Differentiation Of Human Mesenchymal Stem Cells Research Articles

Tailored Physicochemical Cues Direct Human Mesenchymal Stem Cell Differentiation through Epigenetic Regulation Using Colloidal Self-Assembled Patterns.

The extracellular matrix (ECM) shapes the stem cell fate during differentiation by exerting relevant biophysical cues. However, the mechanism of stem cell fate decisions in response to ECM-backed complex biophysical cues has not been fully understood due to the lack of versatile ECMs. Here, we designed two versatile ECMs using colloidal self-assembly technology to probe the mechanisms of their effects on mechanotransduction and stem cell fate regulation. Binary colloidal crystals (BCC) with a hexagonally close-packed structure, composed of silica (5 μm) and polystyrene (0.4 μm) particles as well as a polydimethylsiloxane-embedded BCC (BCCP), were fabricated. They have defined surface chemistry, roughness, stiffness, ion release, and protein adsorption properties, which can modulate the cell adhesion, proliferation, and differentiation of human adipose-derived stem cells (hASCs). On the BCC, hASCs preferred osteogenesis at an early stage but showed a higher tendency toward adipogenesis at later stages. In contrast, the results of BCCP diverged from those of BCC, suggesting a unique regulation of ECM-dependent mechanotransduction. The BCC-mediated cell adhesion reduced the size of the focal adhesion complex, accompanying an ordered spatial organization and cytoskeletal rearrangement. This morphological restriction led to the modulation of mechanosensitive transcription factors, such as c-FOS, the enrichment of transcripts in specific signaling pathways such as PI3K/AKT, and the activation of the Hippo signaling pathway. Epigenetic analyses showed changes in histone modifications across different substrates, suggesting that chromatin remodeling participated in BCC-mediated mechanotransduction. This study demonstrates that BCCs are versatile artificial ECMs that can regulate human stem cells' fate through unique biological signaling, which is beneficial in biomaterial design and stem cell engineering.

Increased Intracellular Insulin from Differentiated Stem Cells to Insulin- Producing Cells Incorporating Β-Carotene

Analyzes the effect of β-Carotene (BC) in the processes of differentiation of adipose tissue Human Mesenchymal Stem Cells (hADMSCs) and dental pulp Human Mesenchymal Stem Cells (hDPMSCs) for obtaining Insulin-Producing Cells (IPCs). The BC present in the methanolic extract was analyzed and quantified by UV-VIS, TLC and HPLC methods. The cell viability test was done by flow cytometry, a concentration of 1.5 μM of BC or AP extract no cytotoxic effect on stem cells, so this concentration was used for the Human Mesenchymal Stem Cells (hMSCs) differentiation protocols for obtaining IPCs. The differentiation tests were done with the AP extract or β-carotene standard addition which demonstrates the increased cell differentiation percentages by the presence of intracellular insulin. This was more outstanding in the hMSCs that were stimulated with the AP in stage 3 and applied at all stages mainly in hDPMSCs, reaching up to 74% of the insulin-positive population respect to 11% where the molecules were not added. RT-PCR was performed for the level of gene expression like MAFA, PDX1 and NKX6.1 and those are found well expressed on differentiated cells. This could be relevant in cell therapy in diabetes, where inclusion of AP extracts or BC may improve yield of IPCs.

Manipulating Stem Cell Fate with Disordered Bioactive Cues on Surfaces: The Role of Bioactive Ligand Selection.

