Science shines in a new virtual SY-Stem.

The third 'Symposium for the Next Generation of Stem Cell Research' (SY-Stem) was held virtually on 3-5 March 2021, having been cancelled in 2020 due to the COVID-19 pandemic. As in previous years, the meeting highlighted the work of early career researchers, ranging from postgraduate students to young group leaders working in developmental and stem cell biology. Here, we summarize the excellent work presented at the Symposium, which covered topics ranging from pluripotency, species-specific aspects of development and emerging technologies, through to organoids, single-cell technology and clinical applications.

Assessment of Mitochondrial Reactive Oxygen Species and Redox Regulation in Stem Cells.

Mitochondrial reactive oxygen species (mtROS) and redox regulation play an important role in stem cell maintenance and cell fate decisions. Although changes in mtROS and redox homeostasis represent a physiological mechanism to drive stem cell commitment and differentiation, dysregulation of this system can lead to defects in stem cell maintenance and regenerative capacity. This chapter explains the methods used to assess mitochondrial superoxide levels and redox regulation in stem cell populations.

Sequential Scalp Assessment in Hair Regeneration Therapy Using an Adipose-Derived Stem Cell-Conditioned Medium.

BACKGROUND: An adipose-derived stem cell-conditioned medium (ADSC-CM) reportedly exerts skin-rejuvenating and hair growth-promoting effects. In the therapeutic application of ADSC-CM for alopecia, changes to the interfollicular scalp remain unclear although some evidence has indicated hair growth-promoting effects. OBJECTIVE: To evaluate the effects of ADSC-CM not only on hair follicles, but also on the interfollicular scalp. METHODS: Forty patients (21 men, 19 women; age range, 23-74 years) with alopecia were treated by intradermal injection of ADSC-CM every month for 6 months. Eighty fixed sites on patients were investigated by trichograms, physiological examinations, and ultrasonographic examinations at 4 time points (before treatment and 2, 4, and 6 months after the initial treatment). RESULTS: Hair density and anagen hair rate increased significantly. As physiological parameters, transepidermal water loss value gradually increased, with significant differences at 4 and 6 months after the initial treatment, but hydration state of the stratum corneum and skin surface lipid level showed no obvious changes. As ultrasonographic parameters, dermal thickness and dermal echogenicity were increased significantly. CONCLUSION: Intradermal administration of ADSC-CM on the scalp has strong potential to provide regenerative effects for hair follicles and the interfollicular scalp. An adipose-derived stem cell-conditioned medium offers a promising prospect as an alternative treatment for alopecia.

Knockdown of the thyroid hormone transporter MCT8 in chicken retinal precursor cells hampers early retinal development and results in a shift towards more UV/blue cones at the expense of green/red cones.

Thyroid hormones (THs) play a crucial role in coordinating brain development in vertebrates. They fine-tune processes like cell proliferation, migration, and differentiation mainly by regulating the transcriptional activity of many essential genes. Regulators of TH availability thereby define the cellular concentration of the bioactive 3,5,3'-triiodothyronine, which binds to nuclear TH receptors. One important regulator, the monocarboxylate transporter 8 (MCT8), facilitates cellular TH uptake and is known to be necessary for correct brain development, but data on its potential role during retinal development is lacking. The retinal cyto-architecture has been conserved throughout vertebrate evolution, and we used the chicken embryo to study the need for MCT8 during retinal development. Its external development allows easy manipulation, and MCT8 is abundantly expressed in the retina from early stages onwards. We induced MCT8 knockdown by electroporating a pRFP-MCT8-RNAi vector into the retinal precursor cells (RPCs) at embryonic day 4 (E4), and studied the consequences for early (E6) and late (E18) retinal development. The empty pRFP-RNAi vector was used as a control. RPC proliferation was reduced at E6. This resulted in cellular hypoplasia and a thinner retina at E18 where mainly photoreceptors and horizontal cells were lost, the two predominant cell types that are born around the stage of electroporation. At E6, differentiation into retinal ganglion cells and amacrine cells was delayed. However, since the proportion of a given cell type within the transfected cell population at E18 was similar in knockdown and controls, the partial loss of some cell types was most-likely due to reduced RPC proliferation and not impaired cell differentiation. Photoreceptors displayed delayed migration at first, but had successfully reached the outer nuclear layer at E18. However, they increasingly differentiated into short wavelength-sensitive cones at the expense of medium/long wavelength-sensitive cones, while the proportion of rods was unaltered. Improperly formed sublaminae in the inner plexiform layer additionally suggested defects in synaptogenesis. Altogether, our data echoes effects of hypothyroidism and the loss of some other regulators of TH availability in the developing zebrafish and rodent retina. Therefore, the expression of MCT8 in RPCs is crucial for adequate TH uptake during cell type-specific events in retinal development.

