Biophysical factors in the regulation of asymmetric division of stem cells

Stem cells are a promising cell source for regenerative medicine due to their characteristics of self‐renewal and differentiation. The intricate balance between these two cell fates is maintained by precisely controlled symmetric and asymmetric cell divisions. Asymmetric division has a fundamental importance in maintaining tissue homeostasis and in the development of multi‐cellular organisms. For example, during development, asymmetric cell divisions are responsible for the formation of the body axis. Mechanistically, mitotic spindle dynamics determine the assembly and separation of chromosomes and regulate the orientation of cell division. Interestingly, symmetric and asymmetric cell division is not mutually exclusive and a range of factors are involved in such cell‐fate decisions, the measurement of which can provide efficient and reliable information on the regenerative potential of a cell. The balance between self‐renewal and differentiation in stem cells is controlled by various biophysical and biochemical cues. Although the role of biochemical factors in asymmetric stem cell division has been widely studied, the effect of biophysical cues in stem‐cell self‐renewal is not comprehensively understood. Herein, we review the biological relevance of stem‐cell asymmetric division to regenerative medicine and discuss the influences of various intrinsic and extrinsic biophysical cues in stem‐cell self‐renewal. This review particularly aims to inform the clinical translation of efforts to control the self‐renewal ability of stem cells through the tuning of various biophysical cues.     

Comparative osteogenic transcription profiling of various fetal and adult mesenchymal stem cell sources

Human mesenchymal stem cells (MSC) from adult and fetal tissues are promising candidates for cell therapy but there is a need to identify the optimal source for bone regeneration. We have previously characterized MSC populations in first trimester fetal blood, liver, and bone marrow and we now evaluate their osteogenic differentiation potential in comparison to adult bone marrow MSC. Using quantitative real-time RT-PCR, we demonstrated that 16 osteogenic-specific genes (OC, ON, BSP, OP, Col1, PCE, Met2A, OPG, PHOS1, SORT, ALP, BMP2, CBFA1, OSX, NOG, IGFII) were expressed in both fetal and adult MSC under basal conditions and were up-regulated under osteogenic conditions both in vivo and during an in vitro 21-day time-course. However, under basal conditions, fetal MSC had higher levels of osteogenic gene expression than adult MSC. Upon osteogenic differentiation, fetal MSC produced more calcium in vitro and reached higher levels of osteogenic gene up-regulation in vivo and in vitro. Second, we observed a hierarchy within fetal samples, with fetal bone marrow MSC having greater osteogenic potential than fetal blood MSC, which in turn had greater osteogenic potential than fetal liver MSC. Finally, we found that the level of gene expression under basal conditions was positively correlated with both calcium secretion and gene expression after 21 days in osteogenic conditions. Our findings suggest that stem cell therapy for bone dysplasias such as osteogenesis imperfecta may benefit from preferentially using first trimester fetal blood or bone marrow MSC over fetal liver or adult bone marrow MSC.     

Osteogenic potential: comparison between bone marrow and adipose-derived mesenchymal stem cells

Bone tissue engineering(BTE) is now a promising re-search issue to improve the drawbacks from traditional bone grafting procedure such as limited donor sources and possible complications. Stem cells are one of the major factors in BTE due to the capability of self re-newal and multi-lineage differentiation. Unlike embry-onic stem cells, which are more controversial in ethical problem, adult mesenchymal stem cells are considered to be a more appropriate cell source for BTE. Bone marrow mesenchymal stem cells(BMSCs) are the ear-liest-discovered and well-known stem cell source using in BTE. However, the low stem cell yield requiring long expansion time in vitro, pain and possible morbidities during bone marrow aspiration and poor proliferation and osteogenic ability at old age impede its' clinical ap-plication. Afterwards, a new stem cell source coming from adipose tissue, so-called adipose-derived stemcells(ASCs), is found to be more suitable in clinical ap-plication because of high stem cells yield from lipoaspi-rates, faster cell proliferation and less discomfort and morbidities during harvesting procedure. However, the osteogenic capacity of ASCs is now still debated be-cause most papers described the inferior osteogenesis of ASCs than BMSCs. A better understanding of the osteogenic differences between ASCs and BMSCs is crucial for future selection of cells in clinical application for BTE. In this review, we describe the commonality and difference between BMSCs and ASCs by cell yield, cell surface markers and multiple-differentiation poten-tial. Then we compare the osteogenic capacity in vitro and bone regeneration ability in vivo between BMSCs and ASCs based on the literatures which utilized both BMSCs and ASCs simultaneously in their articles. The outcome indicated both BMSCs and ASCs exhibited the osteogenic ability to a certain extent both in-vitro and in-vivo. However, most in-vitro study papers verified the inferior osteogenesis of ASCs; conversely, in-vivo research reviews revealed more controversies in this issue. We expect the new researchers can have a quick understanding of the progress in this filed and design a more comprehensive research based on this review.     

