A hybrid construct of decellularized matrix and fibrin for differentiating adipose stem cells into insulin-producing cells, an optimized in vitro assessment.

The generation of insulin-producing cells (IPCs) is an attractive approach for replacing damaged ß cells in diabetic patients. In the present work, we introduced a hybrid platform of decellularized amniotic membrane (dAM) and fibrin encapsulation for differentiating adipose tissue-derived stem cells (ASCs) into IPCs. ASCs were isolated from healthy donors and characterized. Human AM was decellularized, and its morphology, DNA, collagen, glycosaminoglycan (GAG) contents, and biocompatibility were evaluated. ASCs were subjected to four IPC differentiation methods, and the most efficient method was selected for the experiment. ASCs were seeded onto dAM, alone or encapsulated in fibrin gel with various thrombin concentrations, and differentiated into IPCs according to a method applying serum-free media containing 2-mercaptoethanol, nicotinamide, and exendin-4. PDX-1, GLUT-2 and insulin expression were evaluated in differentiated cells using real-time PCR. Structural integrity and collagen and GAG contents of AM were preserved after decellularization, while DNA content was minimized. Cultivating ASCs on dAM augmented their attachment, proliferation, and viability and enhanced the expression of PDX-1, GLUT-2, and insulin in differentiated cells. Encapsulating ASCs in fibrin gel containing 2 mg/ml fibrinogen and 10 units/ml thrombin increased their differentiation into IPCs. dAM and fibrin gel synergistically enhanced the differentiation of ASCs into IPCs, which could be considered an appropriate strategy for replacing damaged ß cells.

Rutin-Zn(II) complex promotes bone formation - A concise assessment in human dental pulp stem cells and zebrafish.

We have assessed the molecular role of Rutin and rutin-Zn(II) complex on osteoblast differentiation and mineralization in human dental pulp cells and zebrafish model. The biocompatibility of the rutin-Zn(II) complex was determined using MTT and chick embryotoxicity assays. Alizarin red staining and ALP measurements were performed to study the osteogenic role of Rutin and rutin-Zn(II) complex at the cellular level in hDPSCs. At molecular level, following rutin and rutin-Zn(II) exposure, the mRNA expression profile of osteoblast markers such Runx2, type 1 col, OC, and ON were investigated. In addition to this, the expression of negative regulators of osteoblast development such Smad7, Smurf1, and HDAC7 waere studied by Real time RT-PCR analysis. The osteogenic role of prepared complex under in vivo was studied by an in-house zebrafish scale model followed by osteoblast differentiation markers expression profiling and Ca:P level measurement by ICP-MS. Rutin and the rutin-Zn(II) complex were found to be non-toxic till 10 µM and increased the expression of osteoblast differentiation marker genes. It also enhanced calcium deposition in both in vitro and in vivo models. Osteogenic property of rutin-Zn(II) in hDPSCs was found be mediated by Smad7, Smurf1, and HDAC7 and enhancing Runx2 expression. Our study warrants the possible use of rutin-Zn(II) as naïve agent or in combination with other bone scaffolding systems/materials for bone tissue engineering applications.

Assessment of electromechanically stimulated bone marrow stem cells seeded acellular cardiac patch in a rat myocardial infarct model.

In this study, we evaluated cardiomyogenic differentiation of electromechanically stimulated rat bone marrow-derived stem cells (rt-BMSCs) on an acellular bovine pericardium (aBP) and we looked at the functioning of this engineered patch in a rat myocardial infarct (MI) model. aBP was prepared using a detergent-based decellularization procedure followed by rt-BMSCs seeding, and electrical, mechanical, or electromechanical stimulations (3 millisecond pulses of 5 V cm-1at 1 Hz, 5% stretching) to enhance cardiomyogenic differentiation. Furthermore, the electromechanically stimulated patch was applied to the MI region over 3 weeks. After this period, the retrieved patch and infarct region were evaluated for the presence of calcification, inflammatory reaction (CD68), patch to host tissue cell migration, and structural sarcomere protein expressions. In conjunction with any sign of calcification, a higher number of BrdU-labelled cells, and a low level of CD68 positive cells were observed in the infarct region under electromechanically stimulated conditions compared with static conditions. More importantly, MHC, SAC, Troponin T, and N-cad positive cells were observed in both infarct region, and retrieved engineered patch after 3 weeks. In a clear alignment with other results, our developed acellular patch promoted the expression of cardiomyogenic differentiation factors under electromechanical stimulation. Our engineered patch showed a successful integration with the host tissue followed by the cell migration to the infarct region.

