Characterization of cell signaling,morphology, and differentiation potential of human mesenchymal stem cells based on cell adhesion mechanism

It is essential to characterize the cellular properties of mesenchymal stem cell populations to maintain quality specifications and control in regenerative medicine. Biofunctional materials have been designed as artificial matrices for the stimulation of cell adhesion and specific cellular functions. We have developed recombinant maltose-binding protein (MBP)-fused proteins as artificial adhesion matrices to control human mesenchymal stem cell (hMSC) fate by using an integrin-independent heparin sulfate proteoglycans-mediated cell adhesion. In this study, we characterize cell adhesion-dependent cellular behaviors of human adipose-derived stem cells (hASCs) and human bone marrow stem cells (hBMSCs). We used an MBP-fused basic fibroblast growth factor (MF)-coated surface and fibronectin (FN)-coated surface to restrict and support, respectively, integrin-mediated adhesion. The cells adhered to MF exhibited restricted actin cytoskeleton organization and focal adhesion kinase phosphorylation. The hASCs and hBMSCs exhibited different cytoplasmic projection morphologies on MF. Both hASCs and hBMSCs differentiated more dominantly into osteogenic cells on FN than on MF. In contrast, hASCs differentiated more dominantly into adipogenic cells on MF than on FN, whereas hBMSCs differentiated predominantly into adipogenic cells on FN. The results indicate that hASCs exhibit a competitive differentiation potential (osteogenesis vs. adipogenesis) that depends on the cell adhesion matrix, whereas hBMSCs exhibit both adipogenesis and osteogenesis in integrin-mediated adhesion and thus hBMSCs have noncompetitive differentiation potential. We suggest that comparing differentiation behaviors of hMSCs with the diversity of cell adhesion is an important way to characterize hMSCs for regenerative medicine.     

Bone tissue formation from human embryonic stem cells in vivo

Although the use of embryonic stem cells in the assisted repair of musculoskeletal tissues holds promise, a direct comparison of this cell source with adult marrow-derived stem cells has not been undertaken. Here we have compared the osteogenic differentiation potential of human embryonic stem cells (hESC) with human adult-derived stem cells in vivo. hESC lines H7, H9, the HEF-1 mesenchymal-like, telomerized H1 derivative, the human embryonic kidney epithelial cell line HEK293 (negative control), and adult human mesenchymal stem cells (hMSC) were either used untreated or treated with osteogenic factors for 4 days prior to injection into diffusion chambers and implantation into nude mice. After 11 weeks in vivo chambers were removed, frozen, and analyzed for evidence of bone, cartilage, and adipose tissue formation. All hESCs, when pretreated with osteogenic (OS) factors gave rise exclusively to bone in the chambers. In contrast, untreated hESCs (H9) formed both bone and cartilage in vivo. Untreated hMSCs did not give rise to bone, cartilage, or adipose tissue in vivo, while pretreatment with OS factors engendered both bone and adipose tissue. These data demonstrate that hESCs exposed to OS factors in vitro undergo directed differentiation toward the osteogenic lineage in vivo in a similar fashion to that produced by hMSCs. These findings support the potential future use of hESC-derived cells in regenerative medicine applications.     

Culturing of human mesenchymal stem cells as three-dimensional aggregates induces functional expression of CXCR4 that regulates adhesion to endothelial cells

Culture-expanded human mesenchymal stem cells (hMSCs) are increasingly used in a variety of preclinical and clinical studies. However, these cells have a low rate of engraftment to bone marrow or damaged tissues. Several laboratories have shown that during isolation and subculturing mesenchymal stem cells quickly lose the expression of CXCR4, the key receptor responsible for lymphocytes and hematopoietic stem cell homing. Here we show that culturing of hMSCs as three-dimensional aggregates (hMSC spheroids) restores CXCR4 functional expression. Expression of CXCR4 inversely correlates with the secretion of SDF-1 by hMSCs. Cells from hMSC spheroids up-regulate expression of CD49b, the alpha2 integrin subunit, and suppress the expression of CD49d, the alpha4 integrin subunit. Transfer of cells from the spheroids back to a monolayer suppresses the expression of CXCR4 and CD49b and restores the expression of CD49d. Treatment of cells from the spheroids with SDF-1 leads to CXCR4 internalization and activation of ERK-1,2. Adhesion of hMSCs to human umbilical vein endothelial cells (HUVECs) was investigated. SDF-1, AMD-3100, or exposure of HUVECs to hypoxia did not affect adhesion of hMSCs from a monolayer to HUVECs. Adhesion of cells from hMSC spheroids to HUVECs was stimulated by SDF-1, AMD-3100, or by exposure of HUVECs to hypoxia. Stimulatory effects of hypoxia and addition of SDF-1 or AMD-3100 were not additive. Overall, our data indicate that the expression of CXCR4 by hMSCs regulates hMSC adhesion to endothelial cells.     

