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A combinatorial cell-laden gel microarray for inducing osteogenic differentiation of human mesenchymal stem cells

A combinatorial cell-laden gel microarray for inducing osteogenic differentiation of human mesenchymal stem cells

In this work, we present a 3D cell-laden gel microarray platform for combinatorial screening of human mesenchymal stem cells (hMSCs) differentiation in response to multiple ECM and growth factors components. An automated printing strategy, which utilizes 1000-fold less materials and cells, compared to conventional multi- well-based assays was employed to generate arrays of miniaturized cell-laden hydrogel constructs. Each microgel unit, composed of methacrylated gelatin (GE), contained living hMSCs along with ECM proteins and was exposed to osteogenic bone morphogenic proteins (BMPs). From the microarray analysis, we identified ECM combinations, which induced a 2-fold increase in Alkaline Phosphatase (ALP) expression. Furthermore, we evaluated the rel- evance of our platform within macroscale settings to investigate its translational potential. By utilizing our 3D microarray platforms, it is possible to efficiently screen ECM and growth factor combinations, which promote stem cell differentiation. We envision that, our cell- laden gel microarray platform could potentially accelerate the development of innovative and biomimetic materials for a wide range of applications from tissue engineering to stem cell bioengi- neering and drug screening.
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Double porous poly (Ɛ-caprolactone)/chitosan membrane scaffolds as niches for human mesenchymal stem cells

Double porous poly (Ɛ-caprolactone)/chitosan membrane scaffolds as niches for human mesenchymal stem cells

Oxygen consumption A B S T R A C T In this paper, we developed membrane scaffolds to mimic the biochemical and biophysical properties of human mesenchymal stem cell (hMSC) niches to help direct self-renewal and proliferation providing to cells all ne- cessary chemical, mechanical and topographical cues. The strategy was to create three-dimensional membrane scaffolds with double porosity, able to promote the mass transfer of nutrients and to entrap cells. We developed poly (Ɛ-caprolactone) (PCL)/chitosan (CHT) blend membranes consisting of double porous morphology: (i) surface macrovoids (big pores) which could be easily accessible for hMSCs invasion and proliferation; (ii) in- terconnected microporous network to transfer essential nutrients, oxygen, growth factors between the macro- voids and throughout the scaffolds. We varied the mean macrovoid size, effective surface area and surface morphology by varying the PCL/CHT blend composition (100/0, 90/10, 80/20, 70/30). Membranes exhibited macrovoids connected with each other through a microporous network; macrovoids size increased by increasing the CHT wt%. Cells adhered on the surfaces of PCL/CHT 100/0 and PCL/CHT 90/10 membranes, that are characterized by a high effective surface area and small macrovoids while PCL/CHT 80/20 and PCL/CHT 70/30 membranes with large macrovoids and low effective surface area entrapped cells inside macrovoids.
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Intravenous administration of 99mTc-HMPAO-labeled human mesenchymal stem cells after stroke: in vivo imaging and biodistribution.

Intravenous administration of 99mTc-HMPAO-labeled human mesenchymal stem cells after stroke: in vivo imaging and biodistribution.

technically easier than a surgical procedure in the setting 7. Bindslev, L.; Haack-Sorensen, M.; Bisgaard, K.; Kragh, of pilot clinical trials (4). Clinical studies using intrace- L.; Mortensen, S.; Hesse, B.; Kjaer, A.; Kastrup, J. Label- rebral grafts in a few patients after stroke reported lim- ling of human mesenchymal stem cells with indium-111 for SPECT imaging: Effect on cell proliferation and dif-

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Titania–hydroxyapatite nanocomposite coatings support human mesenchymal stem cells osteogenic differentiation

Titania–hydroxyapatite nanocomposite coatings support human mesenchymal stem cells osteogenic differentiation

bioactivity a coating with higher bond and cohesive strengths as well as chemical stability under high mechani- cal solicitations is needed. Since the early 1990s, there has been significant research to establish the correct biological stimuli required to obtain osteoblasts from human mesenchymal stem cells (hMSC) by the use of supplemented media. 1,6,7 More recently, researchers have focused on 2D substrate-induced hMSC osteogenesis and substrate-induced bioactivity. 2,7–11 However, the current lack of knowledge about substrate characteristics such as surface chemistry and topography control over hMSC behaviour and differentiation has largely restricted hMSC osteogenic differentiation to biological intervention in the form of growth factors and cytokines. Despite current efforts, the optimal bone implant coatings capable of influencing and accommodating the growth and osteogenic differentiation of hMSCs are yet to be identified.
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Role of stromal-derived factor-1 in the hematopoietic-supporting activity of human mesenchymal stem cells.

