Induced Stem Cell Differentiation Research Articles

A Slotted Decellularized Osteochondral Scaffold With Layer-Specific Release of Stem Cell Differentiation Stimulators Enhances Cartilage and Bone Regeneration in Osteochondral Defects in a Rabbit Model

Background: Owing to the disappointing regenerative ability of osteochondral tissue, without treatment an osteochondral defect would progress to osteoarthritis. This situation motivates the need for new strategies to enhance the regeneration of osteochondral defects. Purpose: To develop a tissue-engineering scaffold by tethering bone morphogenetic protein 2 (BMP2) and transforming growth factor beta 3 (TGFβ3) in a layer-specific manner on a slotted decellularized osteochondral matrix (SDOM) and to evaluate the efficacy of this scaffold for osteochondral regeneration. Study Design: Controlled laboratory study. Methods: Normal osteochondral tissue from the rabbit patellofemoral groove was sectioned into a slot shape and decellularized for fabricating an SDOM. The collagen-binding domain (CBD) was fused into the N-terminus of BMP2 or TGFβ3 to synthesize 2 recombinant growth factors (GFs) (CBD-BMP2 or CBD-TGFβ3), which were tethered to the bone layer and cartilage layer, respectively, of the SDOM to prepare a tissue-engineering scaffold (namely, CBD-GFs/SDOM). After examining the influence of the CBD-GFs/SDOM on the viability and layer-specific differentiation of bone marrow mesenchymal stem cells in vitro, we determined the regeneration potential of the CBD-GFs/SDOM on osteochondral regeneration in a rabbit model. A total of 72 New Zealand White rabbits with a cylindrical osteochondral defect in the patellofemoral groove were randomly assigned to 3 groups: defect only (control [CTL] group), defect patched with an SDOM (SDOM group), and defect patched with the CBD-GFs/SDOM (CBD-GFs/SDOM group). At 6 or 12 weeks postoperatively, the rabbits were euthanized to harvest the knee joint, which was then evaluated via gross observation, micro–computed tomography, histological staining, and mechanical testing. Results: In vitro, the CBD-GFs/SDOM was noncytotoxic, showed high biomimetics with normal osteochondral tissue, was suitable for cell adhesion and growth, and had good layer-specific ability in inducing stem cell differentiation. Macroscopic images showed that the CBD-GFs/SDOM group had significantly better osteochondral regeneration than the CTL and SDOM groups had. Micro–computed tomography demonstrated that much more bony tissue was formed at the defect sites in the CBD-GFs/SDOM group compared with the defect sites in the CTL or SDOM group. Histological analysis showed that the CBD-GFs/SDOM group had a significant enhancement in osteochondral regeneration at 6 and 12 weeks postoperatively in comparison with the CTL or SDOM group. At 12 weeks postoperatively, the mechanical properties of reparative tissue were significantly better in the CBD-GFs/SDOM group than in the other groups. Conclusion: The CBD-GFs/SDOM is a promising scaffold for osteochondral regeneration. Clinical Relevance: The findings of this study indicated that the CBD-GFs/SDOM is an excellent candidate for reconstructing osteochondral defects, which may be translated for clinical use in the future.

A high spatial resolution osteogenic differentiation in human mesenchymal stem cells induced by femtosecond laser.

A variety of physical and chemical methods have been developed in research laboratories for the induction of stem cell differentiation. However, the use of exogenous chemicals and materials may limit their widespread utility in clinics. To develop a clean and precise induction approach with minimal invasion, we reported here that 1-second stimulation by a tightly focused femtosecond laser (fsL) (140 mW/μm2 , 200 fs) can modulate the signaling systems in human mesenchymal cells, such as intracellular calcium and reactive oxygen species. Upon stimulation on an automatic platform, hMSCs were found to express osteoblastic markers and form calcium-rich deposits. Moreover, tissue mineralization was observed when the fsL-illuminated hMSCs were ectopically transplanted into nude mice. Collectively, we described a novel and non-contact optical stimulation method for cell differentiation with high spatiotemporal resolution.