The development of 2D or 3D bioactive platforms for rapidly isolating pure populations of cells from adult stem cells holds promise for advancing the understanding of cellular mechanisms, drug testing, and tissue engineering. Over the years, methods have emerged to synthesize bioactive micro- and nanostructured 2D materials capable of directing stem cell fate. We introduce a novel method for randomly micro- or nanopatterning any protein/peptide onto both 2D and 3D scaffolds via spray technology. Our goal is to investigate the impact of arranging bioactive micropatterns (ordered vs disordered) on surfaces to guide human mesenchymal stem cell (hMSC) differentiation. The spray technology efficiently coats materials with controlled, cost-effective bioactive micropatterns in various sizes and shapes. BMP-2 mimetic peptides were covalently grafted, individually or in combination with RGD peptides, onto activated polyethylene terephthalate (PET) surfaces through a spraying process, incorporating nano/microscale parameters like size, shape, and composition. The study explores different peptide distributions on surfaces and various peptide combinations. Four surfaces were homogeneously functionalized with these peptides (M1 to M4 with various densities of peptides), and six surfaces with disordered micro- and nanopatterns of peptides (S0 to S5 with different sizes of peptide patterns) were synthesized. Fluorescence microscopy assessed peptide distribution, followed by hMSC culture for 2 weeks, and evaluated osteogenic differentiation via immunocytochemistry and RT-qPCR for osteoblast and osteocyte markers. Cells on uniformly peptide-functionalized surfaces exhibited cuboidal forms, while those on surfaces with disordered patterns tended toward columnar or cuboidal shapes. Surfaces S4 and S5 showed dendrite-like formations resembling an osteocyte morphology. S5 showed significant overexpression of osteoblast (OPN) and osteocyte markers (E11, DMP1, and SOST) compared to control surfaces and other micropatterned surfaces. Notably, despite sharing an equivalent quantity of peptides with a homogeneous functionalized surface, S5 displayed a distinct distribution of peptides, resulting in enhanced osteogenic differentiation of hMSCs.

Dysregulated lncRNAs regulate human umbilical cord mesenchymal stem cell differentiation into insulin-producing cells by forming a regulatory network with mRNAs

ObjectiveIn recent years, cell therapy has emerged as a new research direction in the treatment of diabetes. However, the underlying molecular mechanisms of mesenchymal stem cell (MSC) differentiation necessary to form such treatment have not been clarified.MethodsIn this study, human umbilical cord mesenchymal stem cells (HUC-MSCs) isolated from newborns were progressively induced into insulin-producing cells (IPCs) using small molecules. HUC-MSC (S0) and four induced stage (S1–S4) samples were prepared. We then performed transcriptome sequencing experiments to obtain the dynamic expression profiles of both mRNAs and long noncoding RNAs (lncRNAs).ResultsWe found that the number of differentially expressed lncRNAs and mRNAs trended downwards during differentiation. Gene Ontology (GO) analysis showed that the target genes of differentially expressed lncRNAs were associated with translation, cell adhesion, and cell connection. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the NF-KB signalling pathway, MAPK signalling pathway, HIPPO signalling pathway, PI3K–Akt signalling pathway, and p53 signalling pathway were enriched in these differentially expressed lncRNA-targeting genes. We also found that the coexpression of the lncRNA CTBP1-AS2 with PROX1 and the lncRNAs AC009014.3 and GS1-72M22.1 with JARID2 mRNA was related to the development of pancreatic beta cells. Moreover, the coexpression of the lncRNAs: XLOC_ 050969, LINC00883, XLOC_050981, XLOC_050925, MAP3K14- AS1, RP11-148K1.12, and CTD2020K17.3 with p53, regulated insulin secretion by pancreatic beta cells.ConclusionIn this study, HUC-MSCs combined with small molecule compounds were successfully induced into IPCs. Differentially expressed lncRNAs may regulate the insulin secretion of pancreatic beta cells by regulating multiple signalling pathways. The lncRNAs AC009014.3, Gs1-72m21.1, and CTBP1-AS2 may be involved in the development of pancreatic beta cells, and the lncRNAs: XLOC_050969, LINC00883, XLOC_050981, XLOC_050925, MAP3K14-AS1, RP11-148K1.12, and CTD2020K17.3 may be involved in regulating the insulin secretion of pancreatic beta cells, thus providing a lncRNA catalogue for future research regarding the mechanism of the transdifferentiation of HUC-MSCs into IPCs. It also provides a new theoretical basis for the transplantation of insulin-producing cells into diabetic patients in the future.Graphical

PBF-LB fabrication of microgrooves for induction of osteogenic differentiation of human mesenchymal stem cells