Cost saving, patient centered algorithm for progenitor cell mobilization for autologous hematopoietic cell transplantation.

Administration of plerixafor with granulocyte-colony stimulating factor (G-CSF) mobilizes CD34+ cells much more effectively than G-CSF alone, but cost generally limits plerixafor use to patients at high risk of insufficient CD34+ cell collection based on low peripheral blood (PB) CD34+ counts following 4 days of G-CSF. We analyzed costs associated with administering plerixafor to patients with higher day 4 CD34+ cell counts to decrease apheresis days and explored the use of a fixed split dose of plerixafor instead of weight-based dosing. We analyzed 235 patients with plasma cell disorders or non-Hodgkin's lymphoma who underwent progenitor cell mobilization and autologous hematopoietic cell transplantation (AHCT) between March 2014 and December 2017. Two hundred ten (89%) received G-CSF plus Plerixafor and 25 (11%) received G-CSF alone. Overall, 180 patients (77%) collected in 1 day, 53 (22%) in 2 days and 2 (1%) in 3 days. Based on our data, we present a probabilistic algorithm to identify patients likely to require more than one day of collection using G-CSF alone. CD34+ cell yield, ANC and platelet recovery were not significantly different between fixed and standard dose plerixafor. Plerixafor enabled collection in 1 day and with estimated savings of $5000, compared to patients who did not receive plerixafor and required collection for three days. While collection and processing costs and patient populations vary among institutions, our results suggest re-evaluation of current algorithms.

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo.

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

Ex Vivo Manufacture of Megakaryocytes and Platelets from Stem Cells: Recent Advances Toward Transfusion in Humans.

The generation of ex vivo functional megakaryocytes (MK) and platelets is an important issue in transfusion medicine as donor dependence implies in limitations, such as shortage of eligible volunteers. Indeed, platelet transfusion is still a procedure that saves the lives of patients with defective platelet production. Recent technological development has enabled the isolation and expansion of stem cells that can be used as a source for the production of functional platelets for transfusion. In this review, we discuss recent approaches of in vitro or ex vivo production of MK and platelets, suggesting that, in the near future, donor-independent sources may become a possibility. The feasibility of using these cells in the clinic may be safer, and in vitro manipulation could generate universally compatible products, solving problems related to platelet refractoriness. However, functionality and survival testing of these products in human beings are scarce; therefore, additional studies are needed to consolidate this purpose.

Uncovering the cell fate decision in lysis-lysogeny transition and stem cell development via Markov state modeling.

Understanding the behavior of a complex gene regulatory network is a fundamental but challenging task in systems biology. How to reduce the large number of degrees of freedom of a specific network and identify its main biological pathway is the key issue. In this paper, we utilized the transition path theory (TPT) and Markov state modeling (MSM) framework to numerically study two typical cell fate decision processes: the lysis-lysogeny transition and stem cell development. The application of TPT to the lysis-lysogeny decision-making system reveals that the competitions of CI and Cro dimer binding play the major role in determining the cell fates. We also quantified the transition rates from the lysogeny to lysis state under different conditions. The overall computational results are consistent with biological intuitions but with more detailed information. For the stem cell developmental system, we applied the MSM to reduce the original dynamics to a moderate-size Markov chain. Further spectral analysis showed that the reduced system exhibits nine metastable states, which correspond to the refinement of the five known typical cell types in development. We further investigated the dominant transition pathways corresponding to the cell differentiation, reprogramming, and trans-differentiation. A similar approach can be applied to study other biological systems.

Industry updates from the field of stem cell research and regenerative medicine in March 2020.