Role of Stem Cells in the Pathogenesis of Chronic Obstructive Pulmonary Disease and Pulmonary Emphysema.

There are only few human translational studies performed in the area of stem cell research in patients with chronic obstructive pulmonary disease (COPD) and/or pulmonary emphysema. Before progress to clinical trials with stem cells we strongly believe that more human translational studies are essential, otherwise, the clinical rationale would be solely based on limited in vitro and animal studies. In the future, stem cell therapy could be a treatment for this incurable disease. As of now, stem cell therapy is still to be considered as an area of active research, lacking any strong rationale for performing clinical trials in COPD. Although stem cells would be likely to represent a heterogeneous population of cells, the different cell subsets and their importance in the pathogenesis of the different clinical phenotypes need to be fully characterised before progressing to clinical trials. Moreover, the potential side effects of stem cell therapy are underestimated. We should not ignore that some of the most deadly neoplasms are arising from stem cells.

WISP-1 drives bone formation at the expense of fat formation in human perivascular stem cells.

The vascular wall within adipose tissue is a source of mesenchymal progenitors, referred to as perivascular stem/stromal cells (PSC). PSC are isolated via fluorescence activated cell sorting (FACS), and defined as a bipartite population of pericytes and adventitial progenitor cells (APCs). Those factors that promote the differentiation of PSC into bone or fat cell types are not well understood. Here, we observed high expression of WISP-1 among human PSC in vivo, after purification, and upon transplantation in a bone defect. Next, modulation of WISP-1 expression was performed, using WISP-1 overexpression, WISP-1 protein, or WISP-1 siRNA. Results demonstrated that WISP-1 is expressed in the perivascular niche, and high expression is maintained after purification of PSC, and upon transplantation in a bone microenvironment. In vitro studies demonstrate that WISP-1 has pro-osteogenic/anti-adipocytic effects in human PSC, and that regulation of BMP signaling activity may underlie these effects. In summary, our results demonstrate the importance of the matricellular protein WISP-1 in regulation of the differentiation of human stem cell types within the perivascular niche. WISP-1 signaling upregulation may be of future benefit in cell therapy mediated bone tissue engineering, for the healing of bone defects or other orthopaedic applications.

Isolation and functional assessment of mouse skeletal stem cell lineage.

There are limited methods available to study skeletal stem, progenitor, and progeny cell activity in normal and diseased contexts. Most protocols for skeletal stem cell isolation are based on the extent to which cells adhere to plastic or whether they express a limited repertoire of surface markers. Here, we describe a flow cytometry-based approach that does not require in vitro selection and that uses eight surface markers to distinguish and isolate mouse skeletal stem cells (mSSCs); bone, cartilage, and stromal progenitors (mBCSPs); and five downstream differentiated subtypes, including chondroprogenitors, two types of osteoprogenitors, and two types of hematopoiesis-supportive stroma. We provide instructions for the optimal mechanical and chemical digestion of bone and bone marrow, as well as the subsequent flow-cytometry-activated cell sorting (FACS) gating schemes required to maximally yield viable skeletal-lineage cells. We also describe a methodology for renal subcapsular transplantation and in vitro colony-formation assays on the isolated mSSCs. The isolation of mSSCs can be completed in 9 h, with at least 1 h more required for transplantation. Experience with flow cytometry and mouse surgical procedures is recommended before attempting the protocol. Our system has wide applications and has already been used to study skeletal response to fracture, diabetes, and osteoarthritis, as well as hematopoietic stem cell-niche interactions in the bone marrow.

Cerebral organoids: ethical issues and consciousness assessment.

Organoids are three-dimensional biological structures grown in vitro from different kinds of stem cells that self-organise mimicking real organs with organ-specific cell types. Recently, researchers have managed to produce human organoids which have structural and functional properties very similar to those of different organs, such as the retina, the intestines, the kidneys, the pancreas, the liver and the inner ear. Organoids are considered a great resource for biomedical research, as they allow for a detailed study of the development and pathologies of human cells; they also make it possible to test new molecules on human tissue. Furthermore, organoids have helped research take a step forward in the field of personalised medicine and transplants. However, some ethical issues have arisen concerning the origin of the cells that are used to produce organoids (ie, human embryos) and their properties. In particular, there are new, relevant and so-far overlooked ethical questions concerning cerebral organoids. Scientists have created so-called mini-brains as developed as a few-months-old fetus, albeit smaller and with many structural and functional differences. However, cerebral organoids exhibit neural connections and electrical activity, raising the question whether they are or (which is more likely) will one day be somewhat sentient. In principle, this can be measured with some techniques that are already available (the Perturbational Complexity Index, a metric that is directly inspired by the main postulate of the Integrated Information Theory of consciousness), which are used for brain-injured non-communicating patients. If brain organoids were to show a glimpse of sensibility, an ethical discussion on their use in clinical research and practice would be necessary.