Boron Enhances Odontogenic and Osteogenic Differentiation of Human Tooth Germ Stem Cells (hTGSCs) In Vitro

Stem cell technology has been a great hope for the treatment of many common problems such as Parkinson's disease, Alzheimer's disease, diabetes, cancer, and tissue regeneration. Therefore, the main challenge in hard tissue engineering is to make a successful combination of stem cells and efficient inductors in the concept of stem cell differentiation into odontogenic and osteogenic cell types. Although some boron derivatives have been reported to promote bone and teeth growth in vivo, the molecular mechanism of bone formation has not been elucidated yet. Different concentrations of sodium pentaborate pentahydrate (NaB) were prepared for the analysis of cell toxicity and differentiation evaluations. The odontogenic, osteogenic differentiation and biomineralization of human tooth germ stem cells (hTGSCs) were evaluated by analyzing the mRNA expression levels, odontogenic and osteogenic protein expressions, alkaline phosphatase (ALP) activity, mineralization, and calcium deposits. The NaB-treated group displayed the highest ALP activity and expression of osteo- and odontogenic-related genes and proteins compared to the other groups and baseline. In the current study, increased in vitro odontogenic and osteogenic differentiation capacity of hTGSCs by NaB application has been shown for the first time. The study offers considerable promise for the development of new scaffold systems combined with NaB in both functional bone and tooth tissue engineering.     

A unique Golgi apparatus distribution may be a marker for osteogenic differentiation of hDP-MSCs

Stem cell markers are utilized to isolate or identify stem cells. So far, many stem-cell-specific markers have been described, although some of them turned out to be not as specific as it was originally proposed. In this study, we sought to search for a specific stem cell marker that would be phenotypically helpful, characteristically specific, economically affordable and easy to use. Because organelles are one of the major characteristics of eukaryotic cells, we asked the question of whether organelle characteristics might be a useful tool for stem cell characterization. We studied distribution and characteristics of the endoplasmic reticulum, the mitochondria and the Golgi apparatus in human dental-pulp-derived mesenchymal stem cells before and during osteogenic differentiation. Although it was not possible to find a useful macromolecular marker for stem cell characterization, we found that during osteogenic differentiation, the stem cells changed their Golgi characteristics and displayed a unique in vivo pattern. We analysed these unique Golgi structures and proposed a potential osteogenic differentiation marker for human dental-pulp-derived mesenchymal stem cells. This pattern may be used in the evaluation of osteogenic differentiation.     

RUNX2对BMP9诱导的间充质干细胞C3H10T1/2成骨分化的影响

目的:研究和确认RUNX2在骨形态发生蛋白9(BMP9)诱导的间充质干细胞C3H10T1/2成骨分化中的作用。方法:通过Western blot、RT-PCR、荧光素酶活性分析检测BMP9对RUNX2表达的影响;分别在过表达RUNX2和RNA干扰抑制RUNX2表达的情况下,利用碱性磷酸酶(ALP)活性测定和染色、钙盐沉积实验,免疫细胞化学和裸鼠皮下异位成骨实验分析RUNX2对于BMP9诱导的间充质干细胞成骨分化的影响。结果:BMP9可以促进RUNX2的表达;RUNX2体外可促进BMP9诱导的C3H10T1/2的ALP活性和钙盐沉积,却抑制了OCN表达,RUNX2还可促进BMP9诱导的裸鼠皮下异位成骨;而在降低RUNX2表达后,BMP9诱导的C3H10T1/2细胞的ALP活性、钙盐沉积、OCN表达和裸鼠皮下异位成骨均受到抑制。结论:RUNX2可以促进BMP9诱导的间充质干细胞C3H10T1/2细胞成骨分化。     

