Frizzled receptors (FZDs) in Wnt signaling: potential therapeutic targets for human cancers.

Frizzled receptors (FZDs) are key contributors intrinsic to the Wnt signaling pathway, activation of FZDs triggering the Wnt signaling cascade is frequently observed in human tumors and intimately associated with an aggressive carcinoma phenotype. It has been shown that the abnormal expression of FZD receptors contributes to the manifestation of malignant characteristics in human tumors such as enhanced cell proliferation, metastasis, chemotherapy resistance as well as the acquisition of cancer stemness. Given the essential roles of FZD receptors in the Wnt signaling in human tumors, this review aims to consolidate the prevailing knowledge on the specific status of FZD receptors (FZD1-10) and elucidate their respective functions in tumor progression. Furthermore, we delineate the structural basis for binding of FZD and its co-receptors to Wnt, and provide a better theoretical foundation for subsequent studies on related mechanisms. Finally, we describe the existing biological classes of small molecule-based FZD inhibitors in detail in the hope that they can provide useful assistance for design and development of novel drug candidates targeted FZDs.

Immunocompetent PDMS-Free Organ-on-Chip Model of Cervical Cancer Integrating Patient-Specific Cervical Fibroblasts and Neutrophils.

Despite preventive measures and available treatments, cervical cancer still ranks as the fourth most prevalent cancer among women worldwide and remains the leading cause of cancer death in women in many developing countries. To gain further insights into pathogenesis and to develop novel (immuno)therapies, more sophisticated human models recreating patient heterogeneities and including aspects of the tumor microenvironment are urgently required. A novel polydimethylsiloxane-free microfluidic platform, designed specifically for the generation and ccultivation of cervical cancerous tissue, is introduced. The microscale open-top tissue chambers of the cervical cancer-on-chip (CCoC) enable facile generation and long-term cultivation of SiHa spheroids in co-culture with donor-derived cervical fibroblasts. The resulting 3D tissue emulates physiological architecture and allows dissection of distinct effects of the stromal tissue on cancer viability and growth. Treatment with cisplatin at clinically-relevant routes of administration and dosing highlights the platform's applicability for drug testing. Moreover, the model is amenable for integration and recruitment of donor-derived neutrophils from the microvasculature-like channel into the tissue, all while retaining their ability to produce neutrophil extracellular traps. In the future, the immunocompetent CCoC featuring donor-specific primary cells and tumor spheroids has the potential to contribute to the development of new (immuno)therapeutic options.

Past, present, and future of deep transcranial magnetic stimulation: A review in psychiatric and neurological disorders.

Deep transcranial magnetic stimulation (DTMS) is a new non-invasive neuromodulation technique based on repetitive transcranial magnetic stimulation tech-nology. The new H-coil has significant advantages in the treatment and mechanism research of psychiatric and neurological disorders. This is due to its deep stimulation site and wide range of action. This paper reviews the clinical progress of DTMS in psychiatric and neurological disorders such as Parkinson's disease, Alzheimer's disease, post-stroke motor dysfunction, aphasia, and other neurological disorders, as well as anxiety, depression, and schizophrenia.

Rapid Capture of Cancer Extracellular Vesicles by Lipid Patch Microarrays.

Extracellular vesicles (EVs) contain various bioactive molecules such as DNA, RNA, and proteins, and play a key role in the regulation of cancer progression. Furthermore, cancer-associated EVs carry specific biomarkers and can be used in liquid biopsy for cancer detection. However, it is still technically challenging and time consuming to detect or isolate cancer-associated EVs from complex biofluids (e.g., blood). Here, a novel EV-capture strategy based on dip-pen nanolithography generated microarrays of supported lipid membranes is presented. These arrays carry specific antibodies recognizing EV- and cancer-specific surface biomarkers, enabling highly selective and efficient capture. Importantly, it is shown that the nucleic acid cargo of captured EVs is retained on the lipid array, providing the potential for downstream analysis. Finally, the feasibility of EV capture from patient sera is demonstrated. The demonstrated platform offers rapid capture, high specificity, and sensitivity, with only a small need in analyte volume and without additional purification steps. The platform is applied in context of cancer-associated EVs, but it can easily be adapted to other diagnostic EV targets by use of corresponding antibodies.