Piezoelectric charge induced hydrophilic poly(L-lactic acid) nanofiber for electro-topographical stimulation enabling stem cell differentiation and expansion

Cell-traction driven electrical stimulation on piezoelectric nanofibers mediates cell behavior without chemical factors or external stimuli, indicating a promising therapy at cell level. Generally, surface modification is required to achieve favorable cell adhesion for efficient electromechanical signal transmission. Nevertheless, common modification methodologies either easily fade away or destroy the nanostructure of piezoelectric nanofibers. Herein, with the assistance of ultrasound activated surface charges, in situ hydrophilic modification on piezoelectric poly(L-lactic acid) nanofibers is first proposed for cell adhesion. Due to the electrostatic interaction between dopamine and activated piezoelectric nanofiber, the efficient and robust in situ modification is obtained without compromising the nanofibrous structure. Stimulated by the piezoelectric nanofibers with modification, neural stem cells differentiate significantly into nerve cells on day 7 without exogenous growth factors. To our knowledge, the effect of inducing stem cell differentiation far outperforms that in previous reports. In addition, it has been demonstrated that the level of both cell differentiation and directional expansion is consistent with that of cell adhesion condition regardless of the intensity of electrical cue. This connection of cell adhesion and electro-topographical stimulation effects may guide the efforts in the design of electroactive scaffolds.

The Role of Epigenetic in Dental and Oral Regenerative Medicine by Different Types of Dental Stem Cells: A Comprehensive Overview.

Postnatal teeth, wisdom teeth, and exfoliated deciduous teeth can be harvested for dental stem cell (DSC) researches. These mesenchymal stem cells (MSCs) can differentiate and also consider as promising candidates for dental and oral regeneration. Thus, the development of DSC therapies can be considered a suitable but challenging target for tissue regeneration. Epigenetics describes changes in gene expression rather than changes in DNA and broadly happens in bone homeostasis, embryogenesis, stem cell fate, and disease development. The epigenetic regulation of gene expression and the regulation of cell fate is mainly governed by deoxyribonucleic acid (DNA) methylation, histone modification, and noncoding RNAs (ncRNAs). Tissue engineering utilizes DSCs as a target. Tissue engineering therapies are based on the multipotent regenerative potential of DSCs. It is believed that epigenetic factors are essential for maintaining the multipotency of DSCs. A wide range of host and environmental factors influence stem cell differentiation and differentiation commitment, of which epigenetic regulation is critical. Several lines of evidence have shown that epigenetic modification of DNA and DNA-correlated histones are necessary for determining cells' phenotypes and regulating stem cells' pluripotency and renewal capacity. It is increasingly recognized that nuclear enzyme activities, such as histone deacetylases, can be used pharmacologically to induce stem cell differentiation and dedifferentiation. In this review, the role of epigenetic in dental and oral regenerative medicine by different types of dental stem cells is discussed in two new and promising areas of medical and biological researches in recent studies (2010-2022).

Robust temporal map of human in vitro myelopoiesis using single-cell genomics

Myeloid cells are central to homeostasis and immunity. Characterising in vitro myelopoiesis protocols is imperative for their use in research, immunotherapies, and understanding human myelopoiesis. Here, we generate a >470K cells molecular map of human induced pluripotent stem cells (iPSC) differentiation into macrophages. Integration with in vivo single-cell atlases shows in vitro differentiation recapitulates features of yolk sac hematopoiesis, before definitive hematopoietic stem cells (HSC) emerge. The diversity of myeloid cells generated, including mast cells and monocytes, suggests that HSC-independent hematopoiesis can produce multiple myeloid lineages. We uncover poorly described myeloid progenitors and conservation between in vivo and in vitro regulatory programs. Additionally, we develop a protocol to produce iPSC-derived dendritic cells (DC) resembling cDC2. Using CRISPR/Cas9 knock-outs, we validate the effects of key transcription factors in macrophage and DC ontogeny. This roadmap of myeloid differentiation is an important resource for investigating human fetal hematopoiesis and new therapeutic opportunities.

Effect of An Atmospheric Plasma Jet on the Differentiation of Melanoblast Progenitor.

