., cross-linking density of the PF) on BMfMSC survival, morphology and integrin expression. Both two-dimensional (2D) and three-dimensional (3D) culture conditions were employed to examine the BMfMSCs as single cells or as cell spheroids. The hydrogel modulus affected the rate of BMfMSC metabolic activity, the integrin expression levels and the cell morphology, both as single cells and as spheroids. The cell seeding density was also found to be an important parameter of the system in that high densities were favorable in facilitating more cell-to-cell contacts that favored higher metabolic activity. Our findings provide important insight about design of a hydrogel scaffold that can be used to optimize the biological response of BMfMSCs for various tissue engineering applications.

183 Generation of porcine embryonic stem cell lines derived from nuclear transfer embryos reconstructed with induced pluripotent stem cells

To establish a porcine embryonic stem (ES) cell line that not only maintains self-renewing capacity but also exhibits pluripotency [Haraguchi et al. 2012 J. Reprod. Dev. 58, 707-716], 6 synthetic porcine RNAs (Oct4, Sox2, Klf4, c-Myc, Nanog, and Lin28) were chemically transfected into outgrowth cultured cells derived from the inner cell mass of in vitro-produced porcine embryos. Subsequently, cells grew as compact, dome-shaped colonies displaying alkaline phosphatase activity and were cultured for more than 20 passages. Although 13 candidate cell lines were generated (13/43, 30%), none formed teratomas after injection of the cells into SCID (sever combined immunodeficiency) mice. We also observed that when transfection of the exogeneous RNAs was discontinued, the cells no longer maintained a stem cell morphology and began to differentiate (13/13, 100%). This suggests that continuous expression of exogenous reprogramming factors is necessary to maintain induced pluripotency in the pig. Next, we used cloned embryos reconstructed with porcine induced pluripotent stem cells (piPSC), which were created using a recombinant lentivirus expression vector carrying 6 mouse reprogramming factor genes (the same as above) and green fluorescent protein (GFP) (Fukuda et al. 2017 J. Cell Biochem. 118, 537-553]. The piPSC were dispersed to a single cell suspension and electrically fused to cytoplasts prepared following enucleation of in vitro-matured zona-free metaphase II-arrested oocytes. A second cytoplast was then fused to the first reconstruct (double cytoplast nuclear transfer). Reconstructs were electrically activated and cultured in microwells with porcine zygote medium-3 (PZM3). After 5 days, reconstructed embryos developed to GFP-positive blastocysts (10/93, 11%) and 4- to 8-cell stages (25/93, 27%). The blastocysts (10) and 4- to 8-cell-stage embryos (25) were transferred onto mouse embryonic fibroblast feeder cells for outgrowth culture in FCS-based ES cell medium supplemented with 2% polyvinylpyrrolidone. After 24h, the medium was changed to piPSC medium containing CHIR99021, PD0325901, thiazovivin, and GF-109203x. Embryos attached to the feeder cells began to outgrow (8/10 of blastocysts and 6/25 of 4- to 8-cell-stage embryos). To date, 3 ES-like cell lines have been established from blastomeres of embryos (3/25, 12%) but not from blastocysts (0/10, 0%). They show GFP fluorescence and have been maintained continuously in culture for more than 20 passages without any overt changes in morphology. These results suggest that the constant expression of reprogramming factors and the use of combinations of specific small molecule inhibitors largely contribute to the establishment of pluripotent cells in the pig. Further characterisation of the cells is ongoing, including methylation status of the X chromosome and the capacity for in vivo differentiation.

