Mesenchymal Stem Cells Density Research Articles

Volumetric mass density measurements of mesenchymal stem cells in suspension using a density meter

To use regeneratively active cells for cell therapeutic applications, the cells must be isolated from their resident tissues. Different isolation procedures subject these cells to varying degrees of mechanical strain, which can affect the yield of cell number and viability. Knowledge of cell volumetric mass density is important for experimental and numerical optimization of these procedures. Although methods for measuring cell volumetric mass density already exist, they either consume much time and cell material or require a special setup. Therefore, we developed a user-friendly method that is based on the use of readily available instrumentation. The newly developed method is predicated on the linear relationship between the volumetric mass density of the cell suspension and the volumetric mass density, number, and diameter of the cells in the suspension. We used this method to determine the volumetric mass density of mesenchymal stem cells (MSCs) and compared it to results from the established density centrifugation.

Stem Cells and Extrusion 3D Printing for Hyaline Cartilage Engineering.

Hyaline cartilage is deficient in self-healing properties. The early treatment of focal cartilage lesions is a public health challenge to prevent long-term degradation and the occurrence of osteoarthritis. Cartilage tissue engineering represents a promising alternative to the current insufficient surgical solutions. 3D printing is a thriving technology and offers new possibilities for personalized regenerative medicine. Extrusion-based processes permit the deposition of cell-seeded bioinks, in a layer-by-layer manner, allowing mimicry of the native zonal organization of hyaline cartilage. Mesenchymal stem cells (MSCs) are a promising cell source for cartilage tissue engineering. Originally isolated from bone marrow, they can now be derived from many different cell sources (e.g., synovium, dental pulp, Wharton’s jelly). Their proliferation and differentiation potential are well characterized, and they possess good chondrogenic potential, making them appropriate candidates for cartilage reconstruction. This review summarizes the different sources, origins, and densities of MSCs used in extrusion-based bioprinting (EBB) processes, as alternatives to chondrocytes. The different bioink constituents and their advantages for producing substitutes mimicking healthy hyaline cartilage is also discussed.

Inhibited growth of mesenchymal stem cells under simulated microgravity

This study aimed to estimate the effects of simulated microgravity (SMG) on mesenchymal stem cells (MSCs). The 3D clinostat was applied to induce to simulated microgravity on MSC. The results showed that the MSC density in control group was higher than the SMG group. The cell cycle analysis demonstrated that the MSC ratio in G0/G1 phase in SMG group was higher than that of the control group, while the MSC ratio in S phase and G2/M phase in SMG group was lower than those of the control group. The real time quantitative RT-PCR was used to evaluate the expression of cell cycle-related genes, including Cyclin-dependent kinase 2 (Cdk2), Cyclin-dependent kinase 6 (Cdk6), and Cyclin A. The results showed that the transcript expression of Cdk2, Cdk6, and Cyclin A was down-regulated in MSC from SMG group comparing to that of the control group. The flow cytometry was performed to determine the ratio of apoptotic MSCs. There was no significant difference in viability ratio and apoptotic ratio of MSCs between SMG group and control group. The MSCs from SMG group and control group showed similar in Bcl2 and Bax transcript expression.

Mesenchymal Stem Cell Therapy in Cartilage Injuries of Knee – Review

Full-thickness cartilage injuries when left alone would increase the risk of osteoarthritis (OA) with severe associated pain and functional disability. The capability of the mesenchymal stem cells (MSCs) to repair and regenerate cartilage has been widely investigated. Articular cartilage defect may result from direct trauma or chronic degeneration. The subchondral bone plays an important role in healing through the presence of mesenchymal elements. MSCs are the most representative adult stem cells. It was observed that BM-MSCs (Bone Marrow derived MSCs) had superior chondrogenic differentiation capacity as compared to MSCs from other origins. It has been observed that a MSC density of 5 × 107 or 5 × 108 cells per milliliter embedded in a collagen gel had more proteoglycans than lower cell densities, better facilitating cartilage defect healing. The most potent chondrogenic differentiation inducers are transforming growth factor β, bone morphogenic protein, fibroblast growth factor, and insulin-like growth factor 1. Most commonly used delivery system for MSCs are either sealant or scaffold based. Ideal scaffold should have similar characteristics to the native tissue. The scaffold has the advantage of withstanding the in-vivo loading environment and protects the embedded cells. There is contradictory evidence regarding injectable treatment with BM-MSCs suggesting scaffolds may be required for the regeneration of cartilage. There is high quality of evidence to support MSC therapy but further refinement of methodology will be required to support its routine clinical use. There are few options to be explored e.g. MSC exosomes, genetic engineering and epigenetic regulatory mechanisms governing MSC biology