Assessment of the characteristics and biocompatibility of gelatin sponge scaffolds prepared by various crosslinking methods.

This comparative study aims to identify a biocompatible and effective crosslinker for preparing gelatin sponges. Glutaraldehyde (GTA), genipin (GP), 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC), and microbial transglutaminase (mTG) were used as crosslinking agents. The physical properties of the prepared samples were characterized, and material degradation was studied in vitro with various proteases and in vivo through subcutaneous implantation of the sponges in rats. Adipose-derived stromal stem cells (ADSCs) were cultured and inoculated onto the scaffolds to compare the cellular biocompatibility of the sponges. Cellular seeding efficiency and digestion time of the sponges were also evaluated. Cellular viability and proliferation in scaffolds were analyzed by fluorescence staining and MTT assay. All the samples exhibited high porosity, good swelling ratio, and hydrolysis properties; however, material strength, hydrolysis, and enzymolytic properties varied among the samples. GTA-sponge and GP-sponge possessed high compressive moduli, and EDC-sponge exhibited fast degradation performance. GTA and GP sponge implants exerted strong in vivo rejections, and the former showed poor cell growth. mTG-sponge exhibited the optimal comprehensive performance, with good porosity, compressive modulus, anti-degradation ability, and good biocompatibility. Hence, mTG-sponge can be used as a scaffold material for tissue engineering applications.

A supramolecular look at microenvironmental regulation of limbal epithelial stem cells and the differentiation of their progeny / Uma visão supramolecular sobre a regulação microambiental de células tronco epiteliais limbais e a diferenciação de sua progênie

ABSTRACT Various approaches have been taken to improve our knowledge of the microenvironmental regulation of limbal epithelial stem cells. Researchers have extensively investigated the roles of growth factors, survival factors, cytokines, enzymes, and permeable molecules secreted by the limbal cells. However, recent evidence suggests that stem cell fate (i.e., self-renewal or differentiation) can also be influenced by biophysical and mechanical cues related to the supramolecular organization and the liquid crystalline (mesophase) nature of the stromal extracellular matrix. These cues can be sensed by stem cells and transduced into intracellular biochemical and functional responses, a process known as mechanotransduction. The objective of this review is to offer perspectives on the supramolecular microenvironmental regulation of limbal epithelial stem cells and the differentiation of their progeny.

RESUMO Muitas abordagens têm sido utilizadas para ampliar entendimentos sobre a regulação microambiental das células tronco epiteliais limbais. Neste contexto, pesquisadores têm exaustivamente investigado a participação de fatores de crescimento, fatores de sobrevida, citocinas, enzimas e moléculas permeáveis secretadas pelas células limbais. Entretanto, evidências recentes sugerem que o destino (ie. autorrenovação ou recrutamento para a via de diferenciação) das células tronco também sofre influência de estímulos biofísicos ou mecânicos relacionados à organização supramolecular e à natureza liquido-cristalina (mesofases) da matriz extracelular estromal. Esses estímulos podem ser percebidos e traduzidos pelas células tronco em sinais bioquímicos que geram respostas funcionais, através de um processo designado de mecanotransdução. Objetiva-se, com a presente revisão, oferecer ao leitor perspectivas supramoleculares sobre a regulação microambiental das células tronco epiteliais limbais e a diferenciação de sua progênie.

Assessment of Bone Regeneration Using Adipose-Derived Stem Cells in Critical-Size Alveolar Ridge Defects: An Experimental Study in a Dog Model.

PURPOSE: To assess bone regeneration potential of a fibronectin- and adipose-derived stem cell-covered ceramic biomaterial in three-wall critical-size alveolar ridge defects. MATERIALS AND METHODS: In 18 dogs, four dehiscence-type and critical-size defects were created surgically in the edentulous alveolar ridge. Defects were randomly regenerated using biomaterials coated with particulate ß-tricalcium phosphate (ß-TCP), ß-TCP with fibronectin (Fn) (ß-TCP-Fn), and ß-TCP with a combination of Fn and autologous adipose-derived stem cells (ADSCs) (ß-TCP-Fn-ADSCs), leaving one defect as control. The animals were divided into three groups according to the time of euthanasia (1, 2, or 3 months of healing). RESULTS: At the time of sacrifice, statistically significant differences between the four types of defects in the total area of bone regeneration, percentage of neoformed bone matrix, medullary space, or contact between particulate biomaterial and neoformed bone matrix were not found. All defects showed a significant increase in neoformed bone matrix as sacrifice was delayed, but a uniform pattern was not followed. Only defects treated with ß-TCP-Fn-ADSCs showed a significant increase in the bone regeneration area when animals sacrificed at 3 months were compared to those sacrificed at 1 month (P = .006). CONCLUSION: The use of ADSCs in bone regeneration processes of critical-size defects of the alveolar ridge did not entail an advantage regarding greater bone regeneration as compared with other biomaterials. However, the use of ß-TCP coated with a combination of Fn and ADSCs appeared to favor stabilization of the regenerated area, allowing a more efficient maintenance of the space at 3 months of healing.