The possibilities of Cell-IQ phase-contrast microscopy for a continuous real-time observation of multipotent mesenchymal stromal cell culture

Continuous monitoring of multipotent mesenchymal stromal cells (MMSCs) is a promising tool that could be used in cellular biology, environmental research and biotechnology to study in vitro real-time morphology and behavioural response of stem cells alone, as well at contacts with other cells and for controlling a sustainable production of scaffolds for tissue engineering. The in vitro processes of human adipose-derived MMSCs (hAMMSCs) morphology, motility, cell division, and secretion were studied by means of Cell-IQ v2 MLF (CM Technologies Oy, Finland) integrated phase-contrast microscopic platform for a continuous real-time surveillance imaging of living cells. 70 μL suspension (5×104 viable karyocytes) of the cells was applied into the center of the wells of 12-well plastic plates, and cells were allowed to adhere in a moist chamber for 120 min. Nonadherent cells were washed, and the wells were carefully filled with 1.5 mL of a nutrient medium DMEM/F12 (1 : 1) without osteogenic additions. Cells were cultured for 14 days at 100% humidity in a 5% carbondioxide atmosphere at 37°C until a monolayer formation. Digital images of cell culture growth were captured every 45 min. The cells were positively stained with alizarin red (osteoblasts), alcian blue (chondrocytes), or oil red (adipocytes). More than 95% of attached cells expressed CD73, CD90, and CD105 markers, mainly. Thus, the cells corresponded to the morphological criteria of MMSCs. The Cell-IQ system has allowed establishing 182 μm/h linear velocity of free (until the cell contacts) motility of spindle or fibroblast-like cells. Maximum number of cells achieved 136 cells per field of view; 13-24 % of cells divided each 1-3 h until a monolayer was formed. Chemokine cooperation between hAMMSCs and poor macrophages intermixture was proposed. Cell-IQ could be useful for in vitro real-time imaging of cell subpopulations and/or their response to biodegradable scaffolds and/or (micro)environmental factors.DOI: http://dx.doi.org/10.5755/j01.erem.74.3.20548

Isolation and characterization of stem cells from differentially degenerated human lumbar zygapophyseal articular cartilage

The present study aimed to verify the presence of stem cells with multilineage differentiation potential in human lumbar zygapophyseal articular cartilage (LZAC) and to compare the chondrogenic potential of cells obtained from differentially degenerated articular cartilage samples. Surgically obtained human lumbar zygapophyseal joint tissues were classified into the normal, mildly degenerated and severely degenerated groups, according to their pathological characteristics. Primary chondrocytes from these groups were cultured, and stem cells were selected using a monoclonal cell culture method. Differences in stem cell morphology between the three groups were observed using inverted microscopy and phalloidin staining. In addition, stem cell chondrogenic potential was determined through induced differentiation and cellular staining. Gene and protein expression levels of the chondrogenic‑specific markers aggrecan, collagen type‑II and SRY‑related high‑mobility‑group box9 were determined using reverse transcription‑quantitative polymerase chain reaction and western blotting. The clonogenic ability of stem cells in the three groups was determined using a clonogenic assay. It was revealed that stem cells with multilineage differentiation potential were isolated from all three cartilage groups; however, the cells obtained from severely degenerated articular cartilage resulted in severe fibrosis, whilst those obtained from mildly degenerated articular cartilage possessed stronger chondrogenic and clonogenic abilities. Taken together, stem cells with multilineage differentiation potential and clonal properties were identified in human LZAC, and these characteristics were more prominent in mildly degenerated as compared with severely degenerated articular cartilage.

The effect of larger than cell diameter polylactic acid surface patterns on osteogenic differentiation of rat dental pulp stem cells.

Topography of the scaffold is one of the most important factors defining the quality of artificial bone. However, the production of precise micro- and nano-structured scaffolds, which is known to enhance osteogenic differentiation, is expensive and time-consuming. Meanwhile, little is known about macro-patterns (larger than cell diameter) effect on cell fate, while this kind of structures would significantly facilitate the manufacturing of artificial skeleton. Therefore, this research is focused on polylactic acid scaffold's macro-pattern impact on rat's dental pulp stem cells (DPSCs) morphology, proliferation, and osteogenic differentiation. For this study, two types of scaffolds were 3D printed: wavy and porous. Wavy scaffolds consisted of 188 μm wide joined threads, meaning that cells might have been curved on the filament as well as compressed in the groove. Porous scaffolds were designed to avoid groove formation and consisted of 500 μm threads, arranged in the woodpile manner, forming 300 μm diameter pores. We found that both macro-surfaces influenced DPSC morphology compared to control. As a consequence, enhanced DPSC proliferation and increased osteogenic differentiation potential was registered in cells grown on these scaffolds. Finally, our results showed that the construction of an artificial bone did not necessarily require the precise structuring of the scaffold, because both types of macro-topographic PLA scaffolds were sufficient enough to induce spontaneous DPSC osteogenic differentiation. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 174-186, 2019.