Extracellular signal-regulated kinase (ERK) dictates osteogenic and/or chondrogenic lineage commitment of mesenchymal stem cells under dynamic compression

Elucidating the intracellular signaling cascades which lead to differentiation programs can be a daunting but necessary task. Even more so when the nature of the differentiating stimuli can elicit different biochemical responses yet achieve the same functional outcome. In the field of cartilage and bone regeneration the importance of the extracellular signal-regulated kinase (ERK) pathway has been a controversial issue as of late. Whether differentiation results from a soluble chemical induction or a microenvironmental cue on the cells seems to have a determining effect on the role that this pathway plays in ultimate cell fate. Here we explore the role of the ERK1/2 pathway on the mechanical induction of chondrogenesis of bone marrow mesenchymal stem cells (MSC). The cells were encapsulated in fibrin gel scaffolds and subjected to a dynamic mechanical compression stimulus previously demonstrated to induce chondrogenic differentiation of the cells with and without the addition of PD98059, a selective inhibitor for the ERK1/2 pathway. Samples were then analyzed by RT-PCR and histochemical staining for markers of both chondrogenic and osteogenic differentiation. Our results show that dynamic compression induces the chondrogenic differentiation of the cells and that inhibition of the ERK1/2 pathway completely abolishes this chondrogenic response. On the other hand, inhibition of ERK1/2 under dynamic compression augments the osteogenic response of the cells and significantly increases their expression of alkaline phosphatase (ALP), collagen type I (COLI) and osteocalcin (OCN) (P<0.05). These results were confirmed by the histochemical staining where dynamically compressed samples show staining for sulfated glycosaminoglycans (sGAG) while the inhibited and compressed samples show no sGAG but present positive staining for microcalcifications. These results would suggest that the activation of ERK1/2 can determine the ultimate cell fate between the chondrogenic and osteogenic programs in cells stimulated under dynamic unconfined mechanical compression.     

Osteogenic differentiation of human adipose tissue-derived stromal cells (hASCs) in a porous three-dimensional scaffold

Recent studies have shown that liposuction aspirates from rat, rabbit, mouse, and human sources contain pluripotent adipose tissue-derived stromal cells (ASCs) that can differentiate into various mesodermal cell types, including osteoblasts, myoblasts, chondroblasts, and preadipocytes. To develop a research model for autologous bone tissue engineering, we isolated ASCs from human liposuction aspirates (hASCs) and induced their osteogenic differentiation in three-dimensional poly(dl-lactic-co-glycolic acid) (PLGA) scaffolds. Human liposuction aspirates were proteolytically digested and centrifuged to obtain hASCs. After primary culture in control media and expansion to three passages, the cells were seeded in two-dimensional plates or three-dimensional PLGA scaffolds and cultured in osteogenic media for 4 weeks. In two-dimensional culture, osteogenesis was assessed by RT-PCR analysis of the osteogenic-specific bone sialoprotein mRNA, by alkaline phosphatase staining, and by von Kossa staining. In three-dimensional culture, osteogenesis was assessed by von Kossa and alizarine red S staining at 1, 2, and 4 weeks following osteogenic induction. hASCs incubated in two-dimensional osteogenic media stained positively for alkaline phosphatase and with von Kossa stain after 2 weeks of differentiation. Expression of the osteogenesis-specific bone sialoprotein gene was detected by RT-PCR after 2 weeks of differentiation. PLGA scaffolds seeded with hASCs showed multiple calcified extracellular matrix nodules by von Kossa and alizarine red S staining after 2 weeks of differentiation. In conclusion, the authors identified an osteogenic potential of hASCs and demonstrated osteogenic differentiation of hASCs into an osteogenic lineage in three-dimensional PLGA scaffolds.     