Recreating the perivascular niche ex vivo using a microfluidic approach

Stem cell niches are composed of numerous microenvironmental features, including soluble and insoluble factors, cues from other cells, and the extracellular matrix (ECM), which collectively serve to maintain stem cell quiescence and promote their ability to support tissue homeostasis. A hallmark of many adult stem cell niches is their proximity to the vasculature in vivo, a feature common to neural stem cells, mesenchymal stem cells (MSCs) from bone marrow and adipose tissue, hematopoietic stem cells, and many tumor stem cells. In this study, we describe a novel 3D microfluidic device (MFD) as a model system in which to study the molecular regulation of perivascular stem cell niches. Endothelial cells (ECs) suspended within 3D fibrin gels patterned in the device adjacent to stromal cells (either fibroblasts or bone marrow‐derived MSCs) executed a morphogenetic process akin to vasculogenesis, forming a primitive vascular plexus and maturing into a robust capillary network with hollow well‐defined lumens. Both MSCs and fibroblasts formed pericytic associations with the ECs but promoted capillary morphogenesis with distinct kinetics. Biochemical assays within the niche revealed that the perivascular association of MSCs required interaction between their α6β1 integrin receptor and EC‐deposited laminin. These studies demonstrate the potential of this physiologically relevant ex vivo model system to study how proximity to blood vessels may influence stem cell multipotency. Biotechnol. Bioeng. 2010;107: 1020–1028. © 2010 Wiley Periodicals, Inc.     

脂肪间质干细胞研究进展

脂肪间质干细胞,是脂肪组织中一类多能性干细胞。其在体外特定的培养条件下,可诱导分化形成脂肪、骨、软骨、肌肉等组织类型细胞。人体脂肪组织十分丰富,用其分离脂肪间质干细胞可避免分离胚胎干细胞所面临的道德伦理问题和获取极少量骨髓分离骨髓间质干细胞时给供者带来极大痛苦等。因此脂肪间质干细胞可作为组织再生工程的干细胞理想的替代资源。本文重点论述脂肪间质干细胞的研究进展,并探讨其临床应用前景。     

Use of platelet lysate for bone regeneration - are we ready for clinical translation?

Current techniques to improve bone regeneration following trauma or tumour resection involve the use of autograft bone or its substitutes supplemented with osteoinductive growth factors and/or osteogenic cells such as mesenchymal stem cells(MSCs).Although MSCs are most commonly grown in media containing fetal calf serum,human platelet lysate(PL) offers an effective alternative.Bone marrow- derived MSCs grown in PLcontaining media display faster proliferation whilst maintaining good osteogenic differentiation capacity.Limited pre-clinical investigations using PL-expanded MSCs seeded onto osteoconductive scaffolds indicate good potential of such constructs to repair bone in vivo.In an alternative approach,nude PL-coated scaffolds without seeded MSCs have been proposed as novel regenerative medicine devices.Even though methods to coat scaffolds with PL vary,in vitro studies suggest that PL allows for MSC adhesion,migration and differentiation inside these scaffolds.Increased new bone formation and vascularisation in comparison to uncoated scaffolds have also been observed in vivo.This review outlines the state-of-the-art research in the field of PL for ex vivo MSC expansion and in vivo bone regeneration.To minimise inconsistency between the studies,further work is required towards standardisation of PL preparation in terms of the starting material,platelet concentration,leukocyte depletion,and the method of platelet lysis.PL quality control procedures and its "potency" assessment are urgently needed,which could include measurements of key growth and attachment factors important for MSC maintenance and differentiation.Furthermore,different PL formulations could be tailor-made for specific bone repair indications.Such measures would undoubtedly speed up clinical translation of PL-based treatments for bone regeneration.     