Analysis of Different Computational Techniques for Calculating the Polarizability Tensors of Stem Cells with Realistic Three-Dimensional Morphologies.

Recently, the National Institute of Standards and Technology has developed a database of three-dimensional (3D) stem cell morphologies grown in ten different scaffolds to study the effect of the cells' environments on their morphologies. The goal of this work is to study the polarizability tensors of these stem cell morphologies, using three independent computational techniques, to quantify the effect of the environment on the electric properties of these cells. We show excellent agreement between the three techniques, validating the accuracy of our calculations. These computational methods allowed us to investigate different meshing resolutions for each stem cell morphology. After validating our results, we use a fast and accurate Pad' approximation formulation to calculate the polarizability tensors of stem cells for any contrast value between their dielectric permittivity and the dielectric permittivity of their environment. We also performed statistical analysis of our computational results to identify which environment generates cells with similar electric properties. The computational analysis and the results reported herein can be used for shedding light on the response of stem cells to electric fields in applications such as dielectrophoresis and electroporation and for calculating the electric properties of similar biological structures with complex 3D shapes.

Cytotoxicity Evaluation of Ammonia-Modified Graphene Oxide Particles in Lung Cancer Cells and Embryonic Stem Cells

Potential toxicity of graphene oxide (GO) is a subject of increasing research interest in the recent years. Here, we have evaluated the cytotoxicity of ammonia-modified GO (GO-NH2) and pristine GO particles in human lung cancer cells, A549 and embryonic stem cells, Lep3 exposed to different particles concentrations (0.1, 1, 10, 20, and 50 μg/ml) for different times (24 and 48h). Compared with GO, GO-NH2 particles possessed smaller size, positive surface charge and higher thickness. An increased propensity to aggregation in cell cultures was also found for GO-NH2 particles. Cytotoxicity evaluation revealed that GO-NH2 particles are more toxic than pristine GO. Applied at concentrations of 10, 20 and 50 μg/ml for 24h they affect significantly cell morphology of viable embryonic stem cells whereas human lung cancer A549 cells seem to be relatively more resistant to short-time exposure. After 48h exposure however cell proliferation of A549 cells was strongly suppressed in a dose-dependent manner while the proliferation ability of embryonic stem cells was not affected. These results suggested that both GO particles exert different degree of cytotoxicity which is time, dose and cell dependent. In general, ammonia-modified GO particles are more toxic than the pristine GO which should be taken into account for future biomedical applications.

Approaches to improve integration and regeneration of an ex vivo derived temporomandibular joint disc scaffold with variable matrix composition.

Due to their natural biochemical and biomechanical characteristics, using ex vivo tissues as platforms for guided tissue regeneration has become widely accepted, however subsequent attachment and integration of these constructs in vivo is often overlooked. A decellularized porcine temporomandibular joint (TMJ) disc has shown promise as a scaffold to guide disc regeneration and preliminary work has shown the efficacy of surfactant (SDS) treatment within the fibrocartilaginous disc to remove cellular components. The majority of studies focus on the intermediate region of the disc (or disc proper). Using this approach, inherent attachment tissues can be maintained to improve construct stability and integration within the joint. Unlike human disc attachment tissue, the porcine attachment tissues have high lipid content which would require a different processing approach to remove immunogenic components. In order to examine the effect of delipidation on the attachment tissue properties, SDS and two organic solvent mixtures (acetone/ethanol and chloroform/methanol) were compared. Lipid and cellular solubilization, ECM alteration, and seeded human mesenchymal stem cell (MSC) morphology and viability were assessed. Quantitative analysis showed SDS treatments did not effectively delipidate the attachment tissues and cytotoxicity was noted toward MSC in these regions. Acetone/ethanol removed cellular material but not all lipids, while chloroform/methanol removed all visible lipid deposits but residual porcine cells were observed in histological sections. When a combination of approaches was used, no residual lipid or cytotoxicity was noted. Preparing a whole TMJ graft with a combined approach has the potential to improve disc integration within the native joint environment.