Role of stromal-derived factor-1 in the hematopoietic-supporting activity of human mesenchymal stem cells.

Role of stromal-derived factor-1 in the hematopoietic-supporting activity of human mesenchymal stem cells Human bone marrow (BM) mesenchymal stem cells (MSC) have the capacity to give rise to adipocytes, osteocytes and chondrocytes (1). Re- cent findings indicate that they may have a broader differentiation potential and develop into myo- blasts, endothelial cells as well as non-mesodermal cells such as hepatocytes or neural cells (2). Ex vivo- expanded MSC can be reproducibly generated from adult BM and maintained in undifferentiated state for extended periods of time. Interestingly, undif- ferentiated MSC support the proliferation and differentiation of hematopoietic stem/progenitor cells (HSPC) in vitro (3). These findings may be translated into the possible use of autologous MSC
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Differential gene expression profiling of human bone marrow-derived mesenchymal stem cells during adipogenic development.

Differential gene expression profiling of human bone marrow-derived mesenchymal stem cells during adipogenic development.

Methods Isolation and expansion of human MSC Human mesenchymal stem cells were isolated from iliac crest aspirates obtained from informed and consenting patients undergoing orthopedic surgery (Trousseau Hos- pital, Tours, France) or diagnostic examinations (exam- ined to exclude hematopoietic neoplasmas and histologically diagnosed as normal; Charité-Universitäts- medizin, Berlin, Germany), following a procedure approved by the local ethical committees. As previously described [17], nucleated cells were counted and seeded at a density of 5 × 10 4 per cm 2 culture surface in a- MEM (Invitrogen) supplemented with 10% (v/v) screened fetal bovine serum (FBS), 1 ng/ml basic-fibro- blast growth factor (basic-FGF; AbCys, Paris, France), 20 μmol/l L-glutamine (Invitrogen) and 100 units/ml peni- cillin G (Invitrogen). Cells were incubated under stan- dard culture conditions. On day 3, non-adherent cells were removed by changing the medium; thereafter, the medium was changed twice a week. After reaching con- fluence, cells were trypsinized (0.25% v/v trypsin EDTA; Invitrogen), re-suspended in culture media and seeded at 1 × 10 3 cells per cm 2 (passage 1, P1). The isolation of MSC clones was performed as previously described [2].
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Comprehensive transcriptomic and proteomic characterization of human mesenchymal stem cells reveals source specific cellular markers

Comprehensive transcriptomic and proteomic characterization of human mesenchymal stem cells reveals source specific cellular markers

Anja M. Billing, Hisham Ben Hamidane, Shaima S. Dib, Richard J. Cotton, Aditya M. Bhagwat, Pankaj Kumar, Shahina Hayat, Noha A. Yousri, Neha Goswami, Karsten Suhre, Arash Rafii & Johannes Graumann Mesenchymal stem cells (MSC) are multipotent cells with great potential in therapy, reflected by more than 500 MSC-based clinical trials registered with the NIH. MSC are derived from multiple tissues but require invasive harvesting and imply donor-to-donor variability. Embryonic stem cell-derived MSC (ESC-MSC) may provide an alternative, but how similar they are to ex vivo MSC is unknown. Here we performed an in depth characterization of human ESC-MSC, comparing them to human bone marrow- derived MSC (BM-MSC) as well as human embryonic stem cells (hESC) by transcriptomics (RNA-seq) and quantitative proteomics (nanoLC-MS/MS using SILAC). Data integration highlighted and validated a central role of vesicle-mediated transport and exosomes in MSC biology and also demonstrated, through enrichment analysis, their versatility and broad application potential. Particular emphasis was placed on comparing profiles between ESC-MSC and BM-MSC and assessing their equivalency. Data presented here shows that differences between ESC-MSC and BM-MSC are similar in magnitude to those reported for MSC of different origin and the former may thus represent an alternative source for therapeutic applications. Finally, we report an unprecedented coverage of MSC CD markers, as well as membrane associated proteins which may benefit immunofluorescence-based applications and contribute to a refined molecular description of MSC.
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Naproxen affects osteogenesis of human mesenchymal stem cells via regulation of Indian hedgehog signaling molecules