Stem cell differentiation has important implications for biomedical device design and tissue engineering. Recently, inherent material properties, including surface chemistry, stiffness, and topography, have been found to influence stem cell fate. Among these, surface topography is a key regulator of stem cells in contact with materials. The most important aspect of ideal bone tissue engineering is to control the organization of the bone extracellular matrix with fully differentiated osteoblasts. Here, we found that laser powder bed fusion (PBF-LB)-fabricated grooved surface inspired by the microstructure of bone, which induced human mesenchymal stem cell (hMSC) differentiation into the osteogenic lineage without any differentiation supplements. The periodic grooved structure was fabricated by PBF-LB which induced cell elongation facilitated by cytoskeletal tension along the grooves. This resulted in the upregulation of osteogenesis via Runx2 expression. The aligned hMSCs successfully differentiated into osteoblasts and further organized the bone mimetic-oriented extracellular matrix microstructure. Our results indicate that metal additive manufacturing technology has a great advantage in controlling stem cell fate into the osteogenic lineage, and in the construction of bone-mimetic microstructural organization. Our findings on material-induced stem cell differentiation under standard cell culture conditions open new avenues for the development of medical devices that realize the desired tissue regeneration mediated by regulated stem cell functions.

MICRORNAS 125B, 199A-5P, AND 214 AS MODULATORS OF BONE HOMEOSTASIS

Bone homeostasis is a highly regulated process involving pathways in bone as WNT, FGF or BMP, but also requiring support from surrounding tissues as vessels and nerves. In bone diseases, the bone-vessel-nerve triad is impacted. Recently, new players appeared as regulators of bone homeostasis: microRNAs (miRNA). Five miRNAs associated with osteoporotic fractures are already known, among which miR-125b is decreasing bone formation by downregulating human mesenchymal stem cells (hMSCs) differentiation. Other miRNAs, as miR-214 (in cluster with miR-199a), are secreted by osteoclasts to regulate osteoblasts and inhibit bone formation. This forms a very complex regulatory network.hMSCs and osteoblasts (n=3) were transfected with mimic/antagomiR of miR-125b, miR-199a-5p or miR-214, or with a scrambled miRNA (negative control) in osteogenic differentiation calcium-enriched medium (Ca++). Mineralization was assessed by Alizarin Red/CPC staining, miRNA expression by qPCR and protein by western blotting.Exposure of hMSCs or osteoblasts to Ca++ increased mineralization compared to basal medium. hMSCs transfected with miR-125b mimic in Ca++ presented less mineralization compared to scramble. This correlated with decreased levels of BMPR2 and RUNX2. hMSCs transfected with miR-125b inhibitor presented higher mineralization. Interestingly, hMSCs transfected with miR-214 mimic in Ca++ presented no mineralization while miR-214 inhibitor increased mineralization. No differences were observed in hMSCs transfected with miR-199a-5p modulators. On the contrary, osteoblasts transfected with miR-199a-5p mimic present less mineralization than scrambled-transfected and same was observed for miR-214 and miR-125b mimics.We highlight that miR-125b and miR-214 decrease mineralization of hMSCs in calcium-enriched medium. We noticed that miR-199a-5p is able to regulate mineralization in osteoblasts but not in hMSCs suggesting that this effect is cell-specific. Interestingly, the cluster miR-199a/214 is known as modulator of vascular function and could thus contribute to bone remodeling via different ways. With this work we slightly open the door to possible therapeutic approaches for bone diseases.

Resveratrol promotes the differentiation of human umbilical cord mesenchymal stem cells into esophageal fibroblasts via AKT signaling pathway.

Objectives: Resveratrol has been implicated in the differentiation and development of human umbilical cord mesenchymal stem cells. The differentiation of into esophageal fibroblasts is a promising strategy for esophageal tissue engineering. However, the pharmacological effect and underlying mechanism of resveratrol on human umbilical cord mesenchymal stem cells differentiation are unknown. Here, we investigated the effects and mechanism of resveratrol on the differentiation of human umbilical cord mesenchymal stem cells. Methods: Using a transwell-membrane coculture system to culture human umbilical cord mesenchymal stem cells and esophageal fibroblasts, we examined how resveratrol act on the differentiation of human umbilical cord mesenchymal stem cells. Immunocytochemistry, Sirius red staining, quantitative real-time PCR, and Western blotting were performed to examine collagen synthesis and possible signaling pathways in human umbilical cord mesenchymal stem cells. Results: We found that resveratrol promoted collagen synthesis and AKT phosphorylation. However, co-treatment of cells with resveratrol and the PI3K inhibitor LY294002 inhibited collagen synthesis and AKT phosphorylation. We demonstrated that resveratrol down-regulated the expression of IL-6, TGF-β, caspase-9, and Bax by activating the AKT pathway in human umbilical cord mesenchymal stem cell. Furthermore, resveratrol inhibited phosphorylated NF-ĸB in human umbilical cord mesenchymal stem cells. Conclusion: Our data suggest that resveratrol promotes the differentiation of human umbilical cord mesenchymal stem cells into fibroblasts. The underlying mechanism is associated with the downregulation of IL-6 and TGF-β via the AKT pathway and by inhibiting the NF-ĸB pathway. Resveratrol may be useful for esophageal tissue engineering.