骨形态发生蛋白9通过p38激酶途径调控间充质干细胞C3H10T1/2成骨分化

前期研究发现骨形态发生蛋白9(bone morphogenetic protein 9,BMP9)具有较强的诱导间充质干细胞成骨分化的能力.为进一步揭示其诱导和调控间充质干细胞成骨分化的机理,利用BMP9重组腺病毒感染间充质干细胞C3H10T1/2,通过体外细胞实验和体内动物实验,初步分析BMP9是否可通过p38激酶途径调控间充质干细胞成骨分化.结果发现,BMP9可以通过促进p38激酶磷酸化而导致其活化,p38抑制剂SB203580可抑制由BMP9诱导的C3H10T1/2细胞的碱性磷酸酶(alkalinephosphatase,ALP)活性、骨桥蛋白(osteopontin,OPN)表达和钙盐沉积,而且利用抑制剂SB203580抑制p38激酶活性后,BMP9诱导的Smad经典途径的激活也相应受到抑制,RNA干扰导致p38基因沉默同样也可抑制BMP9诱导的ALP活性、OPN表达、钙盐沉积以及裸鼠皮下异位成骨.因此,BMP9可通过活化p38激酶途径调控间充质干细胞C3H10T1/2成骨分化.     

Current evidence on potential of adipose derived stem cells to enhance bone regeneration and future projection

Injuries to the postnatal skeleton are naturally repaired through successive steps involving specific cell types in a process collectively termed “bone regeneration”. Although complex, bone regeneration occurs through a series of well-orchestrated stages wherein endogenous bone stem cells play a central role. In most situations, bone regeneration is successful; however, there are instances when it fails and creates non-healing injuries or fracture nonunion requiring surgical or therapeutic interventions. Transplantation of adult or mesenchymal stem cells (MSCs) defined by the International Society for Cell and Gene Therapy (ISCT) as CD105+CD90+CD73+CD45-CD34-CD14orCD11b-CD79αorCD19-HLA-DR- is being investigated as an attractive therapy for bone regeneration throughout the world. MSCs isolated from adipose tissue, adipose-derived stem cells (ADSCs), are gaining increasing attention since this is the most abundant source of adult stem cells and the isolation process for ADSCs is straightforward. Currently, there is not a single Food and Drug Administration (FDA) approved ADSCs product for bone regeneration. Although the safety of ADSCs is established from their usage in numerous clinical trials, the bone-forming potential of ADSCs and MSCs, in general, is highly controversial. Growing evidence suggests that the ISCT defined phenotype may not represent bona fide osteoprogenitors. Transplantation of both ADSCs and the CD105^- sub-population of ADSCs has been reported to induce bone regeneration. Most notably, cells expressing other markers such as CD146, AlphaV, CD200, PDPN, CD164, CXCR4, and PDGFRα have been shown to represent osteogenic sub-population within ADSCs. Amongst other strategies to improve the bone-forming ability of ADSCs, modulation of VEGF, TGF-β1 and BMP signaling pathways of ADSCs has shown promising results. The U.S. FDA reveals that 73% of Investigational New Drug applications for stem cell-based products rely on CD105 expression as the “positive” marker for adult stem cells. A concerted effort involving the scientific community, clinicians, industries, and regulatory bodies to redefine ADSCs using powerful selection markers and strategies to modulate signaling pathways of ADSCs will speed up the therapeutic use of ADSCs for bone regeneration.     

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.     

Differentiation-dependent secretion of proangiogenic factors by mesenchymal stem cells

Mesenchymal stem cells (MSCs) are a promising cell population for cell-based bone repair due to their proliferative potential, ability to differentiate into bone-forming osteoblasts, and their secretion of potent trophic factors that stimulate angiogenesis and neovascularization. To promote bone healing, autogenous or allogeneic MSCs are transplanted into bone defects after differentiation to varying degrees down the osteogenic lineage. However, the contribution of the stage of osteogenic differentiation upon angiogenic factor secretion is unclear. We hypothesized that the proangiogenic potential of MSCs was dependent upon their stage of osteogenic differentiation. After 7 days of culture, we observed the greatest osteogenic differentiation of MSCs when cells were cultured with dexamethasone (OM+). Conversely, VEGF protein secretion and upregulation of angiogenic genes were greatest in MSCs cultured in growth media (GM). Using conditioned media from MSCs in each culture condition, GM-conditioned media maximized proliferation and enhanced chemotactic migration and tubule formation of endothelial colony forming cells (ECFCs). The addition of a neutralizing VEGF(165/121) antibody to conditioned media attenuated ECFC proliferation and chemotactic migration. ECFCs seeded on microcarrier beads and co-cultured with MSCs previously cultured in GM in a fibrin gel exhibited superior sprouting compared to MSCs previously cultured in OM+. These results confirm that MSCs induced farther down the osteogenic lineage possess reduced proangiogenic potential, thereby providing important findings for consideration when using MSCs for bone repair.     

