Genome-scale CRISPR-Cas9 screening in stem cells: theories, applications and challenges.

Due to the rapid development of stem cell technology, there have been tremendous advances in molecular biological and pathological research, cell therapy as well as organoid technologies over the past decades. Advances in genome editing technology, particularly the discovery of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-related protein 9 (Cas9), have further facilitated the rapid development of stem cell researches. The CRISPR-Cas9 technology now goes beyond creating single gene editing to enable the inhibition or activation of endogenous gene loci by fusing inhibitory (CRISPRi) or activating (CRISPRa) domains with deactivated Cas9 proteins (dCas9). These tools have been utilized in genome-scale CRISPRi/a screen to recognize hereditary modifiers that are synergistic or opposing to malady mutations in an orderly and fair manner, thereby identifying illness mechanisms and discovering novel restorative targets to accelerate medicinal discovery investigation. However, the application of this technique is still relatively rare in stem cell research. There are numerous specialized challenges in applying large-scale useful genomics approaches to differentiated stem cell populations. Here, we present the first comprehensive review on CRISPR-based functional genomics screening in the field of stem cells, as well as practical considerations implemented in a range of scenarios, and exploration of the insights of CRISPR-based screen into cell fates, disease mechanisms and cell treatments in stem cell models. This review will broadly benefit scientists, engineers and medical practitioners in the areas of stem cell research.

A new subtype of artificial cell-derived vesicles from dental pulp stem cells with the bioequivalence and higher acquisition efficiency compared to extracellular vesicles.

Extracellular vesicles (EVs) derived from dental pulp stem cells (DPSC) have been shown an excellent efficacy in a variety of disease models. However, current production methods fail to meet the needs of clinical treatment. In this study, we present an innovative approach to substantially enhance the production of 'Artificial Cell-Derived Vesicles (ACDVs)' by extracting and purifying the contents released by the DPSC lysate, namely intracellular vesicles. Comparative analysis was performed between ACDVs and those obtained through ultracentrifugation. The ACDVs extracted from the cell lysate meet the general standard of EVs and have similar protein secretion profile. The new ACDVs also significantly promoted wound healing, increased or decreased collagen regeneration, and reduced the production of inflammatory factors as the EVs. More importantly, the extraction efficiency is improved by 16 times compared with the EVs extracted using ultracentrifuge method. With its impressive attributes, this new subtype of ACDVs emerge as a prospective candidate for the future clinical applications in regenerative medicine.

Local Spinal Cord Injury Treatment Using a Dental Pulp Stem Cell Encapsulated H2S Releasing Multifunctional Injectable Hydrogel.

Spinal cord injury (SCI) commonly induces nerve damage and nerve cell degeneration. In this work, a novel dental pulp stem cells (DPSCs) encapsulated thermoresponsive injectable hydrogel with sustained hydrogen sulfide (H2S) delivery is demonstrated for SCI repair. For controlled and sustained H2S gas therapy, a clinically tested H2S donor (JK) loaded octysilane functionalized mesoporous silica nanoparticles (OMSNs) are incorporated into the thermosensitive hydrogel made from Pluronic F127 (PF-127). The JK-loaded functionalized MSNs (OMSF@JK) promote preferential M2-like polarization of macrophages and neuronal differentiation of DPSCs in vitro. OMSF@JK incorporated PF-127 injectable hydrogel (PF-OMSF@JK) has a soft consistency similar to that of the human spinal cord and thus, shows a high cytocompatibility with DPSCs. The cross-sectional micromorphology of the hydrogel shows a continuous porous structure. Last, the PF-OMSF@JK composite hydrogel considerably improves the in vivo SCI regeneration in Sprague-Dawley rats through a reduction in inflammation and neuronal differentiation of the incorporated stem cells as confirmed using western blotting and immunohistochemistry. The highly encouraging in vivo results prove that this novel design on hydrogel is a promising therapy for SCI regeneration with the potential for clinical translation.

Inhibitory effects of the nanoscale lysate derived from xenogenic dental pulp stem cells in lung cancer models.