CREB1 Facilitates GABAergic Neural Differentiation of Human Mesenchymal Stem Cells through BRN2 for Pain Alleviation and Locomotion Recovery after Spinal Cord Injury.

The transplantation of GABAergic neuron cells has been reported to alleviate nerve pain and improve motor function after spinal cord injury (SCI). However, human mesenchymal stem cell (hMSC) differentiation into GABAergic neuron cells in a sufficient quantity remains to be accomplished. From a database screening, cAMP-responsive element-binding protein 1 (CREB1) was chosen as a potential modulator due to its critical role in the protein-protein interaction of genes related to GABAergic neural differentiation. Here, CREB1 was overexpressed in transfected hMSCs, where CREB1 could induce differentiation into GABAergic neuron cells with an upregulation of Map2 and GAD1 by 2- and 3.4-fold, respectively. Additionally, GABAergic neural differentiation was enhanced, while Notch signaling was inhibited, and BRN2 transcriptional activation played an important role in neuronal maturation. Moreover, transfected hMSCs injected into immunocompromised mice caused by CsA exhibited the neuronal markers Tuj1 and Map2 via the intraspinal route, suggesting an improvement in survival and neural differentiation. Significantly, improvement in both BMS scores (6.2 ± 1.30 vs. 4 ± 0) and thermal hyperalgesia latency (7.74 ± 2.36 s vs. 4.52 ± 0.39 s) was seen compared with the SCI naïve treatment at 4 weeks post-transplantation. Our study demonstrates that CREB1 is crucial in generating induced GABAergic neuron cells (iGNs) originating from hMSCs. Transplanting iGNs to injured spinal cord provides a promising strategy for alleviating neuropathic pain and locomotion recovery after SCI.

Pueraria lobata-derived exosome-like nanovesicles alleviate osteoporosis by enhacning autophagy

Osteoporosis (OP) is the most common bone disorder worldwide, especially in postmenopausal women. However, many OP drugs are not suitable for long term use due to major adverse effects. Therefore, there is an urgent need to identify more effective and safe therapeutic drugs. Pueraria lobata has been reported to promote osteoblast growth in bone regeneration, but the exact mechanisms still need further exploration. The current study found that Pueraria lobata-derived exosome-like nanovesicles (PELNs) promoting primary human bone mesenchymal stem cells (hBMSCs) differentiation and mineralization both in vitro and in ovariectomized (OVX)-induced osteoporotic rats. Interestingly, the relative abundance of harmful strains significantly decreased in the intestine of the osteoporosis SD rat model administrated PELNs via the regulation of trimethylamine-N-oxide (TMAO), a metabolite of gut microbiota. Moreover, RNA sequencing revealed that the osteogenic activity of PELNs is revealed to autophagy signaling. In vitro and in vivo experiments also showed that the treatment with PELNs promoted the differentiation and function of hBMSCs by elevating autophagy via the degradation of TMAO. Collectively, PELNs demonstrate promise as a therapeutic approach for OP, with TMAO emerging as a potential target of OP treatment.