Molecular Design Principles for Ferroelectrics: Ferroelectrochemistry.

Ferroelectric materials have a variety of technological applications, as transducers, capacitors, sensors, etc. Great interest in molecular ferroelectrics has emerged because of their structural flexibility, tunability, and homochirality. However, the discoveries of molecular ferroelectrics are not abundant. The lack of chemical design is the main challenge in realizing new molecular ferroelectrics. Consequently, chemical design approaches, including the ideas of introducing quasi-spherical theory, homochirality, and H/F substitution, have been developed recently. Through these advanced methodologies, a wide range of ferroelectrics were successfully synthesized, changing the blind search into a targeted chemical design. In this Perspective, we aim to provide insight into the fundamental chemistry and physics of molecular ferroelectrics and propose the concept of "ferroelectrochemistry", concerned with the targeted design and performance optimization of molecular ferroelectrics from the chemical point of view. We start with the basic theories used in the modification of chemical structures for new molecular ferroelectrics, such as the quasi-spherical theory. After that, we focus on the fundamentals of homochirality from the perspective of chemistry and advantages of introducing a homochiral molecule within the scope of ferroelectrics. Further, we explore another design strategy, H/F substitution, as an analogue of the H/D isotope effect. The introduction of a F atom usually does not change the polar point group but may induce a minor structural disruption that enhances physical properties such as Curie temperature and spontaneous polarization. We hope our comprehensive studies on the targeted design and performance optimization strategies for molecular ferroelectrics may build up and enrich the content of ferroelectrochemistry.

The effect of intermittent hypoxia training on migraine: a randomized controlled trial.

OBJECTIVE: To study the effect of intermittent hypoxia training (IHT) for migraine. DESIGN: A single-blind, randomized controlled trial. All participants were recruited from a rehabilitation department in an acute university-affiliated hospital. METHODS: Participants with migraines were randomly assigned to two groups (IHT group and control group). The Migraine Disability Assessment (MIDAS), Visual Analog Scale (VAS), Beck Depression Inventory (BDI), Beck Anxiety Inventory (BAI), Vascular endothelial growth factor (VEGF), calcitonin gene related peptide (CGRP) and cerebrovascular hemodynamic parameters were collected at baseline and end of the 8th week. The attack frequencies of migraines were evaluated at 3 months. RESULTS: Among the 48 subjects, five males and forty-three females, the ages ranged from 19 to 53 years old (mean ± SD = 31.3±7.78). MIDAS, SF-36, VAS, BAI, BDI, VEGF, CGRP and cerebrovascular hemodynamic parameters were improved after IHT intervention. There were significant differences between IHT group and the control group in MIDAS, SF-36, VAS, BAI, BDI, VEGF, CGRP and cerebrovascular hemodynamic parameters at the end of the 8th weeks (P<0.05). Attack frequencies were improved within 3 months after IH training intervention (P<0.01), but not in the control group (P>0.05). No adverse events occurred during the study. CONCLUSION: IHT could improve migraines after intervention up to three months. IHT could be an effective method for relieving a migraine.

Enhanced Stability of Lipid Structures by Dip-Pen Nanolithography on Block-Type MPC Copolymer.

Biomimetic lipid membranes on solid supports have been used in a plethora of applications, including as biosensors, in research on membrane proteins or as interfaces in cell experiments. For many of these applications, structured lipid membranes, e.g., in the form of arrays with features of different functionality, are highly desired. The stability of these features on a given substrate during storage and in incubation steps is key, while at the same time the substrate ideally should also exhibit antifouling properties. Here, we describe the highly beneficial properties of a 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymer for the stability of supported lipid membrane structures generated by dip-pen nanolithography with phospholipids (L-DPN). The MPC copolymer substrates allow for more stable and higher membrane stack structures in comparison to other hydrophilic substrates, like glass or silicon oxide surfaces. The structures remain highly stable under immersion in liquid and subsequent incubation and washing steps. This allows multiplexed functionalization of lipid arrays with antibodies via microchannel cantilever spotting (µCS), without the need of orthogonal binding tags for each antibody type. The combined properties of the MPC copolymer substrate demonstrate a great potential for lipid-based biomedical sensing and diagnostic platforms.