Cyclic strain enhances matrix mineralization by adult human mesenchymal stem cells via the extracellular signal-regulated kinase (ERK1/2) signaling pathway

Physical stimuli play critical roles in the development, regeneration, and pathology of many mesenchymal tissues, most notably bone. While mature bone cells, such as osteoblasts and osteocytes, are clearly involved in these processes, the role of their progenitors in mechanically mediated tissue responses is unknown. In this study, we investigated the effect of cyclic substrate deformation on the proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSCs). Application of equibiaxial cyclic strain (3%, 0.25Hz) to hMSCs cultured in osteogenic media inhibited proliferation and stimulated a 2.3-fold increase in matrix mineralization over unstrained cells. The strain stimulus activated the extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein kinase pathways, but had no effect on c-Jun N-terminal kinase phosphorylation or activity. Strain-induced mineralization was largely mediated by ERK1/2 signaling, as inhibition of ERK1/2 attenuated calcium deposition by 55%. Inhibition of the p38 pathway resulted in a more mature osteogenic phenotype, suggesting an inhibitory role for p38 signaling in the modulation of strain-induced osteogenic differentiation. These results demonstrate that mechanical signals regulate hMSC function, suggesting a critical role for physical stimulation of this specific cell population in mesenchymal tissue formation.     

Quantifying osteogenic cell degradation of silk biomaterials

The degradation of silk protein films by human mesenchymal stem cells (hMSCs), osteoblasts and osteoclasts, cells involved in osteogenic functions in normal and diseased bone, was assessed in vitro. The involvement of specific matrix metalloproteinases (MMPs) and integrin signaling in the degradation process was determined. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to quantitatively compare degradation by the different cell types using surface patterned silk films. Osteoblasts and osteoclasts demonstrated significant degradation of the silk films in vitro in comparison to the hMSCs and the film controls without cells. The osteoclasts degraded the silk films the most and also generated the highest level of MMPs 1 and 2. The osteoblasts upregulated integrins α5 and β1, while the osteoclasts upregulated integrins α2 and β1. There was significant contrast in responses on the silk matrices between osteogenic cells versus undifferentiated hMSCs to illustrate in vitro the role of cell type on matrix remodeling. These are important issues in matching biomaterial matrix features and studies in vitro to remodeling in vivo, in both normal and disease tissue systems. Cell populations and niche factors impact tissue regeneration, wound healing, physiological state, and the ability to better understand the role of different cell types is critical to overall regenerative outcomes.     

Molecular hydrogen inhibits lipopolysaccharide/interferon γ-induced nitric oxide production through modulation of signal transduction in macrophages

Adipose-derived stem cells (ASCs) have been successfully applied in treating bone defects both in animals and humans and promoted osteogenesis in vivo significantly. However, the mechanism of in vivo osteogenesis of ASCs was still little known, we hypothesized that this was mediated in part by interaction between implanted ASCs and local vein endothelial cells. In this study, human adipose-derived stem cells (hASCs) and human umbilical vein endothelial cells (HUVEC) were isolated and characterized. Cells were then either cultured alone or cocultured. Alkaline phosphatase (ALP) staining, quantitative measurement of ALP activity and Alizarin staining of hASCs cultured alone, HUVEC cultured alone and cells cocultured demonstrated that osteogenic differentiation of cocultured cells increased obviously. Osteocalcin (OC) expression of hASCs cocultured with HUVEC showed an obvious raise than hASCs cultured alone. HUVEC cultured alone showed BMP-2 secretion and increased with culturing time. Real-time PCR of the cocultured cells showed four osteogenic differentiation related genes raised with culturing time, while two adipogenic differentiation related genes showed a slightly decrease with culturing time. Results of our study with different culture models showed that in vitro osteogenesis of hASCs was enhanced by coculture with HUVEC which secreted BMP-2. This study not only provided us with an in vitro model of studying interaction between cells, but also helped us to understand the in vivo therapeutic mechanisms of ASCs.     