Synergistic effect of extracellularly supplemented osteopontin and osteocalcin on stem cell proliferation,osteogenic differentiation,and angiogenic properties

A high demand for functional bone grafts is being observed worldwide, especially due to the increased life expectancy. Osteoinductive components should be incorporated into functional bone grafts, accelerating cell recruitment, cell proliferation, angiogenesis, and new bone formation at a defect site. Noncollagenous bone matrix proteins, especially osteopontin (OPN) and osteocalcin (OC), have been reported to regulate some physiological process, such as cell migration and bone mineralization. However, the effects of OPN and OC on cell proliferation, osteogenic differentiation, mineralization, and angiogenesis are still undefined. Therefore, we assessed the exogenous effect of OPN and OC supplementation on human bone marrow mesenchymal stem/stromal cells (hBM MSC) proliferation and osteogenic differentiation. OPN dose-dependently increased the proliferation of hBM MSC, as well as improved the angiogenic properties of human umbilical vein endothelial cells (HUVEC) by increasing the capillary-like tube formation in vitro. On the other hand, OC enhanced the differentiation of hBM MSC into osteoblasts and demonstrated an increase in extracellular calcium levels and alkaline phosphatase activity, as well as higher messenger RNA levels of mature osteogenic markers osteopontin and osteocalcin. In vivo assessment of OC/OPN-enhanced scaffolds in a critical-sized defect rabbit long-bone model revealed no infection, while new bone was being formed. Taken together, these results suggest that OC and OPN stimulate bone regeneration by inducing stem cell proliferation, osteogenesis and by enhancing angiogenic properties. The synergistic effect of OC and OPN observed in this study can be applied as an attractive strategy for bone regeneration therapeutics by targeting different vital cellular processes.     

Osteogenic differentiation of GFP-labeled human umbilical cord blood derived mesenchymal stem cells after cryopreservation

The osteogenic capacity of human umbilical cord blood derived mesenchymal stem cells (UCB-MSCs) has been demonstrated both in vitro and in vivo. Therefore, cell labeling and storage are becoming necessary for researching the potential therapeutic use of UCB-MSCs for bone tissue engineering. The aim of this study was to determine the effect of cryopreservation on the osteogenic differentiation of green fluorescent protein (GFP)-marked UCB-MSCs in vitro. MSCs were isolated from full-term human UCB, expanded, transfected with the GFP gene, and then cryopreserved in liquid nitrogen for 4 weeks. After thawing, cell surface antigen markers and osteogenic potential were analyzed, and the luminescence of these cells was observed by fluorescence microscopy. The results demonstrate that cryopreservation has no effect on the cell phenotype, GFP expression or osteogenic differentiation of UCB-MSCs, showing that cryopreserved GFP-labeled UCB-MSCs might be applied for bone tissue engineering.     

Focal adhesion kinase promotes BMP2-induced osteogenic differentiation of human urinary stem cells via AMPK and Wnt signaling pathways

Human urine-derived stem cells (hUSCs) serve as favorable candidates for bone transplants due to their efficient proliferative and multipotent differentiation abilities, as well as the capacity to secrete a variety of vasoactive agents to facilitate tissue engineering. The present study aimed to explore the role of focal adhesion kinase (FAK) in bone morphogenetic protein 2 (BMP2)-induced osteogenic differentiation of hUSCs and to investigate the underlying mechanism. The degree of osteogenic differentiation and the correlated signals, following BMP2 overexpression and siRNA-mediated silencing of FAK, were determined in vitro. Moreover, hUSCs induced bone formation in a rat model with cranial defects, in vivo. Our findings revealed that alkaline phosphatase production, calcium deposits, osteocalcin and osteopontin expression, and bone formation were upregulated in vitro and in vivo following BMP2-induced osteogenic differentiation, and AMPK and Wnt signaling pathway activation by FAK could effectively regulate BMP2-enhanced osteogenic differentiation of hUSCs. Taken together, these findings indicated that FAK could mediate BMP2-enhanced osteogenic differentiation of hUSCs through activating adenosine 5’-monophosphate-activated protein kinase and Wnt signaling pathways.     