Melanoblasts are the cell source of regeneration for pigment restoration. The ability to differentiate into mature melanocytes is the essential feature of melanoblasts in depigmentation diseases. Cold atmospheric plasma is an ionized gas with near-room temperature and highly reactive species that has been shown to induce stem cell differentiation. The aim of the study was to explore the effect of cold atmospheric plasma on the differentiation of melanoblast progenitor cells. In this study, melanoblasts were exposed to the plasma jet and the cell morphology was observed. The cell cycle and cell proliferation were detected. Furthermore, the cell immunofluorescence and the detection of melanin particle and nitric oxide were carried out to investigate the differentiation of melanoblast progenitor cells. Cells that were treated with the plasma had longer and more synaptic structures, and the G1 phase of cell cycle was prolonged in the treated group. More melanin synthesis-related proteins and melanin particles were produced after plasma treatment. Nitric oxide was one of the active components generated by the plasma jet, and the nitric oxide content in the cell culture medium of the treated group increased. These results indicate that an increase in nitric oxide production caused by a plasma jet can promote cell differentiation. The application of plasma provides an innovative strategy for the treatment of depigmentation diseases.

Stem cell differentiation with consistent lineage commitment induced by a flash of ultrafast-laser activationin vitroand in vivo.

Recent technological advancements on stem cell differentiation induction have been making great progress in stem cell research, regenerative medicine, and therapeutic applications. However, the risk of off-target differentiation limits the wide application of stem cell therapy strategies. Here, we report a non-invasive all-optical strategy to induce stem cell differentiation invitro and invivo that activates individual target stem cells in situ by delivering a transient 100-ms irradiation of a tightly focused femtosecond laser to a submicron cytoplasmic region of primary adipose-derived stem cells (ADSCs). The ADSCs differentiate to osteoblasts with stable lineage commitment that cannot further transdifferentiate because of simultaneous initiation of multiple signaling pathways through specific Ca2+ kinetic patterns. This method can work invivo to direct mouse cerebellar granule neuron progenitors to granule neurons in intact mouse cerebellums through the skull. Hence, this optical method without any genetic manipulations or exogenous biomaterials holds promising potential in biomedical research and cell-based therapies.

Role of the PI3K/Akt signaling pathway in liver ischemia reperfusion injury: a narrative review.

To explore the role of phosphatidylinositol-3-kinase/protein kinase B and alpha serine/threonine protein kinase PI3K/PKB (also known as PI3K/Akt) signaling pathway in liver ischemia reperfusion injury. The PI3K/Akt signaling pathway is one of the major signal transduction pathways that regulates numerous cellular activities in vivo. The main functions of this pathway include induction of stem cell differentiation and metastasis, promotion of cell proliferation, inhibition of apoptosis, and regulation of tissue inflammation, tumor growth, and invasion. Liver ischemia reperfusion injury is an inevitable clinical problem that can occur during liver transplantation, liver resection, and various circulatory shock events, and it is one of the primary reasons for postoperative liver dysfunction, and poor disease outcome and patient prognosis. In recent years, it has been found that PI3K/Akt signaling pathway is closely related to liver ischemia reperfusion injury. In this review, a large number of relevant literatures were collected to explain the biological basis of PI3K/Akt signaling pathway and its role in liver ischemia reperfusion injury. The review was based on a PubMed search using the terms "liver ischemia reperfusion injury", "PI3K/Akt signaling pathway", and "PI3K/Akt signaling pathway AND liver ischemia reperfusion injury", so as to understand the complex interaction between them. Activated PI3K/Akt signaling pathway can exert anti-inflammatory, antioxidant stress, anti-apoptosis and autophagy regulation effects through downstream related targeted pathways and proteins, thus alleviating liver ischemia-reperfusion injury. Therefore, the regulation of PI3K/Akt signaling pathway is expected to become an effective targeted pathway for clinical prevention and alleviation of liver ischemia reperfusion injury.

Coaxial structured drug loaded dressing combined with induced stem cell differentiation for enhanced wound healing

Controllable drug-loaded dressings combined with induced stem cell differentiation have received considerable interest. In this study, a directional core-shell drug-loaded magnetocaloric response PCL/Gelatin-Antibiotics/Fe3O4 multifunctional dressing was developed. Due to the magnetothermal heating effect of magnetic nanoparticles and the contraction of elastic electrospun fibers, the fibers release antibiotics as needed to prevent drug-resistant infection. IV collagenase catalyzes the degradation of gelatin by achieving an optimum reaction temperature, the purpose of which is also to reduce the viscosity of liquid gelatin and promote the release of drugs. With the sacrifice of gelatin, the directional structure of scaffold and the internal steric hindrance promoted stem cell differentiation and wound healing. The expression of Vimentin, VEGF, bFGF, TGF-β, and THY1 was confirmed by fluorescence immunostaining and RT-PCR. Western blot was used to detect expression of Vimentin, collagen, CD34, and CD31 in the (5/5, v/v) PCL/gelatin scaffold incubated with mouse wound. Therefore, the functional fibers can significantly accelerate the healing process.