Evaluation of Microfluidic Ceiling Designs for the Capture of Circulating Tumor Cells on a Microarray Platform.

The capture of circulating tumor cells (CTCs) is still a challenging application for microfluidic chips, as these cells are rare and hidden in a huge background of blood cells. Here, different microfluidic ceiling designs in regard to their capture efficiency for CTCs in model experiments and more realistic conditions of blood samples spiked with a clinically relevant amount of tumor cells are evaluated. An optimized design for the capture platform that allows highly efficient recovery of CTCs from size-based pre-enriched samples under realistic conditions is obtained. Furthermore, the viability of captured tumor cells as well as single cell recovery for downstream genomic analysis is demonstrated. Additionally, the authors' findings underline the importance of evaluating rational design rules for microfluidic devices based on theoretical models by application-specific experiments.

pH-Sensitive Near-IR Emitting Dinuclear Ruthenium Complex for Recognition, Two-Photon Luminescent Imaging, and Subcellular Localization of Cancer Cells.

A dinuclear Ru(II) complex of [(bpy)2Ru(Hdip)Ru(H2bip)](ClO4)4 {bpy is 2,2'-bipyridine, Hdip is 2-(2,6-di(pyridin-2-yl)-pyridin-4-yl)-1H-imidazo[4,5-f]-[1,10]phenanthroline, and H2bip is 2,6-bis(imidazole-2-yl)-pyridine} was synthesized and characterized by elemental analysis, mass spectrometry, and 1H NMR spectroscopy. Spectrophotometric pH titrations in aqueous buffer and in vitro cell experiments indicated the response ability of the complex to pH fluctuations in the physiological pH range (6.0-8.0). The complex was found to be capable of differentiating live HeLa cells from healthy HEK293 cells by selectively accumulating in lysosomes of the HeLa cells. The low cytotoxicity (IC50 > 100 µM), a large Stokes shift (∼200 nm), strong near-IR emission at ∼700 nm, a relatively long excited state lifetime, high photostability, and solubility make this complex considerably promising in real-time tracking and visualization of lysosomes in live cells. More interestingly, the tumor cell-specific two-photon luminescent imaging properties also endow this Ru complex with potential for applications in high-resolution tumor imaging and luminescence-guided tumor resection.

Effect of functional electrical stimulation plus body weight-supported treadmill training for gait rehabilitation in patients with poststroke: a retrospective case-matched study.

BACKGROUND: Functional electrical stimulation (FES) plus body weight-supported treadmill training (BWSTT) provide effective gait training for poststroke patients with abnormal gait. These features promote a successful active motor relearning of ambulation in stroke survivors. AIM: This is a retrospective study to assess the effect of FES plus BWSTT for gait rehabilitation in patients poststroke. DESIGN: A retrospective case-matched study. SETTING: Participants were recruited from a rehabilitation department in an acute university-affiliated hospital POPULATION: Ninety patients poststroke from Yue Bei People's Hospital underwent BWSTT (A: control group) were compared to an equal number of cross-matched patients who received FES plus BWSTT (B: FES plus BWSTT group). METHODS: While B group received FES for 45 minutes plus BSWTT for 30 minutes in the program, group A received time-matched BWSTT alone. The walking speed, step length, step cadence, Fugl-Meyer Lower-Limb Scale (LL-FMA), composite spasticity scale (CSS), 10-Meter Walk Test (10MWT), Tinetti Balance Test (TBT) and nerve physiology testing were collected before and after intervention. RESULTS: One hundred and eighty patients with poststroke abnormal gait were chosen. There were significant differences in walking speed, step length, step cadence, LL-FMA, CSS, TBT, and 10MWT between baseline and postintervention (P<0.05). There were significant differences in walking speed, step length, step cadence, LL-FMA, CSS, TBT, and 10MWT between two groups at the end of the eighth week (P<0.05), but not at baseline (P>0.05). In comparison with group A, the peak of somatosensory evoked potential (SEP) and motor evoked potential (MEP) amplitude increased, the latency was shortened, and the conduction velocity of sensory nerve (SCV) and motor nerve (MCV) was significantly increased in the group B (P<0.05). No adverse events occurred during the study. CONCLUSIONS: This study suggests that FES plus BWSTT could be more effective than BWSTT alone in the improvement of gait, balance, spasticity, and function of the lower limb in patients poststroke. CLINICAL REHABILITATION IMPACT: Introduce effective rehabilitation strategies for poststroke patients with abnormal gait.