Synthetic scaffold coating with adeno-associated virus encoding BMP2 to promote endogenous bone repair

Biomaterial scaffolds functionalized to stimulate endogenous repair mechanisms via the incorporation of osteogenic cues offer a potential alternative to bone grafting for the treatment of large bone defects. We first quantified the ability of a self-complementary adeno-associated viral vector encoding bone morphogenetic protein 2 (scAAV2.5-BMP2) to enhance human stem cell osteogenic differentiation in vitro. In two-dimensional culture, scAAV2.5-BMP2-transduced human mesenchymal stem cells (hMSCs) displayed significant increases in BMP2 production and alkaline phosphatase activity compared with controls. hMSCs and human amniotic-fluid-derived stem cells (hAFS cells) seeded on scAAV2.5-BMP2-coated three-dimensional porous polymer Poly(ε-caprolactone) (PCL) scaffolds also displayed significant increases in BMP2 production compared with controls during 12 weeks of culture, although only hMSC-seeded scaffolds displayed significantly increased mineral formation. PCL scaffolds coated with scAAV2.5-BMP2 were implanted into critically sized immunocompromised rat femoral defects, both with or without pre-seeding of hMSCs, representing ex vivo and in vivo gene therapy treatments, respectively. After 12 weeks, defects treated with acellular scAAV2.5-BMP2-coated scaffolds displayed increased bony bridging and had significantly higher bone ingrowth and mechanical properties compared with controls, whereas defects treated with scAAV2.5-BMP2 scaffolds pre-seeded with hMSCs failed to display significant differences relative to controls. When pooled, defect treatment with scAAV2.5-BMP2-coated scaffolds, both with or without inclusion of pre-seeded hMSCs, led to significant increases in defect mineral formation at all time points and increased mechanical properties compared with controls. This study thus presents a novel acellular bone-graft-free endogenous repair therapy for orthotopic tissue-engineered bone regeneration.     

组织工程中应力对骨髓间充质干细胞分化为成骨细胞的影响

骨髓间充质干细胞具有自我复制、未分化的特点,并可在不同条件下分化为中胚层起源的多种细胞,是一种成体多能干细胞。就组织工程而言,良好的种子细胞是组织工程技术的关键,骨髓间充质干细胞的性质决定了其在骨组织工程领域中的重要地位。此外,骨骼系统属于机体的运动系统,承担体重是骨骼的重要功能之一;而且,人体内几乎所有的细胞都会受到力学因素的影响,故有必要研究力学因素对骨髓间充质干细胞诱导分化为成骨细胞的作用,为骨髓间充质干细胞的体外扩增、诱导分化及培养提供一种新途径。     

Purmorphamine Promotes Matrix Mineralization and Cytoskeletal Changes in Human Umbilical Cord Mesenchymal Stem Cells

Human Umbilical Cord Mesenchymal Stem Cells (hUCMSCs) were subjected to in vitro osteogenic differentiation using a novel combination of signaling molecules including BMP-2 and purmorphamine. Differentiation outcomes were assessed by calcein staining and by microscopic examination of the cytoskeleton. Calcein staining showed appreciable degree of calcium mineralization in cell culture, and changes in the morphological attributes of differentiating cells were observed vis-a-vis the actin cytoskeleton. Finally, positive calcein staining, altered cytoskeletal profile, and stress fiber formation in treated cells demonstrated, for the first time, a potentially synergistic interplay between BMP-2 and the hedgehog agonist, purmorphamine. This study lends support to the notion of combining small doses of potent molecules that can act as safe, less toxic inducers of osteogenic differentiation of human umbilical cord mesenchymal stem cells with respect to bone regeneration.