Wnt1a maintains characteristics of dermal papilla cells that induce mouse hair regeneration in a 3D preculture system

Hair follicle morphogenesis and regeneration depend on intensive but well‐orchestrated interactions between epithelial and mesenchymal components. Therefore, an alternative strategy to reproduce the process of epithelial–mesenchymal interaction in vitro could use a 3D system containing appropriate cell populations. The 3D air–liquid culture system for reproducibly generating hair follicles from dissociated epithelial and dermal papilla (DP) cells combined with a collagen–chitosan scaffold is described in this study. Wnt‐CM was prepared from the supernatant of Wnt1a‐expressing bone marrow mesenchymal stem cells (BM‐MSCs) that maintain the hair‐inducing gene expression of DP cells. The collagen–chitosan scaffold cells (CCS cells) were constructed using a two‐step method by inoculating the Wnt‐CM‐treated DP cells and epidermal (EP) cells into the CCS. The cells in the air–liquid culture formed dermal condensates and a proliferative cell layer in vitro. The CCS cells were able to induce hair regeneration in nude mice. The results demonstrate that Wnt‐CM can maintain the hair induction ability of DP cells in expansion cultures, and this approach can be used for large‐scale preparation of CCS cells in vitro to treat hair loss. Copyright © 2015 John Wiley & Sons, Ltd.     

Influence of in vitro biomimicked stem cell ‘niche’ for regulation of proliferation and differentiation of human bone marrow‐derived mesenchymal stem cells to myocardial phenotypes: serum starvation without aid of chemical agents and prevention of spontaneous stem cell transformation enhanced by the matrix environment

Niche appears important for preventing the spontaneous differentiation or senescence that cells undergo during in vitro expansion. In the present study, it was revealed that human bone marrow‐derived mesenchymal stem cells (hBM‐MSCs) undergo senescence‐related differentiation into the myocardial lineage in vitro without any induction treatment. This phenomenon occurred over the whole population of MCSs, much different from conventional differentiation with limited frequency of occurrence, and was accompanied by a change of morphology into large, flat cells with impeded proliferation, which are the representative indications of MSC senescence. By culturing MSCs under several culture conditions, it was determined that induction treatment with 5‐azacytidine was not associated with the phenomenon, but the serum‐starvation condition, under which proliferation is severely hampered, caused senescence progression and upregulation of cardiac markers. Nevertheless, MSCs gradually developed a myocardial phenotype under normal culture conditions over a prolonged culture period and heterogeneous populations were formed. In perspectives of clinical applications, this must be prevented for fair and consistent outcomes. Hence, the biomimetic 'niche' was constituted for hBM‐MSCs by cultivating on a conventionally available extracellular matrix (ECM). Consequently, cells on ECM regained a spindle‐shape morphology, increased in proliferation rate by two‐fold and showed decreased expression of cardiac markers at both the mRNA and protein levels. In conclusion, the outcome indicates that progression of MSC senescence may occur via myocardial differentiation during in vitro polystyrene culture, and this can be overcome by employing appropriate ECM culture techniques. Copyright © 2013 John Wiley & Sons, Ltd.     

An in vitro model of mesenchymal stem cell targeting using magnetic particle labelling

The specific targeting of cells to sites of tissue damage in vivo is a major challenge precluding the success of stem cell‐based therapies. Magnetic particle‐based targeting may provide a solution. Our aim was to provide a model system to study the trapping and potential targeting of human mesenchymal stem cells (MSCs) during in vitro fluid flow, which ultimately will inform cell targeting in vivo. In this system magnet arrays were used to trap superparamagnetic iron oxide particle‐doped MSCs. The in vitro experiments demonstrated successful cell trapping, where the volume of cells trapped increased with magnetic particle concentration and decreased with increasing flow rate. Analysis of gene expression revealed significant increases in COL1A2 and SOX9. Using principles established in vitro, a proof‐of‐concept in vivo experiment demonstrated that magnetic particle‐doped, luciferase‐expressing MSCs were trapped by an implanted magnet in a subcutaneous wound model in nude mice. Our results demonstrate the effectiveness of using an in vitro model for testing superparamagnetic iron oxide particles to develop successful MSC targeting strategies during fluid flow, which ultimately can be translated to in vivo targeted delivery of cells via the circulation in a variety of tissue‐repair models. Copyright © 2012 John Wiley & Sons, Ltd.     