Effect of Intermittent Hypoxia Training for Dizziness: A Randomized Controlled Trial.

OBJECTIVE: To study the effect of intermittent hypoxia training (IHT) for dizziness. DESIGN: A single-blind, randomized controlled trial. All participants were recruited from a rehabilitation department in an acute university-affiliated hospital. INTERVENTION: Participants with dizziness were randomly assigned to 2 groups (IHT group and control group). The Dizziness Handicap Inventory, Activities-specific Balance Confidence Scale, and Vertigo Visual Analog Scale were conducted at baseline, end of the fourth week. RESULTS: Among 52 subjects, there were18 males and 34 females, ages 35 to 62 years old (mean [SD] = 46.9 [7.93]). Time length since onset ranged from 12 to 34 months (20.2 [7.15] mo). Dizziness Handicap Inventory, Activities-specific Balance Confidence Scale, Vertigo Visual Analog Scale scores, and attack frequencies of dizziness were improved after IHT intervention in the end of the fourth week. There were significant differences between the IHT group and the control group in the Dizziness Handicap Inventory, Activities-specific Balance Confidence Scale, Vertigo Visual Analog Scale scores, and attack frequencies of dizziness at the end of the fourth week (P < .05). No adverse events occurred during the study. CONCLUSION: IHT could improve dizziness after intervention at the end of the fourth week. IHT could be the effective method for treating dizziness.

Evaluation of click chemistry microarrays for immunosensing of alpha-fetoprotein (AFP).

The level of cancer biomarkers in cells, tissues or body fluids can be used for the prediction of the presence of cancer or can even indicate the stage of the disease. Alpha-fetoprotein (AFP) is the most commonly used biomarker for early screening and diagnosis of hepatocellular carcinoma (HCC). Here, a combination of three techniques (click chemistry, the biotin-streptavidin-biotin sandwich strategy and the use of antigen-antibody interactions) were combined to implement a sensitive fluorescent immunosensor for AFP detection. Three types of functionalized glasses (dibenzocyclooctyne- (DBCO-), thiol- and epoxy-terminated surfaces) were biotinylated by employing the respective adequate click chemistry counterparts (biotin-thiol or biotin-azide for the first class, biotin-maleimide or biotin-DBCO for the second class and biotin-amine or biotin-thiol for the third class). The anti-AFP antibody was immobilized on the surfaces via a biotin-streptavidin-biotin sandwich technique. To evaluate the sensing performance of the differently prepared surfaces, fluorescently labeled AFP was spotted onto them via microchannel cantilever spotting (µCS). Based on the fluorescence measurements, the optimal microarray design was found and its sensitivity was determined.

A novel recombinant peptide INSR-IgG4Fc (Yiminsu) restores insulin sensitivity in experimental insulin resistance models.

Type 2 diabetes mellitus (T2DM) is a chronic degenerative endocrine and metabolic disease with high mortality and morbidity, yet lacks effective therapeutics. We recently generated a novel fusion peptide INSR-IgG4Fc, Yiminsu (YMS), to facilitate the high-affinity binding and transportation of insulin. Thus, the aim of the present study was to determine whether the novel recombinant peptide, YMS, could contribute to restoring insulin sensitivity and glycaemic control in insulin resistance models and revealing its underlying mechanism. Palmitic acid (PA)-treated LO2 cells and high fat diet (HFD)-fed mice were treated with YMS. Therapeutic effects of YMS were measured using Western blotting, ELISA, qPCR, Histology and transmission electron microscopy. We observed that YMS treatment effectively improved insulin signaling in PA-treated LO2 cells and HFD-fed mice. Notably, YMS could significantly reduce serum levels of glucose, triglycerides, fatty acids and cholesterol without affecting the serum insulin levels. Moreover, our data demonstrated that YMS could restore glucose and lipid homeostasis via facilitating insulin transportation and reactivating PI3K/Akt signaling in both PA-treated cells and liver, gastrocnemius and brown fat of HFD-fed mice. Additionally, we noticed that the therapeutic effects of YMS was similar as rosiglitazone, a well-recognized insulin sensitizer. Our findings suggested that YMS is a potentially candidate for pharmacotherapy for metabolic disorders associated with insulin resistance, particularly in T2DM.