BACKGROUND: Lung cancer is a highly prevalent malignancy and has the highest mortality rate among all tumors due to lymph node metastasis. Bone marrow and umbilical cord-derived mesenchymal stem cells (MSCs) have demonstrated tumor-suppressive effects on lung cancer. This study investigated the effects of DPSC lysate on proliferation, apoptosis, migration and invasion of cancer cells were studied in vivo and in vitro. METHODS: The proliferation, apoptosis, and migration/metastasis were evaluated by cell counting kit-8 assay, Annexin-V and propidium iodide staining, and the transwell assay, respectively. The expression levels of apoptosis-, cell cycle-, migration-, and adhesion-related mRNA and proteins were measured by qRT-PCR and western blot. The level and mRNA expression of tumor markers carcino embryonic antigen (CEA), neuron-specific enolase (NSE), and squamous cell carcinoma (SCC) were measured by Enzyme-linked immunosorbent assay (ELISA) and qRT-PCR. Finally, a tumor-bearing mouse model was constructed to observe the tumor-suppressive effect of DPSC lysate after intraperitoneal injection. RESULTS: DPSC lysate decreased the viability of A549 cells and induced apoptosis in lung cancer cells. Western blot confirmed that levels of Caspase-3, Bax, and Bad were increased, and Bcl-2 protein levels were decreased in A549 cells treated with DPSC lysate. In addition, DPSC lysate inhibited the migration and invasion of A549 cells; downregulated key genes of the cell cycle, migration, and adhesion; and significantly suppressed tumor markers. Xenograft results showed that DPSC lysate inhibited tumor growth and reduced tumor weight. CONCLUSIONS: DPSC lysate inhibited proliferation, invasion, and metastasis; promoted apoptosis in lung cancer cells; and suppressed tumor growth- potentially providing a cell-based alternative therapy for lung cancer treatment.

The Application of Mesenchymal Stem Cells in Future Vaccine Synthesis.

Vaccines have significant potential in treating and/or preventing diseases, yet there remain challenges in developing effective vaccines against some diseases, such as AIDS and certain tumors. Mesenchymal stem cells (MSCs), a subset of cells with low immunogenicity, high proliferation potential, and an abundant source of extracellular vesicles (EVs), represent one of the novel and promising vaccine platforms. This review describes the unique features and potential mechanisms of MSCs as a novel vaccine platform. We also cover aspects such as the safety and stability of MSCs that warrant future in-depth studies.

Metal-organic framework materials promote neural differentiation of dental pulp stem cells in spinal cord injury.

Spinal cord injury (SCI) is accompanied by loss of Zn2+, which is an important cause of glutamate excitotoxicity and death of local neurons as well as transplanted stem cells. Dental pulp stem cells (DPSCs) have the potential for neural differentiation and play an immunomodulatory role in the microenvironment, making them an ideal cell source for the repair of central nerve injury, including SCI. The zeolitic imidazolate framework 8 (ZIF-8) is usually used as a drug and gene delivery carrier, which can release Zn2+ sustainedly in acidic environment. However, the roles of ZIF-8 on neural differentiation of DPSCs and the effect of combined treatment on SCI have not been explored. ZIF-8-introduced DPSCs were loaded into gelatin methacryloyl (GelMA) hydrogel and in situ injected into the injured site of SCI rats. Under the effect of ZIF-8, axon number and axon length of DPSCs-differentiated neuro-like cells were significantly increased. In addition, ZIF-8 protected transplanted DPSCs from apoptosis in the damaged microenvironment. ZIF-8 promotes neural differentiation and angiogenesis of DPSCs by activating the Mitogen-activated protein kinase (MAPK) signaling pathway, which is a promising transport nanomaterial for nerve repair.

Identifying the key genes of Epstein-Barr virus-regulated tumour immune microenvironment of gastric carcinomas.

The Epstein-Barr virus (EBV) is involved in the carcinogenesis of gastric cancer (GC) upon infection of normal cell and induces a highly variable composition of the tumour microenvironment (TME). However, systematic bioinformatics analysis of key genes associated with EBV regulation of immune infiltration is still lacking. In the present study, the TCGA and GEO databases were recruited to analyse the association between EBV infection and the profile of immune infiltration in GC. The weighted gene co-expression analysis (WGCNA) was applied to shed light on the key gene modules associated with EBV-associated immune infiltration in GC. 204 GC tissues were used to analysed the expression of key hub genes by using the immunohistochemical method. Real-time PCR was used to evaluate the association between the expression of EBV latent/lytic genes and key immune infiltration genes. Our results suggested that EBV infection changed the TME of GC mainly regulates the TIICs. The top three hub genes of blue (GBP1, IRF1, and LAP3) and brown (BIN2, ITGAL, and LILRB1) modules as representative genes were associated with EBV infection and GC immune infiltration. Furthermore, EBV-encoded LMP1 expression is account for the overexpression of GBP1 and IRF1. EBV infection significantly changes the TME of GC, and the activation of key immune genes was more dependent on the invasiveness of the whole EBV virion instead of single EBV latent/lytic gene expression.

MSC based gene delivery methods and strategies improve the therapeutic efficacy of neurological diseases.

Mesenchymal stem cells (MSCs) are promising seed cells for neural regeneration therapy owing to their plasticity and accessibility. They possess several inherent characteristics advantageous for the transplantation-based treatment of neurological disorders, including neural differentiation, immunosuppression, neurotrophy, and safety. However, the therapeutic efficacy of MSCs alone remains unsatisfactory in most cases. To improve some of their abilities, many studies have employed genetic engineering to transfer key genes into MSCs. Both viral and nonviral methods can be used to overexpress therapeutic proteins that complement the inherent properties. However, to date, different modes of gene transfer have specific drawbacks and advantages. In addition, MSCs can be functionalized through targeted gene modification to facilitate neural repair by promoting neural differentiation, enhancing neurotrophic and neuroprotective functions, and increasing survival and homing abilities. The methods of gene transfer and selection of delivered genes still need to be optimized for improved therapeutic and targeting efficacies while minimizing the loss of MSC function. In this review, we focus on gene transport technologies for engineering MSCs and the application of strategies for selecting optimal delivery genes. Further, we describe the prospects and challenges of their application in animal models of different neurological lesions to broaden treatment alternatives for neurological diseases.