Latest developments in the field of stem cell research and regenerative medicine compiled from publicly available information and press releases from nonacademic institutions in March 2020.

Industry updates from the field of stem cell research and regenerative medicine in February 2020.

The latest developments in the field of stem-cell research and regenerative medicine compiled from publicly available information and press releases from nonacademic institutions in February 2020.

Peroxisome Elevation Induces Stem Cell Differentiation and Intestinal Epithelial Repair.

Epithelial-repair-dependent mucosal healing (MH) is associated with a more favorable prognosis for patients with inflammatory bowel disease (IBD). MH is accomplished via repair and regeneration of the intestinal epithelium. However, the mechanism underlying MH is ill defined. We found a striking upregulation of peroxisomes in the injured crypts of IBD patients. By increasing peroxisome levels in Drosophila midguts, we found that peroxisome elevation enhanced RAB7-dependent late endosome maturation, which then promoted stem and/or progenitor-cell differentiation via modulation of Janus Kinase (JAK) and Signal Transducer and Activator of Transcription (STAT)-SOX21A signaling. This in turn enhanced ISC-mediated regeneration. Importantly, RAB7 and SOX21 were upregulated in the crypts of IBD patients. Moreover, administration of drugs that increased peroxisome levels reversed the symptoms of dextran sulfate sodium (DSS)-induced colitis in mice. This study demonstrates a peroxisome-mediated epithelial repair mechanism, which opens a therapeutic avenue for the enhancement of MH in IBD patients.

Australian regulation of autologous human cell and tissue products: implications for commercial stem cell clinics.

In 2018, Australia's Therapeutic Goods Administration introduced regulatory reforms that set stricter criteria around the regulation of products derived from a patient's own cells and tissues, posing significant implications for clinics offering stem cell treatments. We review the regulatory framework and discuss its potential commercial implications, including the ambiguities that may arise from it in practice, as well as the likely impact it will have on product development and advertising practices in the future.

The direct-to-consumer market for stem cell-based interventions in Australia: exploring the experiences of patients.

The prevalence of businesses selling autologous stem cell-based interventions to patients in Australia has raised serious concerns about how weaknesses in regulation have enabled the emergence of an industry that engages in aggressive marketing of unproven treatments to patients. Little is known about how patients experience this marketing and their subsequent interactions with practitioners. This paper reports results from 15 semistructured interviews with patients and carers, and also draws upon discussion conducted with patients, carers and family members (22 participants) in a workshop setting. We explore how Australian patients and carers understand and experience these interventions, and how their presumptions about the ethics of medical practice, and the regulatory environment in Australia have conditioned their preparedness to undergo unproven treatments.

Development and Validation of an Open-Source Grading Tool for Outcome Assessment in Limbal Stem Cell Treatment.

PURPOSE: To design a grading system and validate an open-source tool to improve objective quantification and follow-up of limbal stem cell deficiency (LSCD) after treatment. METHODS: A custom-made web-based grading system was developed for grading stem cell deficient eyes, termed the "Vascularisation, Haze, and Integrity" tool. For validation purposes, 60 corneal slit-lamp images of 30 limbal stem cell deficient eyes were graded by 3 groups of examiners: 3 corneal specialists (group A), 3 ophthalmologists with an expertise other than cornea (group B), and 3 nonclinicians (group C). The intragrader and intergrader agreement was evaluated using Fleiss weighted kappa coefficients and concurrent assessment of interrater and intrarater reliability (IRR) coefficients. RESULTS: The overall intergrader agreement was 0.78, 0.61, and 0.42 for superficial corneal vascularization, corneal haze, and epithelial integrity, respectively. All groups had good agreement for the vascularization parameter with the highest intergrader reliability in group A (IRR = 0.80) and the lowest in group C (IRR = 0.72). When assessing "haze," there was good agreement in groups A (IRR = 0.75) and B (IRR = 0.76) but low agreement in group C (IRR = 0.37). CONCLUSIONS: We report the development and evaluation of a novel method for grading results of limbal stem cell deficient eyes after treatment and provide this system as a free, open-source online tool. The grading tool offers an easy and standardized way of assessing the corneal surface in patients with LSCD, enables evaluation of progression over time, reduces assessment bias, and-if adopted universally-will harmonize outcome being reported between groups.