Isolation and culture of putative mesenchymal stem cells from equine umbilical cord Wharton’s jelly

Despite major progress and knowledge related to the application of adult stem cells, finding alternative sources for bone marrow MSCs has remained a challenge in both humans and animals. In the current study, two protocols namely sequential enzymatic tissue digestion and tissue explant techniques were tried for successful establishment of MSC culture. Umbilical tissues were isolated each time of foaling from five sequential foalings of Marwari mares. Total cell yield, their growth potential and cryopreservation potential were studied. Adherent cell colonies could be established using both isolation methods. Both the cell populations yielded from different protocols performed similarly in terms of population doubling and CFU number value. Additionally, the cells proliferated vigourously and displayed a similar morphology of mesenchymal stem cells. The MSCs were plastic adherent, colonogenic and their morphology was polygonal and fibroblast like. During the proliferation, the cells exhibited density dependent inhibition; analysis of microbial contamination from bacteria, mycoplasma and fungi were negative; the population doubling time of the MSCs isolated was 34.8 h and 40.2 h in enzymatic treatment and tissue explant methods respectively, and diploid chromosome number of the cells was 64, and the diploid frequency was higher than 80%. In conclusion, this study reveals that both the techniques proved to be non-invasive, efficient, simple and quick for isolation and establishment of MSC culture of extra embryonic tissues from equines.

Generation of induced pluripotent stem cells by a polycistronic lentiviral vector in feeder- and serum- free defined culture.

Induced pluripotent stem cells (iPSCs) have great potentials for regenerative medicine. However, serious concerns such as the use of the viral-mediated reprogramming strategies and exposure of iPSCs to animal products from feeder cells and serum-containing medium have restricted the application of iPSCs in the clinics. Therefore, the generation of iPSCs with minimal viral integrations and in non-animal sourced and serum-free medium is necessary. In this report, a polycistronic lentiviral vector carrying Yamanaka's factors was used to reprogram mouse fibroblasts into iPSCs in feeder- and xeno-free culture environment. The generated iPSCs exhibited morphology and self-renewal properties of embryonic stem cells (ESCs), expression of specific pluripotent markers, and potentials to differentiate into the three-major distinct specialized germ layers in vitro. The iPSCs were also shown to have the potential to differentiate into neural precursor and neurons in culture, with greater than 95% expression of nestin, Pax6 and βIII-tubulin. This body of work describes an alternative method of generating iPSCs by using polycistronic lentiviral vector that may minimize the risks associated with viral vector-mediated reprogramming and animal derived products in the culture media.

Recent Advances in Nanocomposites Based on Aliphatic Polyesters: Design, Synthesis, and Applications in Regenerative Medicine

In the last decade, biopolymer matrices reinforced with nanofillers have attracted great research efforts thanks to the synergistic characteristics derived from the combination of these two components. In this framework, this review focuses on the fundamental principles and recent progress in the field of aliphatic polyester-based nanocomposites for regenerative medicine applications. Traditional and emerging polymer nanocomposites are described in terms of polymer matrix properties and synthesis methods, used nanofillers, and nanocomposite processing and properties. Special attention has been paid to the most recent nanocomposite systems developed by combining alternative copolymerization strategies with specific nanoparticles. Thermal, electrical, biodegradation, and surface properties have been illustrated and correlated with the nanoparticle kind, content, and shape. Finally, cell-polymer (nanocomposite) interactions have been described by reviewing analysis methodologies such as primary and stem cell viability, adhesion, morphology, and differentiation processes.