Fibroblasts share mesenchymal phenotypes with stem cells, but lack their differentiation and colony-forming potential

Background information. Although MSCs (mesenchymal stem cells) and fibroblasts have been well studied, differences between these two cell types are not fully understood. We therefore comparatively analysed antigen and gene profiles, colony‐forming ability and differentiation potential of four human cell types in vitro: commercially available skin‐derived fibroblasts [hSDFs (human skin‐derived fibroblasts)], adipose tissue‐derived stem cells [hASCs (human adipose tissue‐derived stem cells)], embryonic lung fibroblasts (WI38) and dermal microvascular endothelial cells [hECs (human dermal microvascular endothelial cells)]. Results. hSDFs, hASCs and WI38 exhibited a similar spindle‐like morphology and expressed same antigen profiles: positive for MSC markers (CD44, CD73 and CD105) and fibroblastic markers [collagen I, HSP47 (heat shock protein 47), vimentin, FSP (fibroblast surface protein) and αSMA (α smooth muscle actin)], and negative for endothelial cell marker CD31 and haemopoietic lineage markers (CD14 and CD45). We further analysed 90 stem cell‐associated gene expressions by performing real‐time PCR and found a more similar gene expression pattern between hASCs and hSDFs than between hSDFs and WI38. The expression of embryonic stem cell markers [OCT4, KLF4, NANOG, LIN28, FGF4 (fibroblast growth factor 4) and REST] in hASCs and hSDFs was observed to differ more than 2.5‐fold as compared with WI38. In addition, hSDFs and hASCs were able to form colonies and differentiate into adipocytes, osteoblasts and chondrocytes in vitro, but not WI38. Moreover, single cell‐derived hSDFs and hASCs obtained by clonal expansion were able to differentiate into adipocytes and osteoblasts. However, CD31 positive hECs did not show differentiation potential. Conclusions. These findings suggest that (i) so‐called commercially available fibroblast preparations from skin (hSDFs) consist of a significant number of cells with differentiation potential apart from terminally differentiated fibroblasts; (ii) colony‐forming capacity and differentiation potential are specific important properties that discriminate MSCs from fibroblasts (WI38), while conventional stem cell properties such as plastic adherence and the expression of CD44, CD90 and CD105 are unspecific for stem cells.     

预血管化细胞膜片的构建

为探索新的体外获得毛细血管样网络结构来解决工程化组织预血管化的问题,该研究将人骨髓间充质干细胞（humanmesenchymalstemcells,hMSCs）以9×10^4／cm。细胞密度体外连续培养形成细胞膜片,将培养的脐静脉血内皮细胞（humanumbilicalveinendothelialcells,HUVECs）v25x104／cm2细胞密度接种到上述间充质干细胞膜片上,并培养在内皮细胞培养介质中。在设计的时间点用倒置相差显微镜观察,发现内皮细胞在膜片上迁移,细胞重排,膜片上的基质蛋白也发生重排．导致微槽和空泡出现。CD31免疫荧光染色观察到进行性管腔形成的过程;CD90免疫荧光染色显示膜片上的hMSCs围绕着HUVECs周边排列,说明hMSCs作为周细胞支持了HUVECs的生长;在培养第10d可见少量的α-SMA的表达,暗示着在此种培养模式下,hMSCs具有较低的向肌细胞分化的潜能。这些结果表明,将内皮细胞接种在未分化干细胞膜片上,可以在体外形成具有血管网络结构的预血管化膜片,为构建血管化工程化组织提供了新的思路。     

Mechanisms for osteogenic differentiation of human mesenchymal stem cells induced by fluid shear stress

Mechanical stimuli can improve bone function by promoting the proliferation and differentiation of bone cells and osteoblasts. As precursors of osteoblasts, human mesenchymal stem cells (hMSCs) are sensitive to mechanical stimuli. In recent years, fluid shear stress (FSS) has been widely used as a method of mechanical stimulation in bone tissue engineering to induce the osteogenic differentiation of hMSCs. However, the mechanism of this differentiation is not completely clear. Several signaling pathways are involved in the mechanotransduction of hMSCs responding to FSS, such as MAPK, NO/cGMP/PKG and Ca²⁺ signaling pathway. Here, we briefly review how hMSCs respond to fluid flow stimuli and focus on the signal molecules involved in this mechanotransduction.     