Parvovirus B19 Infection May Potentially Determine the Fate of Hematopoiesis by Altering the Human Bone Marrow Mesenchymal Stem Cells Differentiation

Background: Human bone marrow mesenchymal stem cells (hBM-MSCs), as supporters for hematopoiesis, differentiate into osteoblasts and adipocytes. Studies showed that infection of hBM-MSCs by Parvovirus B19 (B19V) can affect the differentiation capability of hBM-MSCs. This study aims to evaluate B19V effects on the differentiation of hBM-MSCs.
 Materials and Methods: In this experimental study hBM-MSCs were cultured up to passage 3. Nucleofection was subsequently employed to deliver a plasmid containing the B19V genome into the cells. The transfected cells were then differentiated into osteoblast and adipocyte lineages. qRT-PCR was then performed to analyze the differentiation 14 days after transfection.
 Results: On the 14th day after induction the findings demonstrated a significant increase in adipocyte-specific (PPARγ and LPL) gene expression compared to the control group (p<0.05) and a slight but not statistically significant decrease in the expression of the osteocyte-specific genes (RUNX2 and osteocalcin) (p>0.05).
 Conclusion: The results suggest that B19V infection can promote the differentiation of hBM-MSCs towards adipocytes and affect the bone marrow microenvironment as well as hematopoiesis

Regulation of Osteoimmune Microenvironment and Osteogenesis by 3D-Printed PLAG/black Phosphorus Scaffolds for Bone Regeneration.

The treatment of bone defects remains a significant challenge to be solved clinically. Immunomodulatory properties of orthopedic biomaterials have significance in regulating osteoimmune microenvironment for osteogenesis. A lactic acid-co-glycolic acid (PLGA) scaffold incorporates black phosphorus (BP) fabricated by 3D printing technology to investigate the effect of BP on osteoimmunomodulation and osteogenesis in site. The PLGA/BP scaffold exhibits suitable biocompatibility, biodegradability, and mechanical properties as an excellent microenvironment to support new bone formation. The studies' result also demonstrate that the PLGA/BP scaffolds are able to recruit and stimulate macrophages M2polarization, inhibit inflammation, and promote human bone marrow mesenchymal stem cells (hBMSCs) proliferation and differentiation, which in turn promotes bone regeneration in the distal femoral defect region of steroid-associated osteonecrosis (SAON) rat model. Moreover, it is screened and demonstrated that PLGA/BP scaffolds can promote osteogenic differentiation by transcriptomic analysis, and PLGA/BP scaffolds promote osteogenic differentiation and mineralization by activating PI3K-AKTsignaling pathway in hBMSC cells. In this study, it is shown that the innovative PLGA/BP scaffolds are extremely effective in stimulating bone regeneration by regulating macrophage M2 polarization and a new strategy for the development of biomaterials that can be used to repair bone defects is offered.

Human alveolar bone-derived mesenchymal stem cell cultivation on a 3D-printed PDLLA scaffold for bone formation

This study aimed to assess effects of 3-dimensionally (3D) printed poly-d,l-lactin (PDLLA) on human alveolar bone-derived mesenchymal stem cell (h-ABMSC) osteogenic proliferation and differentiation. Human ABMSCs were cultured and identified using flow cytometry and morphological analysis. Control and PDLLA experimental groups were assessed using a Cell Counting Kit-8 (CCK-8) to detect cellular cytotoxicity and proliferative activity. Real-time quantitative polymerase chain reaction was used to determine expression levels of osteogenesis genes including alkaline phosphatase (ALP), Runt-related transcription factor 2 (Runx-2), osteopontin (OPN), and osteocalcin (OCN). The results showed that h-ABMSCs were successfully cultured and revealed by microscopic observation. Human ABMSCs were spindle-shaped, with clustered and fish-like primary cells. Cell surface markers were negative for CD34 and positive for CD44 and CD90. PDLLA had no cytotoxicity. Human ABMSCs proliferated normally, and osteogenic differentiation of the cells was observed on the surface of PDLLA. Cellular proliferative activity and expression levels of osteogenesis-related genes of PDLLA and control groups showed no significant difference, including ALP, Runx-2, OPN, and OCN. These results suggest that 3D-printed PDLLA has good cell compatibility and biological activity.

Non-Specific Inhibition of Dipeptidyl Peptidases 8/9 by Dipeptidyl Peptidase 4 Inhibitors Negatively Affects Mesenchymal Stem Cell Differentiation.