Wnt Modulating Agents Inhibit Human Cytomegalovirus Replication

Infection with human cytomegalovirus (HCMV) continues to be a threat for pregnant women and immunocompromised hosts. Although limited anti-HCMV therapies are available, development of new agents is desired. The Wnt signaling pathway plays a critical role in embryonic and cancer stem cell development and is targeted by gammaherpesviruses, Epstein-Barr virus (EBV), and Kaposi''s sarcoma-associated herpesvirus (KSHV). HCMV infects stem cells, including neural progenitor cells, during embryogenesis. To investigate the role of Wnt in HCMV replication in vitro, we tested monensin, nigericin, and salinomycin, compounds that inhibit cancer stem cell growth by modulating the Wnt pathway. These compounds inhibited the replication of HCMV Towne and a clinical isolate. Inhibition occurred prior to DNA replication but persisted throughout the full replication cycle. There was a significant decrease in expression of IE2, UL44, and pp65 proteins. HCMV infection resulted in a significant and sustained decrease in expression of phosphorylated and total lipoprotein receptor-related protein 6 (pLRP6 and LRP6, respectively), Wnt 5a/b, and β-catenin and a modest decrease in Dvl2/3, while levels of the negative regulator axin 1 were increased. Nigericin decreased the expression of pLRP6, LRP6, axin 1, and Wnt 5a/b in noninfected and HCMV-infected cells. For all three compounds, a correlation was found between expression levels of Wnt 5a/b and axin 1 and HCMV inhibition. The decrease in Wnt 5a/b and axin 1 expression was more significant in HCMV-infected cells than noninfected cells. These data illustrate the complex effects of HCMV on the Wnt pathway and the fine balance between Wnt and HCMV, resulting in abrogation of HCMV replication. Additional studies are required to elucidate how HCMV targets Wnt for its benefit.     

Bone marrow‐on‐a‐chip: Long‐term culture of human haematopoietic stem cells in a three‐dimensional microfluidic environment

Multipotent haematopoietic stem and progenitor cells (HSPCs) are the source for all blood cell types. The bone marrow stem cell niche in which the HSPCs are maintained is known to be vital for their maintenance. Unfortunately, to date, no in vitro model exists that accurately mimics the aspects of the bone marrow niche and simultaneously allows the long‐term culture of HSPCs. In this study, a novel three‐dimensional coculture model is presented, based on a hydroxyapatite coated zirconium oxide scaffold, comprising of human mesenchymal stromal cells (MSCs) and cord blood derived HSPCs, enabling successful HSPC culture for a time span of 28 days within the microfluidic multiorgan chip. The HSPCs were found to stay in their primitive state (CD34⁺CD38^?) and capable of granulocyte, erythrocyte, macrophage, megakaryocyte colony formation. Furthermore, a microenvironment was formed bearing molecular and structural similarity to the in vivo bone marrow niche containing extracellular matrix and signalling molecules known to play an important role in HSPC homeostasis. Here, a novel human in vitro bone marrow model is presented for the first time, capable of long‐term culture of primitive HSPCs in a microfluidic environment.     

Characterization of biomaterial‐free cell sheets cultured from human oral mucosal epithelial cells

The purpose of this study was to report the characteristics of biomaterial‐free sheets cultured from human oral mucosal epithelial cells without fibrin support, in vitro and after transplantation to limbal‐deficient models. Human oral mucosal epithelial cells and limbal epithelial cells were cultured for 2 weeks, and the colony‐forming efficiency (CFE) rates were compared. Markers of stem cells (p63), cell proliferation (Ki‐67) and epithelial differentiation (cytokeratin; K1, K3, K4, K13) were observed in colonies and in biomaterial‐free sheets. Biomaterial‐free sheets which had been detached with 1% dispase or biomaterial‐free sheets generated by fibrin support were transplanted to 12 limbal‐deficient rabbit models. In vitro cell viability, in vivo stability and cytokeratin characteristics of biomaterial‐free sheets were compared with those of sheets formed by fibrin‐coated culture 1 week after transplantation. Mean CFE rate was significantly higher in human oral mucosal epithelial cells (44.8%) than in human limbal epithelial cells(17.7%). K3 and K4 were well expressed in both colonies and sheets. Biomaterial‐free sheets had two to six layers of stratified cells and showed an average of 79.8% viable cells in the sheets after detachment. Cytokeratin expressions of biomaterial‐free sheets were comparable to those of sheets cultured by fibrin support, in limbal‐deficient models. Both p63 and Ki‐67 were well expressed in colonies, isolated sheets and sheets transplanted to limbal‐deficient models. Our results suggest that biomaterial‐free sheets cultured from human oral mucosal epithelial cells without fibrin support can be an alternative option for cell therapy in use for the treatment of limbal‐deficient diseases. Copyright © 2014 John Wiley & Sons, Ltd.     