Differentiation of Porcine Mesenchymal Stem Cells to Lung Epithelial Cells Using Media Conditioned by Human Primary Small Airway Epithelial Cells

Mesenchymal stem cells (MSCs) under defined conditions, can differentiate into many different cell types, and may be the key to treating a multitude of ailments including chronic lung injury. Although MSCs can reduce inflammation and alter immune response when injected into sites of injury, differentiating MSCs in vitro prior to injection may enhance their therapeutic actions. The aim of this study was to develop a simple and reliable method to differentiate porcine adipose‐derived (pAMSCs) and bone marrow‐derived mesenchymal stem cells (pBMSCs) to small airway epithelium (SAE) for future testing in a pig model of lung injury. Traditional induction of MSCs to SAE involves treatment with an artificial combination of factors that mimic the local milieu in vivo. A simpler and more physiologically‐relevant approach is to use naturally conditioned media that may yield more biologically active cells. We hypothesized that media conditioned with human primary small airway epithelial cells (hSAECs) can induce differentiation of pAMSCs and pBMSCs to SAE. hSAECs (ATCC) were seeded in serum‐free M199 media to create conditioned media which was collected 4 days after seeding for immediate use. pAMSCs and pBMSCs were isolated in‐house from juvenile Yorkshire pigs, and expanded to passage 3 prior to start of experiments. Fibronectin‐coated, 8‐well slides (for immunohistochemistry) or T75 flasks (to examine morphology) were seeded with either pAMSCs or pBMSCs (10,000 cells/cm2). Every 4 days for 16 days, a subset of cells were labeled with stem cell markers (anti‐FOXA1, anti‐CD44, anti‐CD105), small airway cell markers (anti‐SCGB3A2, anti‐RAGE), and neurofilament markers (anti‐neurofilament heavy polypeptide, anti‐68kDa neurofilament)), and media was exchanged with either conditioned media or unconditioned media. After 5 days, pBMSCs exposed to conditioned media transitioned to cuboidal and epithelial‐like cells. pBMSCs exposed to unconditioned media remained elongated and stem cell‐like. pAMSCs proliferated faster than pBMSCs, but yielded spindly overgrown cells in high density that did not differentiate in either conditioned or unconditioned media. Expression of proteins did not correlate with the morphological changes in pBMSCs. Although RAGE expression increased initially in response to conditioned media, levels were reduced after day 12. SCGB3A2 expression also increased over time, but was not different between the conditioned and unconditioned groups. The distinct change in morphology observed in pBMSCs exposed to conditioned media indicates a promising phenotypic change. Verification of the epithelial cell phenotype induced by the conditioned media with semi‐quantitative gene and protein expression of cell markers for small airway cells and their functional ability to produce surfactant remains to be determined. Our study indicates that conditioned media is able to induce differentiation of MSCs into epithelial cells. Further, our results revealed that the source of MSCs (adipose versus bone marrow), and the density of MSCs may be important factors in successful differentiation of MSCs.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Simulating localised cellular inflammation and substrate properties in a strain energy density based bone remodelling algorithm for use in modelling trauma

Bone responds to mechanical stimulus and a range of pre-existing finite element models have been suggested to reproduce the internal physiological structure of bone. Inflammation effects are not included in these models, yet inflammation is a key component of bone repair in trauma. Therefore, a model is proposed and tested here that extends these methods to include parameters that could be considered to represent the behaviour of bone remodelling when influenced by inflammation. The proposed model regulates remodelling based on findings from recent studies into the nature of heterotopic ossification, the formation of heterotopic bone, which have revealed information about the nature of bone after high levels of trauma. These parameters include consideration of the distance from the zone of trauma, the density of mesenchymal stem cells, and substrate stiffness as a trigger for cells becoming osteogenic. The method is tested on a two-dimensional plate model and shows that the new extended algorithm can produce a range of structures depending on inputs that could be used in the future to replicate physiological scenarios.