Naproxen affects osteogenesis of human mesenchymal stem cells via regulation of Indian hedgehog signaling molecules

Omar Salem 1 , Hong Tian Wang 1 , Abdulrahman M Alaseem 1 , Ovidiu Ciobanu 1 , Insaf Hadjab 1,3 , Rahul Gawri 1,4,5 , John Antoniou 1,2 and Fackson Mwale 1,2* Abstract Introduction: We previously showed that type X collagen, a marker of late stage chondrocyte hypertrophy (associated with endochondral ossification), is constitutively expressed by mesenchymal stem cells (MSCs) from osteoarthritis patients and this may be related to Naproxen (Npx), a nonsteroidal anti-inflammatory drug used for therapy. Hedgehog (HH) signaling plays an important role during the development of bone. We tested the hypothesis that Npx affected osteogenic differentiation of human MSCs through the expression of Indian hedgehog (IHH), Patched-1 (PTC1) and GLI family members GLI1, GLI2, GLI3 in vitro.
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Chondrogenic, hypertrophic, and osteochondral differentiation of human mesenchymal stem cells on three‐dimensionally woven scaffolds

Chondrogenic, hypertrophic, and osteochondral differentiation of human mesenchymal stem cells on three‐dimensionally woven scaffolds

6 Center of Regenerative Medicine, Washington University, St. Louis, St. Louis, MO 63110 USA Abstract The development of mechanically functional cartilage and bone tissue constructs of clinically relevant size, as well as their integration with native tissues, remain important challenges for regenerative medicine. The objective of this study was to assess adult human mesenchymal stem cells (MSCs) in large, three dimensionally woven poly( ε-caprolactone) (PCL) scaffolds in proximity to viable bone, both in a nude rat subcutaneous pouch model and under simulated conditions in vitro. In Study I, various scaffold permutations: PCL alone, PCL-bone, “point-of- care” seeded MSC-PCL-bone, and chondrogenically pre-cultured Ch-MSC-PCL-bone constructs were implanted in a dorsal, ectopic pouch in a nude rat. After eight weeks, only cells in the Ch- MSC-PCL constructs exhibited both chondrogenic and osteogenic gene expression profiles. Notably, while both tissue profiles were present, constructs that had been chondrogenically pre- cultured prior to implantation showed a loss of glycosaminoglycan (GAG) as well as the presence of mineralization along with the formation of trabecula-like structures. In Study II of the study, the GAG loss and mineralization observed in Study I in vivo were recapitulated in vitro by the presence of either nearby bone or osteogenic culture medium additives but were prevented by a
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Double porous poly (Ɛ-caprolactone)/chitosan membrane scaffolds as niches for human mesenchymal stem cells

Double porous poly (Ɛ-caprolactone)/chitosan membrane scaffolds as niches for human mesenchymal stem cells