Downregulated Calcium-Binding Protein S100A16 and HSP27 in Placenta-Derived Multipotent Cells Induce Functional Astrocyte Differentiation

Little is known about genes that induce stem cells differentiation into astrocytes. We previously described that heat shock protein 27 (HSP27) downregulation is directly related to neural differentiation under chemical induction in placenta-derived multipotent stem cells (PDMCs). Using this neural differentiation cell model, we cross-compared transcriptomic and proteomic data and selected 26 candidate genes with the same expression trends in both omics analyses. Those genes were further compared with a transcriptomic database derived from Alzheimer’s disease (AD). Eighteen out of 26 candidates showed opposite expression trends between our data and the AD database. The mRNA and protein expression levels of those candidates showed downregulation of HSP27, S100 calcium-binding protein A16 (S100A16) and two other genes in our neural differentiation cell model. Silencing these four genes with various combinations showed that co-silencing HSP27 and S100A16 has stronger effects than other combinations for astrocyte differentiation. The induced astrocyte showed typical astrocytic star-shape and developed with ramified, stringy and filamentous processes as well as differentiated endfoot structures. Also, some of them connected with each other and formed continuous network. Immunofluorescence quantification of various neural markers indicated that HSP27 and S100A16 downregulation mainly drive PDMCs differentiation into astrocytes. Immunofluorescence and confocal microscopic images showed the classical star-like shape morphology and co-expression of crucial astrocyte markers in induced astrocytes, while electrophysiology and Ca2+ influx examination further confirmed their functional characteristics. In conclusion, co-silencing of S100A16 and HSP27 without chemical induction leads to PDMCs differentiation into functional astrocytes.Graphical abstract

Fluoxetine regulates the neuronal differentiation of neural stem cells transplanted into rat brains after stroke by increasing the 5HT level

Fluoxetine, a 5-HT uptake inhibitor, has been adopted for the treatment of post-stroke depression in recent years. It has been confirmed to induce neuronal regeneration in vivo, but its effect on inducing stem cell differentiation after transplantation has not yet been verified. To evaluate its regulatory effect on stem cell differentiation, fluoxetine was used in this study to treat rats with cerebral ischemia after neural stem cell (NSC) transplantation. The results showed that the proportion of NSCs differentiating into neurons significantly increased after fluoxetine treatment. In NSC adherent culture, the addition of 5-HT but not of fluoxetine significantly increased the neuronal differentiation ratio of NSCs. Moreover, the addition of 5-HT2A or 5-HT3A antagonists inhibited this effect. In addition, Western blotting revealed that the increase in 5-HT inhibited ERK2 phosphorylation and upregulated neurogenin1 expression. In conclusion, fluoxetine increased the 5-HT level and promoted neuronal differentiation, thereby upregulating neurogenin1 expression and downregulating ERK2 phosphorylation.

Potential Applications of Low-intensity Extracorporeal Shock-Wave Therapy in Urological Diseases via Activation of Tissue Resident Stem Cells

For many years, low-intensity extracorporeal shock-wave therapy (Li-ESWT) has been clinically applied as a noninvasive therapeutic method, for urological diseases. The major corresponding biological molecular mechanisms of Li-ESWT are to induce stem cell differentiation, neural regeneration, and angiogenesis. This narrative review aims to present an overview of the potential utility of Li-ESWT and its effects on stem cell therapies. Recent studies have also shown that the combination treatment of Li-ESWT and stem cell therapies can be a new option for the treatment of erectile dysfunction (ED), urinary incontinence, bladder dysfunction, and other diseases. The potential contributions of Li-ESWT on stem cell therapies for these diseases are studied, highlighting the influence of Li-ESWT on proliferation, viability, and differentiation capacity of certain stem cells. The potential mechanisms, including the increased expression of vascular endothelial growth factor, chemokine CXC motif ligand 5, and transforming growth factor-β1 are described herein. Li-ESWT can also activate many cellular signaling pathways. The combination of Li-ESWT and stem cell therapies is a promising strategy for urological diseases. However, a much greater understanding of the mechanisms by which Li-ESWT enhances the efficacy of stem cell therapy is still needed before this combined treatment can be recommended for large-scale clinical application.