DPP4 may play a relevant role in MSC differentiation into osteoblasts or adipocytes. Dipeptidyl peptidase 4 (DPP4) inhibitors (DPP4i), such as sitagliptin and vildagliptin, are used as antidiabetic drugs. However, vildagliptin is not a specific DPP4i and also inhibits DPP8/9, which is involved in energy metabolism and immune regulation. The aim of this study is to evaluate how sitagliptin, vildagliptin or 1G244 (a DPP8/9 specific inhibitor) may influence cell viability, as well as osteogenic and adipogenic differentiation in human mesenchymal stem cells (MSC). Viability, apoptosis, osteoblastogenesis and adipogenesis markers, as well as protein synthesis of β-catenin, were studied in MSC cultures induced to differentiate into osteoblasts or adipocytes in the presence or absence of sitagliptin, vildagliptin or 1G244. The two tested DPP4i did not affect MSC viability, but 1G244 significantly decreased it in MSC and osteoblast-induced cells. Additionally, 1G244 and vildagliptin inhibited osteogenesis and adipogenesis, unlike sitagliptin. Therefore, inhibition of DPP4 did not affect MSC viability and differentiation, whereas inhibition of DPP8/9 negatively affected MSC. To the best of our knowledge, these results show for the first time that DPP8/9 have an important role in the viability and differentiation of human MSC. This data can be considered for human clinical use of drugs affecting DPP8/9 activity.

Histone Modification of Osteogenesis Related Genes Triggered by Substrate Topography Promotes Human Mesenchymal Stem Cell Differentiation.

The clinical success of orthopedic implants is closely related to their integration in the bone tissue promoted by rough device surfaces. The biological response of precursor cells to their artificial microenvironments plays a critical role in this process. In this study, we elucidated the relation between cell instructivity and surface microstructure of polycarbonate (PC)-based model substrates. The rough surface structure (hPC) with an average peak spacing (Sm) similar to the trabecular spacing of trabecular bone improved osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs), as compared to the smooth surface (sPC) and the surface with a moderate Sm value (mPC). The hPC substrate promoted the cell adhesion and assembling of F-actin and enhanced cell contractile force by upregulating phosphorylated myosin light chain (pMLC) expression. The increased cell contractile force led to YAP nuclear translocation and the elongation of cell nuclei, presenting higher levels of active form of Lamin A/C. The nuclear deformation alternated the histone modification profile, particularly the decrease of H3K27me3 and increase of H3K9ac on the promoter region of osteogenesis related genes (ALPL, RUNX2, and OCN). Mechanism study using inhibitors and siRNAs elucidated the role of YAP, integrin, F-actin, myosin, and nuclear membrane proteins in such a regulatory process of surface topography on stem cell fate. These mechanistical insights on the epigenetic level give a new perspective in understanding of the interaction of substrate and stem cells as well as provide valuable criteria for designing bioinstructive orthopedic implants.

Tauroursodeoxycholic Acid Enhances Osteogenic Differentiation through EGFR/p-Akt/CREB1 Pathway in Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are pluripotent stromal cells that are among the most appealing candidates for regenerative medicine and may aid in the repair and regeneration of skeletal disorders through multiple mechanisms, including angiogenesis, differentiation, and response to inflammatory conditions. Tauroursodeoxycholic acid (TUDCA) has recently been used in various cell types as one of these drugs. The mechanism of osteogenic differentiation by TUDCA in hMSCs remains unknown. Cell proliferation was performed by the WST-1 method, and alkaline phosphatase activity and alizarin red-sulfate staining were used to confirm the osteogenic differentiation indicator. Expression of genes related to bone differentiation and specific genes related to signaling pathways was confirmed by quantitative real-time polymerase chain reaction. We found that cell proliferation was higher as the concentration increased, and showed that the induction of osteogenic differentiation was significantly enhanced. We also show that osteogenic differentiation genes were upregulated, with the expression of the epidermal growth factor receptor (EGFR) and cAMP responsive element binding protein 1 (CREB1) being specifically high. To confirm the participation of the EGFR signaling pathway, the osteogenic differentiation index and expression of osteogenic differentiation genes were determined after using an EGFR inhibitor. As a result, EGFR expression was remarkably low, and that of CREB1, cyclin D1, and cyclin E1 was also significantly low. Therefore, we suggest that TUDCA-induced osteogenic differentiation of human MSCs is enhanced through the EGFR/p-Akt/CREB1 pathway.

Controlling differentiation of stem cells via bioactive disordered cues.