Adipocyte Regeneration after Free Fat Transplantation: Promotion by Stromal Vascular Fraction Cells

Our objective was to explore the mechanism of cell-assisted adipose transplantation by using freshly isolated human stromal vascular fraction (SVF) cells and to observe the dynamic changes of the graft after transplantation. The SVF was isolated from human liposuction aspirates, and 0.5 ml adipose tissue was mixed with 1 × 10(6) SVF cells or culture medium then injected to nude mice subcutaneously. At 1, 4, 7, 14, 30, 60, and 90 days after transplantation, samples were harvested for 1) general observation and retention rate; 2) whole-mount stain; 3) H&E stain; 4) immunohistochemical staining for S100, CD68, and CD34; 5) ELISA for VEGF and bFGF; 6) peroxisome proliferator-activated receptor-γ (PPARγ) fluorescence in situ hybridization. The retention rate in the experiment group was markedly higher than that in the control group. Whole-mount stain shows most of the cells in the center of the graft could not survive the ischemia until day 14. Histology showed new vessels on the surface of the graft at 3 days. However, in the control group, fewer newly formed vessels were detected until day 7. In the late stage of transplantation, gradual fibrosis was found in the graft, and the tissue was divided into a grid-like structure. A large number of round neonatal adipocytes with big nuclei in the center were found surrounding the new vessels, which were S100 and CD34 positive and CD68 negative. In the late stage of transplantation, most of the neonatal adipocytes were human PPARγ positive. Moreover, the SVF group showed a higher level of VEGF and bFGF. SVF assisting adipose transplantation could increase the retention rate of the graft through promoting adipose tissue regeneration via secretion of growth factors, promotion of angiogenesis, and increasing the density of mesenchymal stem cells.

Effect of Imatinib on Proliferation, Apoptosis and Expression of Platelet Derived Growth Factor Receptor By Mesenchymal Stem Cells

IntroductionImatinib Mesylate (IM) is a tyrosine kinase inhibitor (TKI), which targets Platelet Derived Growth Factor Receptor (PDGF-R), c-kit and BCR-ABL and is used in the treatment of Gastrointestinal Stromal Tumors (GIST) and Chronic Myeloid Leukemia (CML). Patients using Imatinib for a long time display abnormalities in bone metabolism (Vandyke et al. J Clin Endocrinol Metab 2013). In vitro, IM affects osteoblastic differentiation through inhibition of PDGF/PDGF-Rb signaling, as well as osteoclast function/differentiation through modulation of PDGF/PDGF-Ra signaling (Berman et al. Leuk Res 2013). Conflicting reports have shown differential effects on in vivo bone marrow density. Confounding factors include patient age (pediatric ve adult), type of bone (osteochondral vs trabecular), duration and dose of IM treatment. Mesenchymal Stem Cells (MSCs) are cells with great regenerative potential and differentiate into adipogenic, osteogenic cells and multiple other cell lineages (Bianco et al. Cell Stem Cell 2008). MSCs express high cell surface levels of PDGF-Rb and intermediate levels of PDGF-Ra and are therefore likely to be affected by IM treatment. Here, we wanted to assess the effect of IM on proliferation and apoptosis of MSCs as well as on surface expression of PDGF-Rb and PDGF-Ra.MethodsHealthy human bone marrow MSCs were isolated using Ficoll and plastic adherence and cultured up till passage 3. Proliferation assays were performed using Real Time Cell Analysis (XCELLigence, Roche) to determine optimal in vitro doses of IM (Novartis). IM was used at doses from 2,5 uM to 20 uM and found to be optimal at 5 uM. PDGF-Rb (CD140b) and PDGF-Ra (CD140a) surface expression were measured using monoclonal antibodies and read using a FACSARIA (Becton Dickinson). Apoptosis was assessed using Annexin-V and Propidium Iodide. Adipogenic and osteogenic differentiation was evaluated after 21 days in differentiation media with or without IM, using spectrophotometric quantitation of levels of Oil Red O (Adipogenic differentiation) and Calcium Phosphate (Osteogenic differentiation).ResultsIM inhibited proliferation of MSCs in a dose-dependent fashion, with doses > 5 uM resulting in severe suppression of proliferation. Inhibition of proliferation by IM could be overcome by increasing cell densities of MSCs, but not by addition of PDGF-BB. Co-treatment with IM and PDGF-BB resulted in more pronounced suppression of MSC proliferation. Treatment with IM resulted in a decrease of cell surface expression of both PDGF-Rb from 97,4±2,36% to 77,3±0,13% (n=3, p<0.02) and PDGF-Ra from 18,4% to 6,4% (n=1). Addition of PDGF-BB resulted in a further decrease in cell surface expression of PDGF-Rb, but had no effect on expression of PDGF-Ra. IM increased apoptosis levels (Annexin-V positive cells) about twofold. Addition of 5 or 10 ng/mL PDGF could completely abrogate this effect. Treatment of K562, a BCR-ABL positive CML cell line, with 5 uM IM suppressed proliferation of K562 four-fold, but had no obvious effect on levels of apoptosis. Treatment of MSCs with IM during differentiation revealed no clear effect on adipogenesis, but did increase osteogenic differentiation, as measured as an increase in Calcium-Phosphate.ConclusionsImatinib Mesylate inhibits PDGF/PDGF-R signaling through interference with tyrosine kinases. Here, we found that 5 uM IM not only affects MSC proliferation through inhibition of PDGF-receptor signaling, but also through downregulation of PDGF-Rb and PDGF-Ra cell surface expression. This dose is very close to the maximal plasma concentration of 4.6 uM observed in patients (Druker et al. New England J Med 2001). Addition of PDGF-BB enhanced the effects of IM on suppression of MSC proliferation, likely through further downregulation of surface PDGF-Rb expression, thus decreasing PDGF/PDGF-R signaling. PDGF signaling has been implicated in regulation of invasiveness of cancers and TKI have been used in the treatment of several types of cancer. Whether or not combination treatment of IM with PDGF-BB similarly affects proliferation/invasiveness of BCR-ABL+ CML cells, and PDGF-Ra positive GIST cells, remains to be investigated. Prolonged treatment with IM in patients has been shown to affect bone remodeling and bone densities. Our current results suggest that treatment with IM impacts BM-resident MSCs, supporting increased osteogenesis. DisclosuresNo relevant conflicts of interest to declare.