Assessment of Post-thaw Quality of Dental Mesenchymal Stromal Cells After Long-Term Cryopreservation by Uncontrolled Freezing.

Cryopreservation abilities of dental tissue-derived mesenchymal stromal cells (DMSCs) including dental pulp stem cells (DPSCs) and dental follicle stem cells (DFSC) play an important role in the applications of these cells in clinical settings. In this context, we checked whether storage at - 80 °C in 10% DMSO for a longer period has any adverse effect on the functionality and genetic stability. We carried our studies on DPSC and DFSC samples that were revived after a maximum of 5 years of cryopreservation. We observed that even after long-term uncontrolled freezing at - 80 °C, these cells survived and proliferated efficiently. The assessment was made based on their post-thaw morphology, immunophenotypes, differentiation potential, growth kinetics, and genetic features. These cells retained the expression of stemness markers, differentiation ability and maintained their normal karyotype. Our results indicated no significant morphological or immunophenotypic differences between the cryopreserved DMSCs and the fresh DMSCs. Our study implies that mesenchymal stromal cells derived from the dental tissue origin are very robust and do not require any sophisticated preservation protocols. Thus, these can be an ideal source for research, stem cell banking, as well as successful clinical applications in tissue engineering and cell-based therapeutics. Graphical Abstract Schematic diagram showing the cryopreservation of DMSCs by uncontrolled freezing at -80 c has no adverse effects on their functionality and genetic stability.

Innovative tailor made dextran based membranes with excellent non-inflammatory response: In vivo assessment.

In this work, dextran based membranes with potential to be used as implantable devices in Tissue Engineering and Regenerative Medicine (TERM) were prepared by a straightforward strategy. Briefly, two polymers approved by the Food and Drug Administration, viz. dextran and poly(ε-caprolactone) (PCL) were functionalized with methacrylate moieties, and subjected to photocrosslinking. Employing different weight ratios of each polymer in the formulations allowed to obtain transparent membranes with tunable physicochemical properties and low adverse host tissue response. Independently of the material, all formulations have shown to be thermally stable up to 300 °C whilst variations in the polymer ratio resulted in membranes with different glass transition temperatures (Tg) and flexibility. The swelling capacity ranged from 50% to 200%. On the other hand, in vitro hydrolytic degradation did not show to be material-dependent and all membranes maintained their structural integrity for more than 30 days, losing only 8-12% of their initial weight. Preliminary in vitro biological tests did not show any cytotoxic effect on seeded human dental pulp stem cells (hDPSCs), suggesting that, in general, all membranes are capable of supporting cell adhesion and viability. The in vivo biocompatibility of membranes implanted subcutaneously in rats' dorsum indicate that M100/0 (100%wt dextran) and M25/75 (25 %wt dextran) formulations can be classified as "slight-irritant" and "non-irritant", respectively. From the histological analysis performed on the main tissue organs it was not possible to detect any signs of fibrosis or necrosis thereby excluding the presence of toxic degradation by-products deposited or accumulated in these tissues. In combination, these results suggest that the newly developed formulations hold great potential as engineered devices for biomedical applications, where the biological response of cells and tissues are greatly dependent on the physical and chemical cues provided by the substrate.

Characterizing Direct-to-Consumer Stem Cell Businesses in the Southwest United States.

There are currently hundreds of businesses across the United States offering direct-to-consumer stem cell treatments that have not been through regulatory approval by the Food and Drug Administration (FDA). Here, we provide a detailed characterization of nearly 170 stem cell businesses operating in the Southwest United States. We draw specific attention to two as-yet understudied facets of these businesses. First, we identify differences in the degree to which a given business focuses their practice on stem cell treatments. Second, we compare the stated expertise of the care providers in stem cell businesses with the range of conditions they purport to treat. These findings deepen our knowledge of the growing industry around unapproved stem cell treatments, and are used here to offer suggestions for how the FDA might target its resources with respect to regulatory oversight.