Effect of Amniotic Membrane/Collagen-Based Scaffolds on the Chondrogenic Differentiation of Adipose-Derived Stem Cells and Cartilage Repair.

Objectives: Repairing articular cartilage damage is challenging. Clinically, tissue engineering technology is used to induce stem cell differentiation and proliferation on biological scaffolds to repair defective joints. However, no ideal biological scaffolds have been identified. This study investigated the effects of amniotic membrane/collagen scaffolds on the differentiation of adipose-derived stem cells (ADSCs) and articular cartilage repair. Methods: Adipose tissue of New Zealand rabbits was excised, and ADSCs were isolated and induced for differentiation. An articular cartilage defect model was constructed to identify the effect of amniotic membrane/collagen scaffolds on cartilage repair. Cartilage formation was analyzed by imaging and toluene blue staining. Knee joint recovery in rabbits was examined using hematoxylin and eosin, toluidine, safranine, and immunohistochemistry at 12 weeks post-operation. Gene expression was examined using ELISA, RT-PCR, Western blotting, and immunofluorescence. Results: The adipose tissue was effectively differentiated into ADSCs, which further differentiated into chondrogenic, osteogenic, and lipogenic lineages after 3 weeks’ culture in vitro. Compared with platelet-rich plasmon (PRP) scaffolds, the amniotic membrane scaffolds better promoted the growth and differentiation of ADSCs. Additionally, scaffolds containing the PRP and amniotic membrane efficiently enhanced the osteogenic differentiation of ADSCs. The levels of COL1A1, COL2A1, COL10A1, SOX9, and ACAN in ADSCs + amniotic membrane + PRP group were significantly higher than the other groups both in vitro and in vivo. The Wakitani scores of the ADSC + amniotic membrane + PRP group were lower than that in ADSC + PRP (4.4 ± 0.44**), ADSC + amniotic membrane (2.63 ± 0.38**), and control groups (6.733 ± 0.21) at week 12 post-operation. Osteogenesis in rabbits of the ADSC + amniotic membrane + PRP group was significantly upregulated when compared with other groups. Amniotic membranes significantly promoted the expression of cartilage regeneration-related factors (SOX6, SOX9, RUNX2, NKX3-2, MEF2C, and GATA4). The ADSC + PRP + amniotic membrane group exhibited the highest levels of TGF-β, PDGF, and FGF while exhibiting the lowest level of IL-1β, IL6, and TNF-α in articular cavity. Conclusion: Amniotic membrane/collagen combination-based scaffolds promoted the proliferation and cartilage differentiation of ADSCs, and may provide a new treatment paradigm for patients with cartilage injury.

Narrative review of gene modification: applications in three-dimensional (3D) bioprinting.

ObjectiveThis article focused on the application scenarios of three-dimensional (3D) bioprinting and gene-editing technology in various medical fields, including gene therapy, tissue engineering, tumor microenvironment simulation, tumor model construction, cancer regulation and expression, osteogenesis, and skin and vascular regeneration, and summarizing its development prospects and shortcomings.Background3D bioprinting is a process based on additive manufacturing that uses biological materials as the microenvironment living cells. The scaffolds and carriers manufactured by 3D bioprinting technology provide a safe, efficient, and economical platform for genes, cells, and biomolecules. Gene modification refers to replacing, splicing, silencing, editing, controlling or inactivating genes and delivering new genes. The combination of this technology that changes cell function or cell fate or corrects endogenous mutations and 3D bioprinting technology has been widely used in various medical field.MethodsWe conducted a literature search for papers published up to March 2021 on the gene modification combined with 3D bioprinting in various medical fields via PubMed, Web of Science, China National Knowledge Infrastructure (CNKI). The following medical subject heading terms were included for a MEDLINE search: “3D printing/gene editing”, “3D printing/genetic modification”, “3D printing/seed cell”, “bioprinting/gene editing”, “bioprinting/genetic modification”, “bioprinting/seed cell”, “scaffold/gene editing”, “scaffold/genetic modification”, “scaffold/seed cell”, “gene/scaffold”, “gene/bioprinting”, “gene/3D printing”. Quantitative and qualitative data was extracted through interpretation of each article.ConclusionsWe have reviewed the application scenarios of 3D bioprinting and gene-editing technology in various medical fields, it provides an efficient and accurate delivery system for personalized tumor therapy, enhancing the targeting effect while maintaining the integrity of the fabricated structure. It exhibits significant application potential in developing tumor drugs. In addition, scaffolds obtained via 3D bioprinting provide gene therapy applications for skin and bone healing and repair and inducing stem cell differentiation. It also considers the future development direction in this field, such as the emergence and development of gene printing, 4D printing. The combination of nanotechnology and gene printing may provide a new way for future disease research and treatment.