Ideal bone tissue engineering is to induce bone regeneration through the synergistic integration of biomaterial scaffolds, bone progenitor cells, and bone-forming factors. Biomimetic scaffolds imitate the native extracellular matrix (ECM) and are often utilized in vitro as analogues of the natural ECM to facilitate investigations of cell-ECM interactions and processes. In vivo, the cellular microenvironment has a crucial impact on regulating cell behavior and functions. A PET surface was activated and then functionalized with mimetic peptides to promote human mesenchymal stem cell (hMSC) adhesion and differentiation into an osteogenic lineage. Spray technology was used to randomly micropattern peptides (RGD and BMP-2 mimetic peptides) on the PET surface. The distribution of the peptides grafted on the surface, the roughness of the surfaces and the chemistry of the surfaces in each step of the treatment were ascertained by atomic force microscopy, fluorescence microscopy, time-of-flight secondary ion mass spectrometry, Toluidine Blue O assay, and X-ray photoelectron spectroscopy. Subsequently, cell lineage differentiation was evaluated by quantifying the expression of immunofluorescence markers: osteoblast markers (Runx-2, OPN) and osteocyte markers (E11, DMP1, and SOST). In this article, we hypothesized that a unique combination of bioactive micro/nanopatterns on a polymer surface improves the rate of morphology change and enhances hMSC differentiation. In DMEM, after 14 days, disordered micropatterned surfaces with RGD and BMP-2 led to a higher osteoblast marker expression than surfaces with a homogeneous dual peptide conjugation. Finally, hMSCs cultured in osteogenic differentiation medium (ODM) showed accelerated cell differentiation. In ODM, our results highlighted the expression of osteocyte markers when hMSCs were seeded on PET surfaces with random micropatterns.

Direct 3D printed biocompatible microfluidics: assessment of human mesenchymal stem cell differentiation and cytotoxic drug screening in a dynamic culture system

BackgroundIn vivo-mimicking conditions are critical in in vitro cell analysis to obtain clinically relevant results. The required conditions, comparable to those prevalent in nature, can be provided by microfluidic dynamic cell cultures. Microfluidics can be used to fabricate and test the functionality and biocompatibility of newly developed nanosystems or to apply micro- and nanoelectromechanical systems embedded in a microfluidic system. However, the use of microfluidic systems is often hampered by their accessibility, acquisition cost, or customization, especially for scientists whose primary research focus is not microfluidics.ResultsHere we present a method for 3D printing that can be applied without special prior knowledge and sophisticated equipment to produce various ready-to-use microfluidic components with a size of 100 µm. Compared to other available methods, 3D printing using fused deposition modeling (FDM) offers several advantages, such as time-reduction and avoidance of sophisticated equipment (e.g., photolithography), as well as excellent biocompatibility and avoidance of toxic, leaching chemicals or post-processing (e.g., stereolithography). We further demonstrate the ease of use of the method for two relevant applications: a cytotoxicity screening system and an osteoblastic differentiation assay. To our knowledge, this is the first time an application including treatment, long-term cell culture and analysis on one chip has been demonstrated in a directly 3D-printed microfluidic chip.ConclusionThe direct 3D printing method is tested and validated for various microfluidic components that can be combined on a chip depending on the specific requirements of the experiment. The ease of use and production opens up the potential of microfluidics to a wide range of users, especially in biomedical research. Our demonstration of its use as a cytotoxicity screening system and as an assay for osteoblastic differentiation shows the methods potential in the development of novel biomedical applications. With the presented method, we aim to disseminate microfluidics as a standard method in biomedical research, thus improving the reproducibility and transferability of results to clinical applications.Graphical

Enhanced osteogenic differentiation of human mesenchymal stem cells using size-controlled graphene oxide flakes

Recently, it has been revealed that the physical microenvironment can be translated into cellular mechanosensing to direct human mesenchymal stem cell (hMSC) differentiation. Graphene oxide (GO), a major derivative of graphene, has been regarded as a promising material for stem cell lineage specification due to its biocompatibility and unique physical properties to interact with stem cells. Especially, the lateral size of GO flakes is regarded as the key factor regulating cellular response caused by GO. In this work, GO that had been mechanically created and had an average diameter of 0.9, 1.1, and 1.7 m was produced using a ball-mill process. When size-controlled GO flakes were applied to hMSCs, osteogenic differentiation was enhanced by GO with a specific average diameter of 1.7 μm. It was confirmed that osteogenic differentiation was increased due to the enhanced expression of focal adhesion and the development of focal adhesion subordinate signals via extracellular signal-regulated kinase (ERK)-mitogen-activated protein kinase (MEK) pathway. These results suggest that size-controlled GO flakes could be efficient materials for promoting osteogenesis of hMSCs. Results of this study could also improve our understanding of the correlation between hMSCs and cellular responses to GO.