Effect of therapeutic exercise on knee osteoarthritis after intra-articular injection of botulinum toxin type A, hyaluronate or saline: A randomized controlled trial.

OBJECTIVE: To study the effect of therapeutic exercise on knee osteoarthritis after injection of botulinum toxin type A, hyaluronate or saline. METHODS: Sixty participants with knee osteoarthritis were randomly assigned to 3 groups: injection of saline (control) (group A), botulinum toxin type A (group B), or hyaluronate (group C). All participants received therapeutic exercise. Western Ontario and McMaster Universities Osteoarthritis Index questionnaire score, visual analogue scale pain scale, and Medical Outcomes Study 36-item Health Survey were conducted at baseline, and at the end of the 4th and 8th weeks. RESULTS: At end of the 4th and 8th weeks, the scores on the Western Ontario and McMaster Universities Osteoarthritis Index questionnaire and visual analogue scale were higher in the control group. There were significant differences in Physical Component Summary-36 and Mental Component Summary-36 scores between the 3 groups (p <0.05) at the end of the 4th and 8th weeks, but not between groups A (control) and C (hyaluronate) at the end of the 4th week. No changes were seen on magnetic resonance imaging and X-ray images of the affected knees after the intervention. CONCLUSION: Therapeutic exercise plus botulinum toxin type A or hyaluronate injection can significantly reduce pain and improve knee functioning. Botulinum toxin type A plus therapeutic exercise appears to be more effective.

Chitosan-assisted differentiation of porcine adipose tissue-derived stem cells into glucose-responsive insulin-secreting clusters.

The unique advantage of easy access and abundance make the adipose-derived stem cells (ADSCs) a promising system of multipotent cells for transplantation and regenerative medicine. Among the available sources, porcine ADSCs (pADSCs) deserve especial attention due to the close resemblance of human and porcine physiology, as well as for the upcoming availability of humanized porcine models. Here, we report on the isolation and conversion of pADSCs into glucose-responsive insulin-secreting cells. We used the stromal-vascular fraction of the dorsal subcutaneous adipose from 9-day-old male piglets to isolate pADSCs, and subjected the cells to an induction scheme for differentiation on chitosan-coated plates. This one-step procedure promoted differentiation of pADSCs into pancreatic islet-like clusters (PILC) that are characterized by the expression of a repertoire of pancreatic proteins, including pancreatic and duodenal homeobox (Pdx-1), insulin gene enhancer protein (ISL-1) and insulin. Upon glucose challenge, these PILC secreted high amounts of insulin in a dose-dependent manner. Our data also suggest that chitosan plays roles not only to enhance the differentiation potential of pADSCs, but also to increase the glucose responsiveness of PILCs. Our novel approach is, therefore, of great potential for transplantation-based amelioration of type 1 diabetes.

Controlling multi-function of biomaterials interfaces based on multiple and competing adsorption of functional proteins.

Multifunctional biomaterial surfaces can be created by controlling the competing adsorption of multiple proteins. To demonstrate this concept, bone morphogenetic protein 2 (BMP-2) and fibronectin were adsorbed to the hydrophobic surface of polychloro-para-xylylene. The resulting adsorption properties on the surface depended on the dimensional and steric characteristics of the selected protein molecule, the degree of denaturation of the adsorbed proteins, the associated adsorption of interphase water molecules within the protein layers, and the aggregation of proteins in a planar direction with respect to the adsorbent surface. Additionally, a defined surface composition was formed by the competing adsorption of multiple proteins, and this surface composition was directly linked to the composition of the protein mixture in the solution phase. Although the mechanism of this complex competing adsorption process is not fully understood, the adsorbed proteins were irreversibly adsorbed and were unaffected by the further adsorption of homologous or heterologous proteins. Moreover, synergistic biological activities, including cell osteogenesis and proliferation independently and specifically induced by BMP-2 or fibronectin, were observed on the modified surface, and these biological activities were positively correlated with the surface composition of the multiple adsorbed proteins. These results provide insights and important design parameters for prospective biomaterials and biointerfaces for (multi)functional modifications. The ability to control protein/interface properties will be beneficial for the processing of biomaterials for clinical applications and industrial products.