Sirtuin-1 (SIRT1) Is Required for Promoting Chondrogenic Differentiation of Mesenchymal Stem Cells

Sirtuin-1 (SIRT1), NAD(+)-dependent deacetylase, has been linked to anabolic effects in cartilage, although the mechanisms of SIRT1 signaling during differentiation of mesenchymal stem cells (MSCs) to chondrocytes are poorly understood. Therefore, we investigated the role of SIRT1-mediated signaling during chondrogenic differentiation of MSCs in vitro. High density and alginate cultures of MSCs were treated with chondrogenic induction medium with/without the SIRT1 inhibitor nicotinamide, antisense oligonucleotides against SIRT1 (SIRT1-ASO), IL-1β, and/or resveratrol. Transient transfection of MSCs with SIRT1-antisense oligonucleotides, nicotinamide, and IL-1β inhibited chondrogenesis-induced down-regulation of cartilage-specific proteins, cartilage-specific transcription factor Sox9, and enhanced NF-κB-regulated gene products involved in the inflammatory and degradative processes in cartilage (MMP-9, COX-2, and caspase-3), and NF-κB phosphorylation, acetylation, and activation of IκBα kinase. In contrast, the SIRT1 activator resveratrol or BMS-345541 (inhibitor of IKK) inhibited IL-1β- and NAM-induced suppression of cartilage-specific proteins, Sox9, and up-regulation of NF-κB-regulated gene products. Moreover, SIRT1 was found to interact directly with NF-κB and resveratrol-suppressed IL-1β and NAM but not SIRT1-ASO-induced NF-κB phosphorylation, acetylation, and activation of IκBα kinase. Knockdown of SIRT1 by mRNA abolished the inhibitory effects of resveratrol on inflammatory and apoptotic signaling and Sox9 expression, suggesting the essential role of this enzyme. Finally, the modulatory effects of resveratrol were found to be mediated at least in part by the association between SIRT1 and Sox9. These results indicate for the first time that SIRT1 supports chondrogenic development of MSCs at least in part through inhibition/deacetylation of NF-κB and activation of Sox9.