Investigating developmental effects of PFAS using a 3D human induced pluripotent stem cell differentiation model

Investigating developmental effects of PFAS using a 3D human induced pluripotent stem cell differentiation model

A comprehensive analysis of gene expression changes in a high replicate and open-source dataset of differentiating hiPSC-derived cardiomyocytes

We performed a comprehensive analysis of the transcriptional changes occurring during human induced pluripotent stem cell (hiPSC) differentiation to cardiomyocytes. Using single cell RNA-seq, we sequenced > 20,000 single cells from 55 independent samples representing two differentiation protocols and multiple hiPSC lines. Samples included experimental replicates ranging from undifferentiated hiPSCs to mixed populations of cells at D90 post-differentiation. Differentiated cell populations clustered by time point, with differential expression analysis revealing markers of cardiomyocyte differentiation and maturation changing from D12 to D90. We next performed a complementary cluster-independent sparse regression analysis to identify and rank genes that best assigned cells to differentiation time points. The two highest ranked genes between D12 and D24 (MYH7 and MYH6) resulted in an accuracy of 0.84, and the three highest ranked genes between D24 and D90 (A2M, H19, IGF2) resulted in an accuracy of 0.94, revealing that low dimensional gene features can identify differentiation or maturation stages in differentiating cardiomyocytes. Expression levels of select genes were validated using RNA FISH. Finally, we interrogated differences in cardiac gene expression resulting from two differentiation protocols, experimental replicates, and three hiPSC lines in the WTC-11 background to identify sources of variation across these experimental variables.

Progress in cardiac research: from rebooting cardiac regeneration to a complete cell atlas of the heart.

We review some of the important discoveries and advances made in basic and translational cardiac research in 2020. For example, in the field of myocardial infarction (MI), new aspects of autophagy and the importance of eosinophils were described. Novel approaches, such as a glycocalyx mimetic, were used to improve cardiac recovery following MI. The strategy of 3D bio-printing was shown to allow the fabrication of a chambered cardiac organoid. The benefit of combining tissue engineering with paracrine therapy to heal injured myocardium is discussed. We highlight the importance of cell-to-cell communication, in particular, the relevance of extracellular vesicles, such as exosomes, which transport proteins, lipids, non-coding RNAs, and mRNAs and actively contribute to angiogenesis and myocardial regeneration. In this rapidly growing field, new strategies were developed to stimulate the release of reparative exosomes in ischaemic myocardium. Single-cell sequencing technology is causing a revolution in the study of transcriptional expression at cellular resolution, revealing unanticipated heterogeneity within cardiomyocytes, pericytes and fibroblasts, and revealing a unique subpopulation of cardiac fibroblasts. Several studies demonstrated that exosome- and non-coding RNA-mediated approaches can enhance human induced pluripotent stem cell (iPSC) viability and differentiation into mature cardiomyocytes. Important details of the mitochondrial Ca2+ uniporter and its relevance were elucidated. Novel aspects of cancer therapeutic-induced cardiotoxicity were described, such as the novel circular RNA circITCH, which may lead to novel treatments. Finally, we provide some insights into the effects of SARS-CoV-2 on the heart.