MoNa - A Cost-Efficient, Portable System for the Nanoinjection of Living Cells.

Injection techniques to deliver macromolecules to cells such as microinjection have been around for decades with applications ranging from probing whole organisms to the injection of fluorescent molecules into single cells. A similar technique that has raised recent interest is nanoinjection. The pipettes used here are much smaller and allow for the precise deposition of molecules into single cells via electrokinetics with minimal influence on the cells' health. Unfortunately, the equipment utilized for nanoinjection originates from scanning ion conductance microscopy (SICM) and is therefore expensive and not portable, but usually fixed to a specific microscope setup. The level of precision that these systems achieve is much higher than what is needed for the more robust nanoinjection process. We present Mobile Nanoinjection (MoNa), a portable, cost-efficient and easy to build system for the injection of single cells. Sacrificing unnecessary sub-nanometer accuracy and low ion current noise levels, we were able to inject single living cells with high accuracy. We determined the noise of the MoNa system and investigated the injection conditions for 16 prominent fluorescent labels and fluorophores. Further, we performed proof of concepts by injection of ATTO655-Phalloidin and MitoTracker Deep Red to living human osteosarcoma (U2OS) cells and of living adult human inferior turbinate stem cells (ITSC's) following neuronal differentiation with the MoNa system. We achieved significant cost reductions of the nanoinjection technology and gained full portability and compatibility to most optical microscopes.

Assessment of corneal substrate biomechanics and its effect on epithelial stem cell maintenance and differentiation.

Whilst demonstrated extensively in vitro, the control of cell behaviour via modulation of substrate compliance in live tissues has not been accomplished to date. Here we propose that stem cells can be regulated solely through in situ modulation of tissue biomechanics. By first establishing, via high-resolution Brillouin spectro-microscopy, that the outer edge (limbus) of live human corneas has a substantially lower bulk modulus compared to their centre, we then demonstrate that this difference is associated with limbal epithelial stem cell (LESC) residence and YAP-dependent mechanotransduction. This phenotype-through-biomechanics correlation is further explored in vivo using a rabbit alkali burn model. Specifically, we show that treating the burnt surface of the cornea with collagenase effectively restores the tissue's mechanical properties and its capacity to support LESCs through mechanisms involving YAP suppression. Overall, these findings have extended implications for understanding stem cell niche biomechanics and its impact on tissue regeneration.

Assessment of Gene Function of Mouse Innate Lymphoid Cells for In Vivo Analysis Using Retroviral Transduction.

Retroviral transduction is commonly used to modulate gene expression and is a powerful approach to understand the role of a gene using gain- or loss-of-function strategies. Retroviral vectors can stably integrate non-viral genes into host genomes, providing long-term modulation of gene expression in infected cells and their progeny. Here we describe the generation of retroviral supernatants and the steps to efficiently transduce genes in innate lymphoid cell (ILC) progenitors for subsequent analysis of ILC populations in vivo.

Assessment of PCL/carbon material scaffolds for bone regeneration.

Biomanufacturing is a relatively new research domain focusing on the use of additive manufacturing technologies, biomaterials, cells and biomolecular signals to produce tissue constructs for tissue engineering. For bone regeneration, researchers are focusing on the use of polymeric and polymer/ceramic scaffolds seeded with osteoblasts or mesenchymal stem cells. However, the design of high-performance scaffolds in terms of mechanical, cell-stimulation and biological performance is still required. This is the first paper investigating the use of an extrusion additive manufacturing system to produce poly(ε-caprolactone) (PCL), PCL/graphene nanosheet (GNS) and PCL/carbon nanotube (CNT) scaffolds for bone applications. Scaffolds with regular and reproducible architecture were produced and evaluated from chemical, physical and biological points of view. Results suggest that the addition of both graphene and CNT allow the fabrication of scaffolds with improved properties. It also shows that scaffolds containing graphene present better mechanical properties and high cell-affinity improving cell attachment, proliferation and differentiation.