12/15-Lipoxygenase deficiency reduces densities of mesenchymal stem cells in the dermis of wounded and unwounded skin

Mesenchymal stem cells (MSCs) promote skin healing. 12/15-Lipoxgenase (LOX) is crucial in producing specific lipid mediators in wounded skin. The consequences of 12/15-LOX deficiency in MSC densities in skin are unknown. To determine the effect of 12/15-LOX deficiency in MSC densities in wounded and unwounded dermis. Full-thickness skin incisional wounds were made to 12/15-LOX-deficient (12/15-LOX(-/-) ) and wild-type (WT) C57BL/6 mice. Wounded skin was collected at 3, 8, or 14 days postwounding (dpw). MSCs were analysed in skin sections using histology. 12S- or 15S-hydroxy-eicosatetraenoic acid (HETE) was analysed using a reversed-phase Chiral liquid chromatography-ultraviolet-tandem mass spectrometer. There were more stem cell antigen (Sca)1(+) CD29(+) MSCs (cells/field) at 3, 8, and 14 dpw, more Sca1(+) CD106(+) MSCs at 3 and 14 dpw in the wounded dermis, more MSCs in unwounded dermis of WT mice compared with 12/15-LOX(-/-) mice, and more MSCs in the wounded dermis than in the unwounded dermis. For 12/15-LOX(-/-) dermis, Sca1(+) CD106(+) MSCs peaked and Sca1(+) CD29(+) MSCs reached a flat level at 8 dpw. However, for the WT dermis, MSCs increased from 8 to 14 dpw. There were more Sca1(+) CD106(+) MSCs than Sca1(+) CD29(+) MSCs in the 12/15-LOX(-/-) wounded dermis at 8 dpw. However, there were more Sca1(+) CD29(+) MSCs in the 12/15-LOX(-/-) than Sca1(+) CD106(+) MSCs in the WT wounded dermis at 3 dpw, and Sca1(+) CD106(+) MSCs and Sca1(+) CD29(+) MSCs were at comparable levels in other conditions. 12/15-LOX deficiency suppressed levels of 12/15-LOX protein and their products, 12S-HETE and 15S-HETE, in wounds. 12/15-LOX deficiency reduces MSC densities in the dermis, which correlates with the suppressed 12/15-LOX pathways in wounded and unwounded skin.

Mesenchymal stem cell-collagen microspheres for articular cartilage repair: Cell density and differentiation status

Mesenchymal stem cells (MSC) hold promise for cartilage repair. A microencapsulation technique was previously established to entrap MSC in collagen microspheres, and the collagen fibrous meshwork was found to be an excellent scaffold for supporting MSC survival, growth and differentiation. This study investigates the importance of cell density and differentiation status of MSC–collagen microspheres in cartilage repair. MSC were isolated from rabbit bone marrow and encapsulated in collagen microspheres. The effects of pre-differentiating the encapsulated MSC into chondrogenic lineages and different cell densities on cartilage repair were investigated in rabbits. Implantation of undifferentiated collagen–MSC microspheres formed hyaline-like cartilage rich in type II collagen and glycosaminoglycans (GAG) at 1month post-implantation. By 6months, hyaline cartilage rich in type II collagen and GAG, but negative for type I collagen, and partial zonal organization were found in both undifferentiated and chondrogenically differentiated groups in the high cell density group. The undifferentiated group and high cell density group significantly improved the O’Driscoll histological score. Moreover, the undifferentiated group significantly increased the GAG content. The mechanically differentiated group showed stiffer but thinner cartilage, while the undifferentiated group showed thicker but softer cartilage compared with their respective contra-lateral controls. This work suggests that a higher local cell density favors cartilage regeneration, regardless of the differentiation status of MSC, while the differentiation status of MSC does significantly affect regeneration outcomes.

Induction of Autologous Bone-Marrow Stem Cells by Low-Level Laser Therapy Has Beneficial Effects on the Kidneys Post-Ischemia-Reperfusion Injury in the Rat

Acute renal failure has a 50% - 80% mortality rate. Currently, treatment options for this life-threatening disease are limited. Low-level laser therapy (LLLT) has been found to modulate biological activity. The aim of the present study was to investigate the possible beneficial effects of laser application to stem cells in the bone marrow, on the kidneys of rats that had undergone ischemia-reperfusion injury (IRI). IRI was induced by occlusion of the renal artery to 3- and 7-month-old rats for 15 or 30 minutes. In an additional experiment IRI was applied to both kidneys for 20 min each in 2-3-month-old rats. Rats were then divided randomly into two groups of control and laser-treated. Laser therapy (Ga-Al-As 810 nm, 200 mW output for 2 min) was applied to the bone marrow 1 and 7 days post-IRI to the kidneys, and rats were sacrificed 2 weeks later. Histomorphometry and immunohistochemistry were performed on kidney sections and blood markers for kidney function. Quantitative histomorphometric analysis revealed a reduction in dilatation of the renal tubules, restored structural integrity of the renal tubules, and reduced necrosis in the laser-treated rats as compared to the control, non-laser-irradiated group. C-kit positive cell density in kidneys post-IRI and laser-treatment was significantly (p = 0.015) 3.2-fold higher compared to the control group. Creatinine and blood urea nitrogen content were significantly lower in the laser-treated rats as compared to control. It is concluded that LLLT application to the bone marrow (BM) causes a significant increase in the density of mesenchymal stem cells in the kidneys post-IRI, probably by induction of stem cells in the BM, which subsequently migrate to the IRI kidney, significantly reducing the pathological features of the kidney and increasing kidney function post IRI.