Oxygen consumption A B S T R A C T In this paper, we developed membrane scaffolds to mimic the biochemical and biophysical properties of human mesenchymal stem cell (hMSC) niches to help direct self-renewal and proliferation providing to cells all ne- cessary chemical, mechanical and topographical cues. The strategy was to create three-dimensional membrane scaffolds with double porosity, able to promote the mass transfer of nutrients and to entrap cells. We developed poly (Ɛ-caprolactone) (PCL)/chitosan (CHT) blend membranes consisting of double porous morphology: (i) surface macrovoids (big pores) which could be easily accessible for hMSCs invasion and proliferation; (ii) in- terconnected microporous network to transfer essential nutrients, oxygen, growth factors between the macro- voids and throughout the scaffolds. We varied the mean macrovoid size, effective surface area and surface morphology by varying the PCL/CHT blend composition (100/0, 90/10, 80/20, 70/30). Membranes exhibited macrovoids connected with each other through a microporous network; macrovoids size increased by increasing the CHT wt%. Cells adhered on the surfaces of PCL/CHT 100/0 and PCL/CHT 90/10 membranes, that are characterized by a high effective surface area and small macrovoids while PCL/CHT 80/20 and PCL/CHT 70/30 membranes with large macrovoids and low effective surface area entrapped cells inside macrovoids.
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Global transcriptional profiling of neural and mesenchymal progenitors derived from human embryonic stem cells reveals alternative developmental signaling pathways.

Global transcriptional profiling of neural and mesenchymal progenitors derived from human embryonic stem cells reveals alternative developmental signaling pathways.

The production of mesenchymal precursors (MPC) has not been as widely reported as that of neural precursors. By taking advantages of the recent development of protocols triggering the differentiation of hES toward a near-homogenous amplifiable population of mesenchymal progenitors exhibiting a phenotype of Mesenchymal Stem Cells-like (MSC) can be obtained [18-23]. In this study, we produced highly homogenous cell populations for both neural and mesenchymal precursors by engagement of the hES cells into either the neural or the mesodermal lineages. The analysis of gene expression patterns of these two populations, sharing the same genetic background, compared to the same starting population that were hES cells, using strictly identical procedures for hybridization and statistical analysis, allowed us to select genes that were modulated in opposite directions during commitment to either neural or mesenchymal fates. After this subtractive analysis, selected genes exhibiting modulations specific for either neural or mesenchymal precursors were used to build in silico global gene networks and, using a comparative strategy, to determine their implications as actors in the main signalling pathways involved in early steps of human development.
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Chondrogenic commitment of human umbilical cord blood-derived mesenchymal stem cells in collagen matrices for cartilage engineering

Chondrogenic commitment of human umbilical cord blood-derived mesenchymal stem cells in collagen matrices for cartilage engineering

Tangni Gómez-Leduc 1 , Magalie Hervieu 1 , Florence Legendre 1 , Mouloud Bouyoucef 1 , Nicolas Gruchy 1,2 , Laurent Poulain 3 , Claire de Vienne 4 , Michel Herlicoviez 4 , Magali Demoor 1,* & Philippe Galéra 1,* Umbilical cord blood (UCB) is a promising alternative source of mesenchymal stem cells (MSCs), because UCB-MSCs are abundant and harvesting them is a painless non-invasive procedure. Potential clinical applications of UCB-MSCs have been identified, but their ability for chondrogenic differentiation has not yet been fully evaluated. The aim of our work was to characterize and determine the chondrogenic differentiation potential of human UCB-MSCs (hUCB-MSCs) for cartilage tissue engineering using an approach combining 3D culture in type I/III collagen sponges and chondrogenic factors. Our results showed that UCB-MSCs have a high proliferative capacity. These cells differentiated easily into an osteoblast lineage but not into an adipocyte lineage. Furthermore, BMP-2 and TGF-β1 potentiated chondrogenic differentiation, as revealed by a strong increase in mature chondrocyte- specific mRNA (COL2A1, COL2B, ACAN) and protein (type II collagen) markers. Although growth factors increased the transcription of hypertrophic chondrocyte markers such as COL10A1 and MMP13, the cells present in the neo-tissue maintained their phenotype and did not progress to terminal differentiation and mineralization of the extracellular matrix after subcutaneous implantation in nude mice. Our study demonstrates that our culture model has efficient chondrogenic differentiation, and that hUCB-MSCs can be a reliable source for cartilage tissue engineering.
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Human bone marrow mesenchymal stem cells: a systematic reappraisal via the genostem experience.

Human bone marrow mesenchymal stem cells: a systematic reappraisal via the genostem experience.