The fabrication of biomineralized fiber-aligned PLGA scaffolds and their effect on enhancing osteogenic differentiation of UCMSC cells.

The key factor of scaffold design for bone tissue engineering is to mimic the microenvironment of natural bone extracellular matrix (ECM) and guide cell osteogenic differentiation. The biomineralized fiber-aligned PLGA scaffolds (a-PLGA/CaPs) was developed in this study by mimicking the structure and composition of native bone ECM. The aligned PLGA fibers was prepared by wet spinning and then biomineralized via an alternate immersion method. Introduction of a bioceramic component CaP onto the PLGA fibers led to changes in surface roughness and hydrophilicity, which showed to modulate cell adhesion and cell morphology of umbilical cord mesenchymal stem cells (UCMSCs). It was found that organized actin filaments of UCMSCs cultured on both a-PLGA and a-PLGA/CaP scaffolds appeared to follow contact guidance along the aligned fibers, and those cells grown on a-PLGA/CaP scaffolds exhibited a more polarized cellular morphology. The a-PLGA/CaP scaffold with multicycles of mineralization facilitated the cell attachment on the fiber surfaces and then supported better cell adhesion and contact guidance, leading to enhancement in following proliferation and osteogenic differentiation of UCMSCs. Our results give some insights into the regulation of cell behaviors through design of ECM-mimicking structure and composition and provide an alternative wet-spun fiber-aligned scaffold with HA-mineralized layer for bone tissue engineering application.

Low Cell-Matrix Adhesion Reveals Two Subtypes of Human Pluripotent Stem Cells

SummaryWe show that a human pluripotent stem cell (hPSC) population cultured on a low-adhesion substrate developed two hPSC subtypes with different colony morphologies: flat and domed. Notably, the dome-like cells showed higher active proliferation capacity and increased several pluripotent genes’ expression compared with the flat monolayer cells. We further demonstrated that cell-matrix adhesion mediates the interaction between cell morphology and expression of KLF4 and KLF5 through a serum response factor (SRF)-based regulatory double loop. Our results provide a mechanistic view on the coupling among adhesion, stem cell morphology, and pluripotency, shedding light on the critical role of cell-matrix adhesion in the induction and maintenance of hPSC.

Morphology and contractile gene expression of adipose-derived mesenchymal stem cells in response to short-term cyclic uniaxial strain and TGF-β1.

Previous studies have shown smooth muscle induction in adipose-derived mesenchymal stem cells (ASCs) caused by long-term cyclic stretch. Here we examined the capability of the short-term straining with time steps of 4, 8, 16 and 24 h alone or combined with TGF-β1 on smooth muscle induction of rabbit ASCs. Alterations in cell morphology were quantified through the cell shape index and orientation angle, and expression levels of α-SMA, SM22-α, h-caldesmon and calponin3 markers were examined using the real-time polymerase chain reaction (PCR) method. Moreover, F-actin cytoskeleton organization was observed by fluorescence staining. Mechanical strain either alone or combined with growth factor treatment caused significant up-regulation of both early and intermediate smooth muscle cells (SMCs) specific markers during the initial hours of stimulation peaking in 8 to 16 h. Furthermore, gradual alignment of cells perpendicular to the strain direction during loading time, and cell elongation resembling contractile SMC phenotype, together with alignment and reorganization of F-actin fibers were observed. Considering previously reported protein up-regulation in following days of straining, the effects of short-term cyclic stretch on smooth muscle induction of ASCs were revealed which can be helpful in achieving functional contractile SMCs through synergistic mechano-chemical regulation of ASCs as an appealing cell source for vascular tissue engineering.