Supercharging allografts with mesenchymal stem cells in the operating room during hip revision

Bone marrow derived mesenchymal stem cells (BM-MSCs) have been proposed to improve allografts used during hip revision. However, no study has reported the number of MSCs that could be associated with the allograft and the best technique to load MSCs in allografts. The optimal loading technique should combine methods to increase the initial cell density and create an appropriate environment to accelerate the efficiency of the cell-allograft constructs into clinically applicable grafts. We designed a study to evaluate the number of MSCs in an autograft femoral head considered as the gold standard and to determine the best operating room procedure for loading in allograft with MSCs to approach the same number as in an autograft femoral head. Therefore this study explored a potential of charging whole femoral head allografts with autologous MSCs from iliac crest aspirate for hip revision procedures. First, the study evaluated the total number of mesenchymal stem cells (MSCs) in 1 cc of an average autograft femoral head; this number then serves as a target for loading allografts, in order to achieve the same density of MSCs. For the loading technique itself, several questions were asked and hence several options were investigated. For example, is it better to load the whole allograft or break it up into several fragments? Which way of injecting works best for the whole femoral head allograft (through cartilage or femoral neck)? How concentrated (in terms of MSCs) should the injected iliac crest marrow be? Bone marrow for injection in allografts was obtained from residual marrow from patients undergoing surgical procedures with concentrated bone marrow. With this bone marrow (with and without concentration) we tested different techniques (injection and soaking) to load stem cells in allografts of different sizes: bulk allografts, pieces or blocks (8 or 1 cm(3) blocks) and morselized fragments (from 125 to 8 mm(3)) or particules (1 mm(3)). We also evaluated the release of MSCs from fragments of autografts and allografts loaded with MSCs in cultured medium. The femoral head autografts contained a lower concentration of MSCs than the iliac crests of the same patient. However, in absence of concentration, with bone marrow aspirated from the iliac crest, we were not able to load in the femoral head allograft the same number of MSCs as the number present in an autograft. The loaded volume of bone marrow (and the corresponding number of MSCs) depended on the technique (injecting, soaking) as well as on the volume and shape of the allografts. The seeding efficiency of loading MSCs in allografts increased with the concentration of MSCs in the bone marrow. With concentrated bone marrow, supercharging the allograft with MSCs (as compared with an autograft) was possible in the operating room, and the number of MSCs supercharged in allografts was predictable. The loaded volume of bone marrow depended on the technique (injecting, soaking) as well as on the volume and shape of the allografts. With concentrated bone marrow, the allograft could be charged with a similar or higher number of MSCs than the number present in a femoral head autograft.

Three-dimensional structural niches engineered via two-photon laser polymerization promote stem cell homing

A strategy to modulate the behavior of stem cells in culture is to mimic structural aspects of the native cell/extracellular matrix interaction. We applied femtosecond laser two-photon polymerization (2PP) to fabricate three-dimensional (3-D) microscaffolds, or “niches”, using a hybrid organic–inorganic photoresist called SZ2080. The niches, of sizes fitting in a volume of 100×100×100μm3, were made by an external containment grid of horizontal parallel elements and by an internal 3-D lattice. We developed two niche heights, 20 and 80-100μm, and four lattice pore dimensions (10, 20, 30μm and graded). We used primary rat mesenchymal stem cells (MSCs) to study cell viability, migration and proliferation in the niches, up to 6 culture days. MSCs preferentially stayed on/in the structures once they ran into them through random migration from the surrounding flat surface, invaded those with a lattice pore dimension greater than 10μm, and adhered to the internal lattice while the cell nuclei acquired a roundish morphology. In the niches, the highest MSC density was found in those areas where proliferation was observed, corresponding to the regions where the scaffold surface density available for cell adhesion was highest. The microgeometry inducing the highest cell density was 20μm high with graded pores, in which cell invasion was favored in the central region of large porosity and cell adhesion was favored in the lateral regions of high scaffold surface density. Cell density in the niches, 17±6cells/(100×100μm2), did not significantly differ from that of the flat surface colonies. This implies that MSCs spontaneously homed and established colonies within the 3-D niches. This study brings to light the crucial role played by the niche 3-D geometry on MSC colonization in culture, with potential implications for the design of biomaterial scaffolds for synthetic niche engineering.