Altered osteogenic commitment of human mesenchymal stem cells by ERM protein-dependent modulation of cellular biomechanics

Cellular mechanics is known to play an important role in many cellular functions including adhesion, migration, proliferation, and differentiation. Human mesenchymal stem cells (hMSCs) demonstrate unique mechanical properties distinct from fully differentiated cells. This observation suggests that the stem cell mechanics may be modulated to regulate the hMSCs' lineage commitment. Specifically, ERM (ezrin, radixin, moesin) proteins are known to mediate the membrane-cytoskeleton adhesion, cell elasticity, actin cytoskeleton organization, and therefore could serve as potential targets for modulation of the cellular mechanics. Combining silencing RNA, atomic force microscopy, and laser optical tweezers, the role of the ERM proteins involved in the regulation of stem cell biomechanics and osteogenic differentiation was quantitatively determined. Transient ERM knockdown by RNAi causes disassembly of actin stress fibers and focal adhesions, a decrease in the cell stiffness, and membrane separation from the cytoskeleton. The silencing RNA treatment not only induced mechanical changes in stem cells but impaired biochemically-directed osteogenic differentiation. The intact actin cytoskeleton and focal adhesions of hMSCs appear critical for the osteogenic induction. Thus, ERM knockdown modulates the dynamics of cell mechanical changes during hMSC differentiation and regulates the expression of tissue specific molecular markers. These findings are of particular interest for modulation of the cellular biomechanics to control hMSCs' activities and fate in tissue engineering, regenerative medicine, and other stem cell-based therapeutic applications.     

Extracellular matrix protein mediated regulation of the osteoblast differentiation of bone marrow derived human mesenchymal stem cells

The biomimetic approach of tissue engineering exploits the favorable properties of the extracellular matrix (ECM), to achieve better scaffold performance and tissue regeneration. ECM proteins regulate cell adhesion and differentiation through integrin mediated signal transduction. In the present study, we have examined the role of ECM proteins such as collagen type I, fibronectin, laminin and vitronectin in regulating the proliferation and osteogenic differentiation of bone marrow derived human mesenchymal stem cells (hMSCs). hMSCs were grown on selected ECM protein treated tissue culture plates. The growth kinetics was assessed by calculating the doubling time of the cells on different ECM treated plates. The cells were directed to osteoblast lineage by growing them in osteogenic induction media for 21 day. Differentiation was evaluated at different time points by osteoblast differentiation associated gene expression, alkaline phosphatase (ALP) activity, histochemical staining for mineralized matrix and calcium quantification. The doubling time of hMSCs cultured on collagen type I was significantly low, which was followed by laminin and fibronectin treated plates. However, doubling time of hMSCs cultured on vitronectin treated plate was not significantly different than that of the untreated control. High ALP gene (ALPL) expression and associated enhancement of mineralization were observed on collagen type I, fibronectin and vitronectin treated plates. Collagen type I showed early onset of mineralization with high ALP activity and up-regulation of osteopontin, ALPL, bone sialoprotein and osteocalcin genes. Vitronectin also up-regulated these genes and showed the highest amount of calcium in the secreted mineral matrix. Therefore, we conclude that, ECM proteins indeed modified the growth patterns and induced the osteoblast differentiation of hMSCs. Our findings have significant implication for bone tissue engineering applications.     

Different effects of intermittent and continuous fluid shear stresses on osteogenic differentiation of human mesenchymal stem cells

A reasonable mechanical microenvironment similar to the bone microenvironment in vivo is critical to the formation of engineering bone tissues. As fluid shear stress (FSS) produced by perfusion culture system can lead to the osteogenic differentiation of human mesenchymal stem cells (hMSCs), it is widely used in studies of bone tissue engineering. However, effects of FSS on the differentiation of hMSCs largely depend on the FSS application manner. It is interesting how different FSS application manners influence the differentiation of hMSCs. In this study, we examined the effects of intermittent FSS and continuous FSS on the osteogenic differentiation of hMSCs. The phosphorylation level of ERK1/2 and FAK is measured to investigate the effects of different FSS application manners on the activation of signaling molecules. The results showed that intermittent FSS could promote the osteogenic differentiation of hMSCs. The expression level of osteogenic genes and the alkaline phosphatase (ALP) activity in cells under intermittent FSS application were significantly higher than those in cells under continuous FSS application. Moreover, intermittent FSS up-regulated the activity of ERK1/2 and FAK. Our study demonstrated that intermittent FSS is more effective to induce the osteogenic differentiation of hMSCs than continuous FSS.     