Developmental effects of PFOS, PFOA and GenX in a 3D human induced pluripotent stem cell differentiation model

Polyfluoroalkyl substances (PFASs), including perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA), are persistent pollutants routinely found in human blood. PFASs have been associated with health issues such as decreased birth weight and impaired vaccination response in children. Substitutes to these PFASs, such as ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoate (GenX) have been introduced, although hazard information is limited. Human induced pluripotent stem cell (hiPSC) based models are valuable for studying these compounds, as they mimic human embryonic development. We used our recently developed PluriBeat assay to investigate PFOS, PFOA and GenX for effects on early embryonic development in vitro. In our assay hiPSCs go through the early stages of embryonic development in 3D cultures of embryoid bodies (EBs) that mimic the human blastocyst until they finally form beating cardiomyocytes. Both PFOS and PFOA had a strong effect on cardiomyocyte differentiation at non-cytotoxic concentrations, with PFOS being more potent than PFOA. Moreover, both compounds decreased EB size at the highest test concentrations. GenX induced a weak concentration-dependent effect on differentiation of one hiPSC line, but not of another. Transcriptional analysis of mRNA from the cardiomyocytes showed that PFOS increased expression of the early cardiac marker ISL1, whereas PFOA decreased expression of the cardiomyocyte marker MYH7. This suggest that PFOS and PFOA perturb cardiomyocyte differentiation by disrupting molecular pathways similar to those taking place in the developing embryo. Based on these findings, we conclude that our PluriBeat assay has the potential to become a valuable, sensitive model system for elucidating embryotoxic effects of PFASs in future.

Protocol to Study the Role of Extracellular Vesicles During Induced Stem Cell Differentiation.

Extracellular vesicles (EVs) are vesicles released by cells, which due to their cargo and cell membrane proteins induce changes in the recipient cells. These vesicles can be a novel option to induce stem cell differentiation. Here we described a method to induce mesenchymal stem cell differentiation (MSC) into neuron-like cells using small EVs from neurons. First, we will describe a method based on neurons to induce adipocyte derived stem cells differentiation, a type of MSC, by coculturing both using inserts. Secondly, we will describe a follow-up method by using only isolated neuron-derived small EVs to directly induce ADSC differentiation in neuron-like cells. Importantly, in both methods it is possible to avoid the direct cell-to-cell contact, thus allowing for the study of soluble factors role during stem cell differentiation.

Directed Differentiation of Neural Cells from Human Umbilical Cord Stroma-derived Neural Stem Cells

Objective to investigate the directed differentiation of human umbilical cord stromal-derived stem cells into neural cells. Methods: (1) Human umbilical cord stromal cells (HUMSC-NSCs) were induced to differentiate from primary human umbilical cord stromal cells, and two different methods were used to induce differentiation to neural cells, which were observed under electron microscope. (2) The neural cells induced to differentiate by different induction methods were identified by morphological differences, immunohistochemistry, and Western blot. Results: (1) Some of the suspended NSCs started to adhere to the wall 48-72 h after the addition of the inducer, and elongated protrusions could be seen in 5-7 days with the neurosphere as the center, and cells gradually migrated outward from the neurosphere. 10 days later, several cells with different morphologies could be seen. Some of the cells had increased refractive index and the length of the protrusions increased, showing bipolar growth. In some cases, the protrusions were short and dense, centered on the cytosol, and protruding in a discrete manner. Under electron microscopy, the neurospheres consisted of multiple clonal clusters and two different morphologies of cells, shaped like neurons and astrocytes, could be clearly observed. The number of bipolar neurons was significantly increased in the group with the addition of BDNF.(2) Before induction, 84.5 ± 1.6% and 88.62 ± 1.1% of HUMSC-NSCs expressed stem cell-specific markers: Stro-1 and nestin; neurospheres still expressed 62.7 ± 3.9% of HUMSC-NSCs positive for nestin after 5 d of culture in neuronal cell induction medium. After 10 days of induction, the percentage of immunohistochemically positive stained cells was counted. With the BDMF induction protocol, 38.6 ± 2.9% and 8 ± 1.9% Hoechst33342 positive cells expressed immature (β-tubulin III) and mature (MAP2ab) neuronal cell markers, respectively. Also, 15.8 ± 4.5% and 20.6 ± 4.6% of Hoechst33342-positive cells expressed GFAP (astrocyte marker) and GalC (oligodendrocyte marker). Comparison using a paired t-test revealed that the percentage of β-tubulin III (P<0.001) and MAP2ab (P<0.05) positive cells was significantly higher in the BDNF-induced group than in the general induction group. Western blot results confirmed the immunohistochemical data. Conclusion: After adding BDNF to induce differentiation in the culture group, the number of bipolar neurons was significantly increased, which could promote the induced differentiation of human umbilical cord-derived neural stem cells into neuronal cells.