Fabrication of mesenchymal stem cells-integrated vascular constructs mimicking multiple properties of the native blood vessels

Mesenchymal stem cells (MSCs)-populated small diameter (6 mm) vascular constructs were fabricated. The constructs mimicked the native vessels in multiple levels, i.e. having similar structure and morphology to that of the extracellular matrix in the native blood vessels; recapitulating mechanical properties such as compliance and burst pressure of the native blood vessels; simulating the highly cellularized nature of the native blood vessels; and having an antithrombogenic lumen. The constructs were fabricated by simultaneously assembling poly(ester carbonate urethane) urea nanofibers and MSCs in an electrical field. The nanofibers had a diameter similar to that of the collagen and elastin fibers in the native blood vessels. MSCs were distributed evenly in the constructs. The constructs were highly cellularized when the cell loading density was exceeded 6 million/ml. The vascular constructs were strong and flexible with breaking strains of 144–202%, tensile strengths of 0.80–1.29 MPa, compliances of 13.23–21.96 × 10−4 mmHg−1, stiffness indexes of 7.3–9.8, and burst pressures greater than 1700 mmHg. These mechanical properties were similar to those of the native blood vessels. In vitro platelet deposition experiments showed that platelet adhesion was remarkably decreased in the MSCs-populated constructs compared to that in the construct without MSCs. An increase in MSC density in the constructs further decreased platelet adhesion. When cultured in a spinner flask, MSCs maintained their mitochondria viability and cell number during a two-week culture period, as confirmed by MTT and dsDNA assays. These vascular constructs may hold the potential to regenerate functional small diameter vessels for cardiovascular tissue repair.

Mesenchymal Stem Cells Primed With Valproate and Lithium Robustly Migrate to Infarcted Regions and Facilitate Recovery in a Stroke Model

The migratory efficiency of mesenchymal stem cells (MSC) toward cerebral infarct after transplantation is limited. Valproate (VPA) and lithium enhance in vitro migration of MSC by upregulating CXC chemokine receptor 4 and matrix metalloproteinase-9, respectively. Ability of VPA and lithium to promote MSC homing and to improve functional recovery was assessed in a rat model of cerebral ischemia. MSC primed with VPA (2.5 mmol/L, 3 hours) and/or lithium chloride (2.5 mmol/L, 24 hours) were transplanted into rats 24 hours after transient middle cerebral artery occlusion (MCAO). Neurological function was assessed via rotarod test, Neurological Severity Score, and body asymmetry test for 2 weeks. Infarct volume was analyzed by MRI. The number of homing MSC and microvessel density in the infarcted regions were measured 15 days after MCAO using immunohistochemistry. Priming with VPA or lithium increased the number of MSC homing to the cerebral infarcted regions, and copriming with VPA and lithium further enhanced this effect. MCAO rats receiving VPA-primed and/or lithium-primed MSC showed improved functional recovery, reduced infarct volume, and enhanced angiogenesis in the infarcted penumbra regions. These beneficial effects of VPA or lithium priming were reversed by AMD3100, a CXC chemokine receptor 4 antagonist, and GM6001, a matrix metalloproteinase inhibitor, respectively. Priming with VPA and/or lithium promoted the homing and migration ability of MSC, improved functional recovery, reduced brain infarct volume, and enhanced angiogenesis in a rat MCAO model. These effects were likely mediated by VPA-induced CXC chemokine receptor 4 overexpression and lithium-induced matrix metalloproteinase-9 upregulation.