Genostem (acronym for Adult mesenchymal stem cells engineering for connective tissue disorders. From the bench to the bed side ) “ ” has been an European consortium of 30 teams working together on human bone marrow Mesenchymal Stem Cell (MSC) biological properties and repair capacity. Part of Genostem activity has been dedicated to the study of basic issues on undifferentiated MSCs properties and on signalling pathways leading to the differentiation into 3 of the connective tissue lineages, osteoblastic, chondrocytic and tenocytic. We have evidenced that native bone marrow MSCs and stromal cells, forming the niche of hematopoietic stem cells, were the same cellular entity located abluminally from marrow sinus endothelial cells. We have also shown that culture-amplified, clonogenic and highly-proliferative MSCs were bona fide stem cells, sharing with other stem cell types the major attributes of self-renewal and of multipotential priming to the lineages to which they can differentiate (osteoblasts, chondrocytes, adipocytes and vascular smooth muscle cells/pericytes). Extensive transcription profiling and in vitro and in vivo assays were applied to identify genes involved in differentiation. Thus we have described novel factors implicated in osteogenesis ( FHL2, ITGA5, Fgf18 ), chondrogenesis ( FOXO1A ) and tenogenesis ( Smad8 ). Another part of Genostem activity has been devoted to studies of the repair capacity of MSCs in animal models, a prerequisite for future clinical trials. We have developed novel scaffolds (chitosan, pharmacologically active microcarriers) useful for the repair of both bone and cartilage. Finally and most importantly, we have shown that locally implanted MSCs effectively repair bone, cartilage and tendon.
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Polysaccharide Hydrogels Support the Long-Term Viability of Encapsulated Human Mesenchymal Stem Cells and Their Ability to Secrete Immunomodulatory Factors

Polysaccharide Hydrogels Support the Long-Term Viability of Encapsulated Human Mesenchymal Stem Cells and Their Ability to Secrete Immunomodulatory Factors

and Human HGF Duo Set ELISA were purchased from Cayman Chemical and R&D Systems, respectively. 2.2. Preparation of Particles. Alginate particles were obtained using a dropwise method in CaCl 2 as previously described [21]. First, sodium alginate was sterilized by steaming (134 ° C, 4 minutes) and then dissolved in sterile PBS (2% w/v). Particles were obtained by extruding this solution through a 31G needle into a stirred solution of 100 mM calcium chloride. After 15 minutes, alginate parti- cles were collected by filtration, washed in HEPES buffer, and stored at room temperature. Synthesis of Si-HPMC was performed by grafting 14.24% (w/w) of GPTMS onto HPMC in heterogeneous medium as previously described [22]. Si-HPMC powder was solubilized (3% w/v) in 0.2 M NaOH under constant stirring for 48 h. The solution was then sterilized by steaming (121 ° C, 20 minutes). To initiate the formation of a cross-linked Si-HPMC, the solution was mixed with 0.5 volume of 0.26 M HEPES buffer (pH 3.6). To obtain Si-HPMC particles, an oil dispersion protocol was performed. The solution of Si-HPMC/HEPES was injected into olive oil under stirring. To optimize the encapsulation process, three dispersion parameters were studied: time (1 h or 1 h and 30 min), temperature (room temperature or 37 ° C), and stirring speed (250 rpm or 400 rpm). Si-HPMC particles were collected by filtration, washed in HEPES buffer, and stored at room temperature.
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Proangiogenic and Prosurvival Functions of Glucose in Human Mesenchymal Stem Cells upon Transplantation

Proangiogenic and Prosurvival Functions of Glucose in Human Mesenchymal Stem Cells upon Transplantation