Effect of chitosan conduit under a dynamic culture on the proliferation and neural differentiation of human exfoliated deciduous teeth stem cells

Ex vivo engineering of artificial nerve conduit is a suitable alternative clinical treatment for nerve injuries. Stem cells from human exfoliated deciduous teeth (SHEDs) have been considered as alternative sources of adult stem cells because of their potential to differentiate into multiple cell lineages. These cells, when cultured in six‐well plates, exhibited a spindle fibroblastic morphology, whereas those under a dynamic culture aggregated into neurosphere‐like clusters in the chitosan conduit. In this study, we confirmed that SHEDs efficiently express the neural stem cell marker nestin, the early neural cell marker β‐III‐tubulin, the late neural marker neuron‐specific enolase and the glial cell markers glial fibrillary acidic protein (GFAP) and 2',3'‐cyclic nucleotide‐3'‐phosphodiesterase (CNPase). The three‐dimensional chitosan conduit and dynamic culture system generated fluid shear stress and enhanced nutrient transfer, promoting the differentiation of SHEDs to neural cells. In particular, the gene expressions of GFAP and CNPase increased by 28‐ and 53‐fold, respectively. This study provides evidence for the dynamic culture of SHEDs during ex vivo neural differentiation and demonstrates its potential for cell therapy in neurological diseases. Copyright © 2013 John Wiley & Sons, Ltd.     

Autonomous magnetic labelling of functional mesenchymal stem cells for improved traceability and spatial control in cell therapy applications

Mesenchymal stem cells (MSCs) represent a valuable resource for regenerative medicine treatments for orthopaedic repair and beyond. Following developments in isolation, expansion and differentiation protocols, efforts to promote clinical translation of emerging cellular strategies now seek to improve cell delivery and targeting. This study shows efficient live MSC labelling using silica‐coated magnetic particles (MPs), which enables 3D tracking and guidance of stem cells. A procedure developed for the efficient and unassisted particle uptake was shown to support MSC viability and integrity, while surface marker expression and MSC differentiation capability were also maintained. In vitro, MSCs showed a progressive decrease in labelling over increasing culture time, which appeared to be linked to the dilution effect of cell division, rather than to particle release, and did not lead to detectable secondary particle uptake. Labelled MSC populations demonstrated magnetic responsiveness in vitro through directed migration in culture and, when seeded onto a scaffold, supporting MP‐based approaches to cell targeting. The potential of these silica‐coated MPs for MRI cell tracking of MSC populations was validated in 2D and in a cartilage repair model following cell delivery. These results highlight silica‐coated magnetic particles as a simple, safe and effective resource to enhance MSC targeting for therapeutic applications and improve patient outcomes. © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.     

FGF,TGFβ and Wnt crosstalk: embryonic to in vitro cartilage development from mesenchymal stem cells

Articular cartilage is easily damaged, yet difficult to repair. Cartilage tissue engineering seems a promising therapeutic solution to restore articular cartilage structure and function, with mesenchymal stem cells (MSCs) receiving increasing attention for their promise to promote cartilage repair. It is known from embryology that members of the fibroblast growth factor (FGF), transforming growth factor‐β (TGFβ) and wingless‐type (Wnt) protein families are involved in controlling different differentiation stages during chondrogenesis. Individually, these pathways have been extensively studied but so far attempts to recapitulate embryonic development in in vitro MSC chondrogenesis have failed to produce stable and functioning articular cartilage; instead, transient hypertrophic cartilage is obtained. We believe a better understanding of the simultaneous integration of these factors will improve how we relate embryonic chondrogenesis to in vitro MSC chondrogenesis. This narrative review attempts to define current knowledge on the crosstalk between the FGF, TGFβ and Wnt signalling pathways during different stages of mesenchymal chondrogenesis. Connecting embryogenesis and in vitro differentiation of human MSCs might provide insights into how to improve and progress cartilage tissue engineering for the future. Copyright © 2013 John Wiley & Sons, Ltd.     