Phosphate promotes osteogenic differentiation through non-canonical Wnt signaling pathway in human mesenchymal stem cells.

Phosphate is indispensable in osteogenesis and mineralization. However, mechanisms by which phosphate enhances osteogenic differentiation are not fully understood. In this study, we studied the effect of phosphate on osteogenic differentiation as well as signaling pathways induced by phosphate in the process. Induced human bone marrow-derived mesenchymal stem cells differentiation into osteoblasts by the change of media containing β-glycerophosphate (GP), 1mM inorganic phosphate, or 3mM inorganic phosphate (Pi). The differentiation of osteoblasts was verified by the expression of osteoblast differentiation markers and calcium deposition. RNA sequencing was performed to assess transcriptome in the early stage of osteogenic differentiation. Osteogenic differentiation and mineralization were promoted in the 3mM Pi group compared to those in the GP and 1mM Pi groups on day 7 of culture. RNA sequencing revealed that the gene expressions involved in osteogenesis and the components in the Wnt signaling pathway was increased in 3mM Pi group compared with those in the GP on day 7. Analysis with qPCR and Western blot suggested upregulation of components in the non-canonical Wnt signaling pathway, including WNT5b and phosphorylated-c-Jun in the 3mM Pi group on day 7. WNT11 mRNA expression was increased in the 2 induction groups on day 7. Inhibition of WNT5b by siRNA experiment attenuated the components in non-canonical Wnt signaling expression, including WNT5b, WNT11 and ROR2 mRNA expression and phosphorylated-c-Jun protein expression. In addition, osteogenic differentiation and mineralization were partly decreased in 3mM Pi group on day 7 by the inhibition of WNT5b. Pi promoted osteogenic differentiation through the up-regulation of the non-canonical Wnt signaling pathway, including WNT5b, WNT11, p-c-Jun/c-Jun, in the early stage of differentiation. These findings provide a new perspective into the association of Pi and the non-canonical Wnt signaling pathway during osteogenic differentiation.

High resolution DLP stereolithography to fabricate biocompatible hydroxyapatite structures that support osteogenesis.

Lithography based additive manufacturing techniques, specifically digital light processing (DLP), are considered innovative manufacturing techniques for orthopaedic implants because of their potential for construction of complex geometries using polymers, metals, and ceramics. Hydroxyapatite (HA) coupons, printed using DLP, were evaluated for biological performance in supporting viability, proliferation, and osteogenic differentiation of the human cell line U2OS and human mesenchymal stem cells (MSCs) up to 35 days in culture to determine feasibility for future use in development of complex scaffold geometries. Contact angle, profilometry, and scanning electron microscopy (SEM) measurements showed the HA coupons to be hydrophilic, porous, and having micro size surface roughness, all within favourable cell culture ranges. The study found no impact of leachable and extractables form the DLP printing process. Cells seeded on coupons exhibited morphologies comparable to conventional tissue culture polystyrene plates. Cell proliferation rates, as determined by direct cell count and the RealTime-GloTM MT Cell Viability Assay, were similar on HA coupons and standard tissue culture polystyrene plates). Osteogenic differentiation of human MSCs on HA coupons was confirmed using alkaline phosphatase, Alizarin Red S and von Kossa staining. The morphology of MSCs cultured in osteogenic medium for 14 to 35 days was similar on HA coupons and tissue culture polystyrene plates, with osteogenic (geometric, cuboidal morphology with dark nodules) and adipogenic (lipid vesicles and deposits) features. We conclude that the DLP process and LithaBone HA400 slurry are biocompatible and are suitable for osteogenic applications. Coupons served as an effective evaluation design in the characterization and visualization of cell responses on DLP printed HA material. Results support the feasibility of future technical development for 3D printing of sophisticated scaffold designs, which can be constructed to meet the mechanical, chemical, and porosity requirements of an artificial bone scaffold.