Figure 6. Glucose enhanced hypoxia inducible factor 1a (Hif-1a) bioactivity and vascularization of 3D constructs: (A): Quantification and imaging (at day 14) of Hif-1a expression by human mesenchymal stem cell (hMSC) seeded in poly-acrylonitrile-sodium methallyl sulfonate scaf- folds filled with fibrin gel loaded (black bars) or not (white bars) with glucose. (B): Representative histology results of peripheral vascularization (black arrows) of MSCs containing constructs loaded with glucose after 2 weeks of subcutaneous implantation in mice. Stain: Hematoxylin-Eo- sin-Safran. Magnification: 2 including magnification (10) of region of interest. (C): Representative photograph of the 3D construct when loaded without (left photograph) or with (right photograph) glucose at day 14 postimplantation. (D): Immunostaining of blood vessels using iso- lectin B4, magnification: 10. (E): Peripheral blood vessels quantification of the cell containing constructs filled with either hyaluronic acid or fibrin gel and loaded without or with glucose. A striking increase of peripheral blood vessels was observed in cell-containing constructs loaded with glucose independently of the type of the hydrogel used (i.e., hyaluronic acid or fibrin gel). Data are represented as mean 6 SD. *, p < .05; **, p < .001. Abbreviation: BLI, bioluminescence.
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25-Hydroxyvitamin D₃ induces osteogenic differentiation of human mesenchymal stem cells

25-Hydroxyvitamin D₃ induces osteogenic differentiation of human mesenchymal stem cells

extracted by 50 μ l of 0.1% sodium dodecyl sulphate per well and was stored at − 80 °C. The protein concentration was measured by a BCA Protein Assay Kit (Pierce, Rockford, IL, USA). The ALPL activity was normalised against the total cellular protein concentration and expressed as relative levels against the solvent control. ELISA assay. Cells were cultured and differentiated in 6-well plates for 7, 14, 21, and 28 days. At each time point, the conditioned media were collected and stored in aliquots at − 80 °C for the subsequent determinations of bone gamma-carboxyglutamate protein. The cells were washed once with PBS and lysed with a RIPA buffer containing protease inhibitor cocktail (Pierce, Rockford, IL, USA). The cell lysates were stored in aliquots at − 80 °C for the subsequent measurements of protein concentration and immunoblotting of RUNX2 protein. After day 28, all the conditioned media were diluted 1 to 10 in the sample diluents provided by a Gla-type osteocalcin EIA kit and analysed in duplicate by the Gla-type osteocalcin EIA kit (Zymed Laboratories, Carlsbad, CA, USA) according to the manufactures’ protocol. The readings of the samples in the ELISA measurement were within the range of the standards (0–16 ng/ml). The protein concentrations in the cell lysates were measured in duplicate by a BCA protein assay kit. Subsequently the cell lysates were used in immunoblotting of RUNX2. The bone gamma-carboxyglutamate protein concentrations were normalised against the cellular protein concentration and expressed as ng bone gamma-carboxyglutamate protein per mg cellular protein.
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Hepatocytic Differentiation Potential of Human Fetal Liver Mesenchymal Stem Cells: In Vitro and In Vivo Evaluation

Hepatocytic Differentiation Potential of Human Fetal Liver Mesenchymal Stem Cells: In Vitro and In Vivo Evaluation

3. Results 3.1. Characterization of Isolated Naive FL-MSCs. Four differ- ent fetal livers (GW 11-12) were digested using collagenase and cells were isolated. For all donors, cell viability estimated by trypan blue exclusion always exceeded 90%. After subsequent plating and first enrichment by plastic adherence, cells with fibroblastic shape started proliferating on the third day after plating and confluence was reached after 7–10 days (Figure 1(a)). While emerging cell populations were heteroge- neous before the first detachment, cells presenting mesenchy- mal morphology became predominant after the first passage (Figure 1(a)). The mesenchymal phenotype of the cells was then demonstrated using flow cytometry which revealed their immunopositivity for CD73, CD90, and CD146 and immunonegativity for hematopoietic CD45 and endothelial CD34 and CD31 markers (Figure 1(b)). Using immunofluo- rescence, we also showed that the whole FL-MSC population was immunopositive for vimentin and nestin (type III and VI intermediate filaments, resp.), well described in MSC of other sources (Figure 2). We also demonstrated that smooth muscle (SM) cell markers calponin, 𝛼SM actin, and desmin are positively expressed in all analyzed passages. Upon adi- pogenic differentiation, FL-MSCs accumulated intracellular lipid droplets as revealed by oil red O staining (Figures 3(a) and 3(b)). Upon osteogenic differentiation, differentiated FL- MSCs showed calcium phosphate precipitates as revealed by alkaline phosphatase activity (Figures 3(c) and 3(d)) and alizarin red staining (Figures 3(e) and 3(f)). All these features confirmed the MSC phenotype of the fibroblastoid cells we isolated from fetal liver as previously reported by our team [8].
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Human bone marrow mesenchymal stem cells regulate biased DNA segregation in response to cell adhesion asymmetry.