Culture conditions have an impact on the maturation of traceable,transplantable mouse embryonic stem cell‐derived otic progenitor cells

The generation of replacement inner ear hair cells (HCs) remains a challenge and stem cell therapy holds the potential for developing therapeutic solutions to hearing and balance disorders. Recent developments have made significant strides in producing mouse otic progenitors using cell culture techniques to initiate HC differentiation. However, no consensus has been reached as to efficiency and therefore current methods remain unsatisfactory. In order to address these issues, we compare the generation of otic and HC progenitors from embryonic stem (ES) cells in two cell culture systems: suspension vs. adherent conditions. In the present study, an ES cell line derived from an Atoh1‐green fluorescent protein (GFP) transgenic mouse was used to track the generation of otic progenitors, initial HCs and to compare these two differentiation systems. We used a two‐step short‐term differentiation method involving an induction period of 5 days during which ES cells were cultured in the presence of Wnt/transforming growth factor TGF‐β inhibitors and insulin‐like growth factor IGF‐1 to suppress mesoderm and reinforce presumptive ectoderm and otic lineages. The generated embryoid bodies were then differentiated in medium containing basic fibroblast growth factor (bFGF) for an additional 5 days using either suspension or adherent culture methods. Upon completion of differentiation, quantitative polymerase chain reaction analysis and immunostaining monitored the expression of otic/HC progenitor lineage markers. The results indicate that cells differentiated in suspension cultures produced cells expressing otic progenitor/HC markers at a higher efficiency compared with the production of these cell types within adherent cultures. Furthermore, we demonstrated that a fraction of these cells can incorporate into ototoxin‐injured mouse postnatal cochlea explants and express MYO7A after transplantation. Copyright © 2016 John Wiley & Sons, Ltd.     

Influence of self‐assembling peptide nanofibre scaffolds on retinal differentiation potential of stem/progenitor cells derived from ciliary pigment epithelial cells

Aim of the study was to investigate the influence of the self‐assembling peptide nanofibre scaffolds (SAPNs) on the growth, proliferation and retinal neuronal differentiation of the stem/progenitor cells (SCs) derived from the ciliary pigment epithelium (CPE) of human cadaveric eye. Here SAPNs (RADA16‐I, PM), which is well described in previous studies, commercially available and xeno‐free. The CPE cells isolated were cultured in DMEM/F12 supplemented with N2 and growth factors such as basic fibroblast growth factor and epidermal growth factor, encapsulated in the scaffolds. The entrapped SCs actively expanded and formed clone‐like clusters in the scaffolds. Many cells in the cluster were proliferating, as revealed by 5‐bromo‐2‐deoxyuridine uptake and could be maintained for up to 6 days and expressed neural progenitor markers such as β‐III tubulin, Nestin, Pax6 and Musashi1. Upon differentiation of these cells in conditioned medium, the cells exhibited retinal neuronal markers such as s‐Opsin, rhodopsin and Recoverin. The RT² profiler polymerase chain reaction array experiments showed selective gene expression, possibly involved in neural stem/progenitor cell adhesion and differentiation. These findings suggest the suitability of the three‐dimensional culture system for the proliferation and maintenance of CPE stem/progenitor cells (CPE‐NS) and for possible use in ex vivo studies of small molecules, drug deliveries for retinal diseases and for use in combination with directed stem/progenitor cell differentiation. and ultimately for tissue replacement therapies. Copyright © 2014 John Wiley & Sons, Ltd.     

Dynamic cell–cell interactions between cord blood haematopoietic progenitors and the cellular niche are essential for the expansion of CD34+, CD34+CD38− and early lymphoid CD7+ cells