Assessment of Pulp Regeneration Induced by Stem Cell Therapy by Magnetic Resonance Imaging.

INTRODUCTION: This study was designed to evaluate the usefulness of magnetic resonance imaging (MRI) to assess the regeneration of pulp tissue. METHODS: Mobilized dental pulp stem cells and granulocyte colony-stimulating factor with collagen were transplanted into mature pulpectomized teeth for pulp regeneration (n = 4). The controls consisted of pulpectomized teeth with or without collagen and normal teeth with intact pulp tissue (n = 4, each). The signal intensity (SI) of MRI using T2 sequences was compared after the extraction of teeth in dogs. MRI was correlated with the corresponding histologic findings. RESULTS: Pulp tissue was fully regenerated 90 days after cell transplantation. On the other hand, the root canal was empty in the control collagen-transplanted teeth at 90 days. The SI of the normal teeth was significantly higher than that of nonvital pulpectomized teeth and the controls of collagen transplanted teeth at 90 days. The stem cell transplanted teeth showed a gradual decrease in the SI until 180 days at which time the SI was similar to that in the normal teeth and significantly higher than that in the teeth transplanted with collagen alone without the stem cells. CONCLUSIONS: The changes in the SI of the pulplike tissue were consistent with the histologic findings, showing the potential usefulness of the noninvasive method to serially access the efficacy of pulp regenerative therapy.

How we make cell therapy in Italy.

In the 21st century scenario, new therapeutic tools are needed to take up the social and medical challenge posed by the more and more frequent degenerative disorders and by the aging of population. The recent category of advanced therapy medicinal products has been created to comprise cellular, gene therapy, and tissue engineered products, as a new class of drugs. Their manufacture requires the same pharmaceutical framework as for conventional drugs and this means that industrial, large-scale manufacturing process has to be adapted to the peculiar characteristics of cell-containing products. Our hospital took up the challenge of this new path in the early 2000s; and herein we describe the approach we followed to set up a pharmaceutical-grade facility in a public hospital context, with the aim to share the solutions we found to make cell therapy compliant with the requirements for the production and the quality control of a high-standard medicinal product.

Stem cell patents after the america invents act.

Under the newly passed Leahy-Smith America Invents Act (AIA), the U.S. Patent and Trademark Office may hear new challenges to stem cell patents. Here, we explore how the new law affects challenges to stem cell patents, focusing on two recent cases, and discuss the future of stem cell patent disputes.

Enhancing hair follicle regeneration by nonablative fractional laser: Assessment of irradiation parameters and tissue response.

BACKGROUND AND OBJECTIVE: Identification of methods to enhance anagen entry can be helpful for alopecia. Recently, nonablative laser has been proposed as a potential treatment for alopecia. However, how the laser parameters affect stem cell activity, hair cycles and the associated side effects have not been well characterized. Here we examine the effects of irradiation parameters of 1,550-nm fractional laser on hair cycles. STUDY DESIGN/MATERIALS AND METHODS: The dorsal skin of eight-week-old female C57BL/6 mice with hair follicles in synchronized telogen was shaved and irradiated with a 1,550-nm fractional erbium-glass laser (Fraxel RE:STORE (SR1500) Laser System, Solta Medical, U.S.A.) with varied beam energies (5-35 mJ) and beam densities (500-3500 microthermal zones/cm(2) ). The cutaneous changes were evaluated both grossly and histologically. Hair follicle stem cell activity was detected by BrdU incorporation and changes in gene expression were quantified by real-time PCR. RESULTS: Direct thermal injury to hair follicles could be observed early after irradiation, especially at higher beam energy. Anagen induction in the irradiated skin showed an all-or-non change. Anagen induction and ulcer formation were affected by the combination of beam energy and density. The lowest beam energy of 5 mJ failed to promote anagen entry at all beam densities tested. As beam energy increased from 10 mJ to 35 mJ, we found a decreasing trend of beam density that could induce anagen entry within 7-9 days with activation of hair follicle stem cells. Beam density above the pro-regeneration density could lead to ulcers and scarring followed by anagen entry in adjacent skin. Analysis of inflammatory cytokines, including TNF-α, IL-1ß, and IL-6, revealed that transient moderate inflammation was associated with anagen induction and intense prolonged inflammation preceded ulcer formation. CONCLUSION: To avoid side effects of hair follicle injury and scarring, appropriate combination of beam energy and density is required. Parameters outside the therapeutic window can result in either no anagen promotion or ulcer formation.