Human bone marrow mesenchymal stem cells regulate biased DNA segregation in response to cell adhesion asymmetry.

The observations made on fixed cells were confirmed with live-cell video recording during which rare mitotic events were easier to capture and quantify. Cell divisions were monitored during 20 hr using phase contrast video microscopy ( Figure S1 ). Chromosome alignment along the metaphase plate was visible in phase contrast. On both symmetric and asymmetric micropat- terns, metaphase plates were oriented orthogonally to the long cell axis ( Figure S1 ). This corresponded to an asymmetric orien- tation on asymmetric micropatterns because the two spindle poles were in proximity of parts of the cell cortex that were not symmetrical. In addition, as previously observed, the metaphase plate was off-centered toward the large adhesive area on the asymmetric micropattern (95% confidence interval [CI] was be- tween 3.6 and 9.7 mm away from the center) and centered on symmetric micropatterns (95% CI was between 4.7 and 1.6 mm around the center) ( Figure 1 E). Thus, spindle positioning on asymmetric micropatterns displayed most of the typical fea- tures associated with asymmetric cell division ( Morin and Bel- laı¨che, 2011; Inaba and Yamashita, 2012 ).
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Chip-Based Comparison of the Osteogenesis of Human Bone Marrow- and Adipose Tissue-Derived Mesenchymal Stem Cells under Mechanical Stimulation

Chip-Based Comparison of the Osteogenesis of Human Bone Marrow- and Adipose Tissue-Derived Mesenchymal Stem Cells under Mechanical Stimulation

Recently, microscale engineering has been increasingly used to mimic the cellular microenvironment with high spatiotemporal precision and to present cells with mechanical and biochemical signals [27–29]. These approaches were performed on a chip provide microenvironments that attempt to partially mimic human organs, such as blood vessels, muscles, airways, liver, brain, gut, kidney, and bones. For example, a lung-on-a-chip system was designed to mimic breathing by applying vacuum to side chambers, stretching porous membranes to stimulate cells seeded on the both sides of the membrane [28]. The microdevice replicates dynamic mechanical distortion of the alveolar-capillary interface for inflammatory and toxicology applications. In bone tissue engineering, various static and dynamic mechanical stimuli based on microfabrication technology have been tested with cultured stem cells or precursor cells for understanding osteogenic mechanisms and molecular pathways [30–33]. Micropatterns and structures giving rise to gradients of static mechanical stresses can also be used to pattern lineages (osteogensis in high stress areas and adipogenesis in low stress areas) of stem cells [30]. Osteoblasts on nanotexture under mechanical loading upregulated fibronectin and Cfba expression [31]. A continuous-perfusion microchip enhanced mouse osteoblastic cells in terms of ALP activity with shear stress [32]. A three-dimensional (3D) culture system with poly(ethylene glycol) hydrogel in multilayered polymeric micro- devices, capable of simultaneously applying a range of cyclic, compressive mechanical forces to mouse MSC, was demonstrated [33]. This system has an advantage in conducting mechanically active experiments in 3D culture environments. However, the system requires many complex steps to form cell-loaded cylindrical hydrogels in the microdevice and ultraviolet (UV) exposure, which may decrease cell viability. In our previous studies, we also developed microscale platforms actuated by electromagnetic and pneumatic forces to provide cyclic compressive stimuli to cells, and demonstrated that hMSCs were enhanced in terms of chondro- genic and osteogenic differentiation [22,23]. However, there are still limits in heat generation and the manual closing of the fluidic channels, which prompt the need to continue to improve the utility of such systems, as well as to expand the scope of applications, such as that explored here for stem cell comparative outcomes.
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Human adipose tissue-derived mesenchymal stem cells acquire muscle identity only after spontaneous fusion with myoblasts

Human adipose tissue-derived mesenchymal stem cells acquire muscle identity only after spontaneous fusion with myoblasts

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