Most clinical applications of haematopoietic stem/progenitor cells (HSCs) would benefit from their ex vivo expansion to obtain a therapeutically significant amount of cells from the available donor samples. We studied the impact of cellular interactions between umbilical cord blood (UCB) haematopoietic cells and bone marrow (BM)‐derived mesenchymal stem cells (MSCs) on the ex vivo expansion and differentiative potential of UCB CD34⁺‐enriched cells. UCB cells were cultured: (a) directly in contact with BM MSC‐derived stromal layers (contact); (b) separated by a microporous membrane (non‐contact); or (c) without stroma (no stroma). Highly dynamic culture events occurred in HSC‐MSC co‐cultures, involving cell–cell interactions, which preceded HSC expansion. Throughout the time in culture [18 days], total cell expansion was significantly higher in contact (fold increase of 280 ± 37 at day 18) compared to non‐contact (85 ± 25). No significant cell expansion was observed in stroma‐free cultures. CD34⁺ cell expansion was also clearly favoured by direct contact with BM MSCs (35 ± 5‐ and 7 ± 3‐fold increases at day 18 for contact and non‐contact, respectively). Moreover, a higher percentage of CD34⁺CD38^? cells was consistently maintained during the time in culture under contact (8.1 ± 1.9% at day 18) compared to non‐contact (5.7 ± 1.6%). Importantly, direct cell interaction with BM MSCs significantly enhanced the expansion of early lymphoid CD7⁺ cells, yielding considerably higher (×3–10) progenitor numbers compared to non‐contact conditions. These results highlight the importance of dynamic cell–cell interactions between UCB HSCs and BM MSCs, towards the maximization of HSC expansion ex vivo to obtain clinically relevant cell numbers for multiple settings, such as BM transplantation or somatic cell gene therapy. Copyright © 2009 John Wiley & Sons, Ltd.     

Rotary orbital suspension culture of embryonic stem cell‐derived neural stem/progenitor cells: impact of hydrodynamic culture on aggregate yield,morphology and cell phenotype

Embryonic stem (ES)‐derived neural stem/progenitor cells (ES‐NSPCs) constitute a promising cell source for application in cell therapies for the treatment of central nervous system disorders. In this study, a rotary orbital hydrodynamic culture system was applied to single‐cell suspensions of ES‐NSPCs, to obtain homogeneously‐sized ES‐NSPC cellular aggregates (neurospheres). Hydrodynamic culture allowed the formation of ES‐NSPC neurospheres with a narrower size distribution than statically cultured neurospheres, increasing orbital speeds leading to smaller‐sized neurospheres and higher neurosphere yield. Neurospheres formed under hydrodynamic conditions (72 h at 55 rpm) showed higher cell compaction and comparable percentages of viable, dead, apoptotic and proliferative cells. Further characterization of cellular aggregates provided new insights into the effect of hydrodynamic shear on ES‐NSPC behaviour. Rotary neurospheres exhibited reduced protein levels of N‐cadherin and β‐catenin, and higher deposition of laminin (without impacting fibronectin deposition), matrix metalloproteinase‐2 (MMP‐2) activity and percentage of neuronal cells. In line with the increased MMP‐2 activity levels found, hydrodynamically‐cultured neurospheres showed higher outward migration on laminin. Moreover, when cultured in a 3D fibrin hydrogel, rotary neurospheres generated an increased percentage of neuronal cells. In conclusion, the application of a constant orbital speed to single‐cell suspensions of ES‐NSPCs, besides allowing the formation of homogeneously‐sized neurospheres, promoted ES‐NSPC differentiation and outward migration, possibly by influencing the expression of cell–cell adhesion molecules and the secretion of proteases/extracellular matrix proteins. These findings are important when establishing the culture conditions needed to obtain uniformly‐sized ES‐NSPC aggregates, either for use in regenerative therapies or in in vitro platforms for biomaterial development or pharmacological screening. Copyright © 2016 John Wiley & Sons, Ltd.     

Functional liver tissue engineering by an adult mouse liver‐derived neuro‐glia antigen 2‐expressing stem/progenitor population

Deaths due to end‐stage liver diseases are increasingly registered annually in the world. Liver transplantation is the ultimate treatment for end‐stage liver diseases to date, which has been hampered by a critical shortage of organs. The potential of decellularized liver scaffolds (DLS) derived from solid organs as a three‐dimensional platform has been evolved as a promising approach in liver tissue engineering for translating functional liver organ replacements, but questions still exist regarding the optimal cell population for seeding in DLS and the preparation of the DLS themselves. The aim of our study was to utilize a sodium dodecyl sulfate decellularization procedure in combination with a low concentration of trypsin (0.005%)–ethylenediaminetetraacetic acid (0.002%) process to manufacture DLS from whole mouse livers and recellularized with hepatic stem/progenitors for use in liver tissue engineering and injured liver treatment. Results showed that the DLS generated with all the necessary microstructure and the extracellular components to support seeded hepatic stem/progenitor cell attachment, functional hepatic cell differentiation. Hepatic differentiation from stem/progenitor cells loaded by DLS was more efficient than that of the stem/progenitor cells in the two‐dimensional cell culture model. In summary, the method of DLS loaded by hepatic stem/progenitor cells provided by this study was effective in maintaining DLS extracellular matrix to introduce seeded stem/progenitor cell differentiation, hepatic‐like tissue formation and functional hepatic protein production in vitro that promoted functional recovery and survival in a mouse model of dimethylnitrosamine‐induced liver cirrhosis after auxiliary heterotopic liver transplantation. Copyright © 2016 John Wiley & Sons, Ltd.     