Effect of Pulsed Electromagnetic Fields on Human Mesenchymal Stem Cells Using 3D Magnetic Scaffolds

Alternative bone regeneration strategies that do not rely on harvested tissue or exogenous growth factors are needed. One of the major challenges in tissue reconstruction is recreating the bone tissue microenvironment using the appropriate combination of cells, scaffold, and stimulation to direct differentiation. This study presents a bone regeneration formulation that involves the use of human adipose-derived mesenchymal stem cells (hASCs) and a three-dimensional (3D) hydrogel scaffold based on self-assembled RADA16 peptides containing superparamagnetic iron oxide nanoparticles (NPs). Although superparamagnetic NPs could be used as stimulus to manipulate the cell proliferation and differentiation, in this paper their use is explored for assisting osteogenic differentiation of hASCs in conjunction with direct stimulation by extremely low-frequency pulsed electromagnetic fields (pEMFs). Cellular morphology, proliferation, and viability, as well as alkaline phosphatase activity, calcium deposition, and osteogenic capacity were monitored for cells cultured up to 21 days in the 3D construct. The results show that the pEMFs and NPs do not have any negative effect on cell viability, but instead distinctly induced early differentiation of hASCs to an osteoblastic phenotype, when compared with cells without biophysical stimulation. This effect is attributed to synergy between the pEMFs and NPs, which may have stimulated mechanotransduction pathways, which, in turn activated biochemical signals between cells to differentiate or proliferate. This approach may offer a safe and effective option for the treatment of non-union bone fractures. Bioelectromagnetics. © 2020 The Authors. Bioelectromagnetics published by Wiley Periodicals, Inc.     

Maintenance of human adipose derived stem cell (hASC) differentiation capabilities using a 3D culture

In this study, 3D culture system for human adipose-derived stem cell (hASC) using a BioLevitator as the bioreactor for microcarrier-based cultures was established. During the culturing period, hASCs preferred to grow in crevices between microcarriers and a high viability was maintained even when reaching confluency. Adipogenic or osteogenic differential medium was used to induce hASCs and differential potentials of these cells were compared between 2D and 3D environments via RT-PCR and staining quantifications. CEBP/α gene expression was significant higher in 3D condition at day 21 (P < 0.05). Staining quantification indicates that cells cultured in 3D condition have significant better differentiation potential from day 14 to 21 for both adipogenic and osteogenic lineages (P < 0.01).     

Donepezil hydrochloride as a novel inducer for osteogenic differentiation of mesenchymal stem cells on PLLA scaffolds in vitro

Over the past decades, bone defects caused by illness or trauma have been the most common traumatic injuries in humans and treatment of orthopedic infections has always been a serious challenge to experts in the world. In this project, poly L-lactic acid (PLLA) nanofibrous scaffolds were synthesized as a nontoxic, eco-friendly, and cost-effective scaffold by the electrospinning technique. Then, the impact of PLLA on the cell proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSCs) was assayed in the presence and absence of donepezil hydrochloride (DH) which was prescribed in patients with Alzheimer's disease. Also, hMSCs were seeded on PLLA scaffold in the presence (PLLA-DH) and absence of 1 μg mL^-1 of DH under osteogenic induction media. Osteogenic differentiation of hMSCs was assessed by specific bone-related tests including alkaline phosphatase (ALP) activity, Alizarin red and von Kossa staining, calcium content assay. Also, Osteocalcin and osteopontin were evaluated as osteogenic proteins as well as ALP, osteonectin, osteocalcin, collagen type I (Col-I) and Runx2 as osteogenic genes via immunocytochemistry (ICC) and Real-time PCR analyses. The obtained data showed the higher ALP enzyme activity and biomineralization, more intensity during von Kossa staining as well as the increase in the expression rate of osteogenic related gene and protein markers in differentiated hMSCs on PLLA-DH. In conclusion, the present study revealed that the combination of PLLA scaffold with DH provides a scope to develop a suitable matrix in bone tissue engineering applications.