Chondrogenic differentiation and immunological properties of mesenchymal stem cells in collagen type I hydrogel

Allogeneic mesenchymal stem cells (MSCs) are regarded as promising seed cells for engineering cartilage. However, few researches have covered the immune properties of seeded MSCs. Collagen has been considered as good scaffold, whether it has inherent chondrogenic inducibility for MSCs is still in debate. In this study, engineering grafts are constructed by neonatal rabbit MSCs and collagen Type I hydrogel. After periods of culture, the appearance of chondroid tissue in the grafts and the cartilage matrix-specific genes expressions of seeded cells prove the inducibility of collagen hydrogel, even if the growth factors are absence. With the differentiation, immunological properties of MSCs are changing. The expressions of main histocompatibility complex (MHC) molecules increase and the ability to inhibit the proliferation of activated lymphocytes may be declined. But to a large extent, it keeps the low stimulating to allogeneic lymphocytes and the small absolute value of MHCs. The changes are adverse for avoiding inflammation and rejection. Therefore, suitable scaffold and engineering strategies should be selected. For the grafts based on Collagen I hydrogel and MSCs, a longer culture period might not be necessary. To maintain the immune regulation, a higher initial MSCs density in engineering grafts may be more meaningful.

Regulation of suppressing and activating effects of mesenchymal stem cells on the encephalitogenic potential of MBP 68–86-specific lymphocytes

Mesenchymal stem cells (MSCs) possess immunosuppressive potential and are proposed as a tool for cell therapy, however under certain circumstance they could change to become immunostimulating. It is therefore of great importance to better understand the MSCs' immune plasticity. Here we used different doses of MSCs to co-culture with MBP 68 – 86-specific T cells and found that MSCs exerted a suppressive effect on the encephalitogenic ability of MBP 68 – 86-specific T cells at high MSC density (MSC/effector ratio ≥ 1:10). Whereas at lower MSC/effector ratios (≤ 1:50), MSCs shifted towards stimulatory activity. Thus the limited amounts of MSCs for application should be considered.

Dexamethasone inhibition of confluence‐induced apoptosis in human mesenchymal stem cells

Human mesenchymal stem cells (MSCs) have been extensively characterized with respect to their in vitro expansion and differentiation potential, especially with respect to osteogenesis. Dexamethasone (Dex) is a well-known inducer of osteogenic differentiation of MSCs, but little is known about the effect of Dex treatment on apoptosis in MSCs. In this study, apoptosis in MSCs was examined with respect to cell density and Dex supplementation, using DAPI staining and DNA fragmentation ELISA Assay. In MSC cultures initiated at 1.0, 3.0, and 9.0 x 10(3) cells per cm(2), it was found that higher MSC density correlated with increased apoptosis and that this apoptotic effect was diminished in cultures containing 100 nM Dex. MSCs and fibroblasts were co-cultured, along with empty insert controls, and assayed for apoptosis by ELISA and DAPI counts to determine if soluble factors accounted for the cell density-related apoptosis. No difference was seen between MSCs cultured with inserts containing either MSCs, fibroblasts, or empty control. To determine cell contact effects, BrdU-labeled MSCs were cultured alone or with unlabeled chondrocytes at 2x and 8x the number of MSCs, with and without Dex, and apoptosis levels quantified. The results showed increased apoptosis at greater cell densities, and that the amount of apoptosis was greatly diminished in cultures containing Dex. These results show that apoptosis in MSCs is cell density-related, requires direct cell contact, and that Dex treatment reduces or eliminates this density-related apoptosis. These results may impact how MSCs should be cultured for clinical applications.

High inoculation cell density could accelerate the differentiation of human bone marrow mesenchymal stem cells to chondrocyte cells

The effects of the density of human mesenchymal stem cells (MSCs) on their differentiation to chondrocytes in a differentiation medium supplemented with dexamethasone, TGFbeta3, and IGF-1 were investigated for the regenerative therapy of cartilage. The increase in the initial density of MSCs from 0.05 x 10(4) to 0.9 x 10(4) cells/cm(2) accelerated the increase in the expression level of aggrecan mRNA during the differentiation culture for 7 d. The conditioned medium harvested at 7 d from the differentiation culture with an initial MSC density of 0.3 x 10(4) cells/cm(2) accelerated the initial increase in the expression level for 3 d in the differentiation culture with an initial MSC density of 0.3 x 10(4) cells/cm(2), whereas the conditioned medium harvested at 7 d in the differentiation culture with an initial MSC density of 0.05 x 10(4) cells/cm(2) did not. The differentiation culture after 14 d with an initial MSC concentration of 0.3 x 10(4) cells/cm(2) showed an expression level 1.7-fold that in the case of the culture with an initial MSC concentration of 0.05 x 10(4) cells/cm(2). Thus, a high MSC inoculum density might be appropriate for the rapid differentiation of MSCs to chondrocytes.