Advances in biodegradable nanomaterials for photothermal therapy of cancer.

Photothermal cancer therapy is an alternative to chemotherapy, radiotherapy, and surgery. With the development of nanophotothermal agents, this therapy holds immense potential in clinical translation. However, the toxicity issues derived from the fact that nanomaterials are trapped and retained in the reticuloendothelial systems limit their biomedical application. Developing biodegradable photothermal agents is the most practical route to address these concerns. In addition to the physicochemical properties of nanomaterials, various internal and external stimuli play key roles on nanomaterials uptake, transport, and clearance. In this review, we summarized novel nanoplatforms for photothermal therapy; these nanoplatforms can elicit stimuli-triggered degradation. We focused on the recent innovative designs endowed with biodegradable photothermal agents under different stimuli, including enzyme, pH, and near-infrared (NIR) laser.

Enhanced bone morphogenic property of parylene-C.

The ability to induce osteointegration was introduced to a parylene-C surface via the simple and intuitive process of protein adsorption mediated by hydrophobic interactions. In this way, bone morphogenetic protein (BMP)-2, fibronectin, and platelet-rich plasma (PRP) could be immobilized on parylene-C surfaces. This approach alleviates concerns related to the use of potentially harmful substances in parylene-C modification processes. The adsorbed protein molecules were quantitatively characterized with respect to adsorption efficacy and binding affinity, and the important biological activities of the proteins were also examined using both early and late markers of osteogenetic activity, including alkaline phosphatase expression, calcium mineralization and marker gene expression. Additionally, the adsorbed PRP exhibited potential as a substitute for expensive recombinant growth factors by effectively inducing comparable osteogenetic activity. In addition to the excellent biocompatibility of parylene-C and its ability to coat a wide variety of substrate materials, the modification of parylene-C via protein adsorption provides unlimited possibilities for installing specific biological functions, expanding the potential applications of this material to include various biointerface platforms.

3D analysis is superior to 2D analysis for contrast-enhanced ultrasound in revealing vascularity in focal liver lesions - A retrospective analysis of 83 cases.

OBJECTIVE: To examine whether dynamic 3D contrast-enhanced ultrasound (CEUS) is superior to 2D imaging in revealing vascularization of focal liver lesions (FLLs). METHODS: Dynamic 2D and 3D CEUS were used to assess the vascularity of FLLs in 83 patients. The two analyses were carried out sequentially following a bolus intravenous injection of SonoVue contrast agent, one injection of 1.5-2mL was given for each part of the study (one for the 2D and another for the 3D). Due to the large data volume for 3D analysis, only the arterial phase (20-25s) was recorded and analyzed. The data were analyzed by two independent analysts. RESULTS: The current study included a total of 83 patients with FLL. The diagnosis was established based on histopathology, Computed Tomography (CT), and magnetic resonance imaging (MRI). Among the 83 cases, 61 were hepatocellular carcinomas (include 10 metastasis, 51 HCCs), and the remaining 22 were benign lesions: hemangiomas (n=15), focal nodular hyperplasias (n=4), and inflammatory lesions (n=3). The overall analysis showed similar patterns of enhancement between the 2D and 3D CEUS in the following features: morphological characteristics, and spatial relationships. In comparison to 2D imaging, 3D CEUS reveal more details of the boundary and feeding arteries of the lesions, as well as distorted features of supply vessels of hepatocellular carcinomas. CONCLUSIONS: Dynamic 3D-CEUS is superior to 2D-CEUS in displaying the spatial relationship of FLLs and their vascularity patterns, and simultaneous imaging of FLL perfusion and anatomic features.

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues.

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.