持续 Wnt 信号通路激活促进胚胎干细胞源造血干细胞的增殖简

背景：如何提高胚胎干细胞诱导效率、促进胚胎干细胞源造血干细胞体外增殖成为目前急需解决的课题。目的：以外源性Wnt3a作为诱导剂,激活培养中的小鼠胚胎干细胞Wnt/β-catenin信号通路,观察该通路的激活是否促进胚胎干细胞向造血祖细胞的定向分化。方法：用外源性wnt3a（100μg/L）持续作用ES-E14TG2a小鼠胚胎干细胞21 d,通过细胞免疫荧光及蛋白免疫印迹检测细胞内β-catenin蛋白含量,QRT-PCR检测Wnt下游靶标基因的表达量来确定经典Wnt/β-catenin信号通路是否被激活,然后采用单层贴壁培养法诱导其向造血干细胞分化,流式细胞仪检测造血发育相关表面标志CD34＋/Sca-1＋,同时以QRT-PCR法检测造血相关基因的表达情况。结果与结论：ES-E14TG2a小鼠胚胎干细胞经wnt3a（100μg/L）连续培养21 d后发现β-catenin蛋白在细胞内积累;Wnt信号通路的下游靶标基因Pitx2、Frizzled、Sox17、Oct4的表达量均出现不同程度的增加,可见经典 Wnt/β-catenin 信号通路有被激活;单层贴壁培养法诱导其向造血干细胞分化的过程中检测到CD34＋/Sca-1＋细胞含量在14 d时占总细胞量高达20.2%,而对照组的仅占11.9%。造血相关基因骨形态发生蛋白4、FLK2及CD34的表达量均增加,而Smad5的表达则明显受到抑制。说明Wnt3a持续作用可激活Wnt/β-catenin信号通路,并促进ES-E14TG2a小鼠胚胎干细胞向造血干细胞的定向分化。     

Wnt‐4 potently inhibits capillary outgrowth from rat aorta in 3D culture

Regulation of angiogenesis involves tight cell‐to‐cell and cell‐to‐extracellular‐matrix interactions. Various reports demonstrate that the Wnt signaling pathways participate in this regulation. Using a three‐dimensional aortic ring culture combined with an ex vivo retroviral infection approach, we evaluated the effects of two Wnt growth factors, Wnt‐1 and Wnt‐4, on the formation and growth of new capillaries. Our results show that Wnt‐1 had no effect, whereas Wnt‐4 was a potent inhibitor of capillary outgrowth in vitro.     

Pdx1 and controlled culture conditions induced differentiation of human amniotic fluid‐derived stem cells to insulin‐producing clusters

This study investigated the differentiation of human amniotic fluid‐derived stem cells (hAFSCs) into insulin‐producing clusters in vitro. Adenovirally‐delivered mouse Pdx1 (Ad‐Pdx1) induced human Pdx1 expression in hAFSCs and enhanced the coordinated expression of downstream β‐cell markers. When Ad‐Pdx1‐transduced hAFSCs were sequentially treated with activin A, bFGF and nicotinamide and the culture plate surface coated with poly‐l ‐ornithine, the expression of islet‐associated human mRNAs for Pdx1, Pax6, Ngn3 and insulin was increased. C‐peptide ELISA confirmed that Ad‐Pdx1‐transduced hAFSCs processed and secreted insulin in a manner consistent with that pathway in pancreatic β‐cells. To sustain the β‐cell‐like phenotype and investigate the effect of three‐dimensional (3D) conformation on the differentiation of hAFSCs, Pdx1‐transduced cells were encapsulated in alginate and cultured long‐term under serum‐free conditions. Over 2 weeks, partially differentiated hAFSC clusters increased in size and increased insulin secretion. Taken together, these data demonstrate that ectopic Pdx1 expression initiates pancreatic differentiation in hAFSCs and that a β‐cell‐like phenotype can be augmented by culture conditions that mimic the stromal components and 3D geometry associated with pancreatic islets. Copyright © 2012 John Wiley & Sons, Ltd.