MBRS-46. JERANTININE: A NOVEL TUMOUR-SPECIFIC ALKALOID FOR THE TREATMENT OF PAEDIATRIC MEDULLOBLASTOMA

Medulloblastoma is the most common malignant paediatric brain tumour. Standard treatment includes a combination of surgery, craniospinal radiotherapy and adjuvant chemotherapy. The chemotherapy agent Vincristine causes potent inhibition of tumour growth, however, high levels of toxicity and intrinsic drug resistance mechanisms, limit its clinical use. Jerantinine is a novel aspidosperma indole alkaloid that, like Vincristine, works by destabilising microtubule growth and hence blocks cell division. Here, we tested the hypothesis that Jerantinine would be an effective alternative to Vincristine in medulloblastoma. Jerantinine, like Vincristine, caused a dose-dependent decrease in cell viability and colony formation in medulloblastoma cell lines. Cytotoxicity of Vincristine could be potentiated with the ABCB1 inhibitor Verapamil, however there was no potentiation of Jerantinine’s activity indicating that Jerantinine functions independently of intrinsic ABCB1 expression and is able to circumvent this drug resistance mechanism. The effect of Jerantinine treatment was also assessed in 3D spheroid culture. Spheroid viability decreased with increasing concentrations of Jerantinine, as measured using the CellTiter-Glo® 3D cell viability assay. Automated imaging analysis (Celigo® cytometer) also showed an increase in cell death at a range of Jerantinine concentrations over 48 hours. Confocal microscopy showed comparable effects of Jerantinine and Vincristine in medulloblastoma cell lines, including cytoskeletal defects, inhibition of tubulin polymerisation and defective cell division. However unlike Vincristine, Jerantinine caused no visible disruption to cellular morphology of neural stem cells. These results suggest that Jerantinine is an effective, tumour-specific alternative to Vincristine in the treatment of paediatric medulloblastoma.

Substrate micropatterns produced by polymer demixing regulate focal adhesions, actin anisotropy, and lineage differentiation of stem cells

Stem cells are adherent cells whose multipotency and differentiation can be regulated by numerous microenvironmental signals including soluble growth factors and surface topography. This study describes a simple method for creating distinct micropatterns via microphase separation resulting from polymer demixing of poly(desaminotyrosyl-tyrosine carbonate) (PDTEC) and polystyrene (PS). Substrates with co-continuous (ribbons) or discontinuous (islands and pits) PDTEC regions were obtained by varying the ratio of PDTEC and sacrificial PS. Human mesenchymal stem cells (MSCs) cultured on co-continuous PDTEC substrates for 3 days in bipotential adipogenic/osteogenic (AD/OS) induction medium showed no change in cell morphology but exhibited increased anisotropic cytoskeletal organization and larger focal adhesions when compared to MSCs cultured on discontinuous micropatterns. After 14 days in bipotential AD/OS induction medium, MSCs cultured on co-continuous micropatterns exhibited increased expression of osteogenic markers, whereas MSCs on discontinuous PDTEC substrates showed a low expression of adipogenic and osteogenic differentiation markers. Substrates with graded micropatterns were able to reproduce the influence of local underlying topography on MSC differentiation, thus demonstrating their potential for high throughput analysis. This work presents polymer demixing as a simple, non-lithographic technique to produce a wide range of micropatterns on surfaces with complex geometries to influence cellular and tissue regenerative responses. Statement of SignificanceA better understanding of how engineered microenvironments influence stem cell differentiation is integral to increasing the use of stem cells and materials in a wide range of tissue engineering applications. In this study, we show the range of topography obtained by polymer demixing is sufficient for investigating how surface topography affects stem cell morphology and differentiation. Our findings show that co-continuous topographies favor early (3-day) cytoskeletal anisotropy and focal adhesion maturation as well as long-term (14-day) expression of osteogenic differentiation markers. Taken together, this study presents a simple approach to pattern topographies that induce divergent responses in stem cell morphology and differentiation.