Progress on the cell therapy strategy of mesenchymal stem cells / 口腔疾病防治

@#Cell therapy based on mesenchymal stem cells (MSCs) has been a hot research topic in recent years, including the traditional cell therapy strategy based on living cells and the new cell-free therapy strategy based on soluble proteins or bioactive molecules such as extracellular vesicles (EVs). At present, MSC-induced cells have mature functions and specific structures, and insitu transplantation combined with biomaterials or organic technology has greatly improved the settlement rate and function. On the other hand, as the large-scale culture technique and EVs separation technique evolve, it is possible to obtain a large number of pure EVs, and EVs are gradually becoming a hot spot of current research. An increasing number of studies have shown that the therapeutic effect of MSCs not only occurs by implantation and differentiation but also manifests as the paracrine effect of MSCs. In this review, we discuss the emerging outcomes of cell therapies and acellular therapies to alleviate these pathological conditions.

Characteristics of culture-condition stimulated exosomes or their loaded hydrogels in comparison with other extracellular vesicles or MSC lysates.

Recently, it has become popular to study the use of extracellular vesicles (EVs) secreted by stem cells to repair damaged tissues or lost cells. Various cell types and physiological fluids release EVs, and they play an important role in cell-to-cell communication. Moreover, EVs have been implicated in important processes, such as immune responses, homeostasis maintenance, coagulation, inflammation, cancer progression, angiogenesis, and antigen presentation. Thus, EVs participate in both physiological and pathological progression. The main classes of EVs include exosomes, microvesicles (MVs), and apoptotic bodies (ApoBDs). Exosomes, which carry a mass of signal molecules such as RNA, DNA, proteins, and lipids, are the most important of these EVs subsets. Currently, exosomes are generating substantial interest in the scientific community. Exosomes loaded hydrogels or under different cultural environments exhibit different properties and functions. Therefore, the exosomes obtained from different sources and conditions are worth reviewing. More importantly, no review article has compared the different EVs, such as exosomes, MVs, ApoBDs, and mesenchymal stem cell (MSC) lysates, which are special soluble substances. The differentiation between EVs and MSC lysates is a logical approach. Accordingly, this review provides an update on the latest progress in studying the roles of culture-condition stimulated exosomes or their loaded hydrogels and the differentiation between exosomes, MVs, ApoBDs, and MSC lysates. Published studies were retrieved from the PubMed® database for review.

Generation of a luciferase-expressing human embryonic stem cell line: NERCe002-A-2.

The human embryonic stem cell line NERCe002-A-2 was generated by transduction of NERCe002-A cells with an expression vector carrying the luciferase gene. The stem cells labelled with luciferase can be transplanted into animals and detected by the bioluminescence imaging technology. This provides optimal prospects of application to in vivo stem cell tracing. Luciferin served as a substrate to detect the activity of luciferase, and luciferase expression was measured by quantitative PCR. Characterization assays suggested that the NERCe002-A-2 cell line expresses typical markers of pluripotency and can form the 3 germ layers in vivo.

Generation of a human embryonic stem cell line, NERCe003-A-1, with lentivirus vector-mediated inducible CTNNB1 overexpression.

The human embryonic stem cell (hESC) line NERCe003-A-1 was generated by introducing lentiviral-vector-mediated tetracycline-inducible ß-catenin expression into a normal hESC line, NERCe003-A. The resulting cell line can overexpress the ß-catenin protein, encoded by the CTNNB1 gene, after exposure to doxycycline (Dox). CTNNB1 gene expression was confirmed by quantitative PCR (qPCR) and immunofluorescence assays. Further characterization confirmed that the NERCe003-A-1 cell line expresses typical pluripotency markers and has the ability to form the three germ layers both in vitro and in vivo.

Establishment and characterization of a human embryonic stem cell line, NERCe002-A-3, with inducible 14-3-3ζ overexpression.

NERCe002-A-3 cells were generated from the normal human embryonic stem cell line NERCe002-A. NERCe002-A-3 cells overexpressed 14-3-3ζ after exposure to doxycycline. 14-3-3ζ protein have the ability to bind a multitude of functionally diverse signalling proteins. The NERCe002-A-3 cell line is considered a model for functional studies of the 14-3-3ζ protein in hESC self-renewal and cell differentiation. Doxycycline-treated NERCe002-A-3 cells showed a>27-fold increase in relative expression of 14-3-3ζ as compared with un-induced cells. Characterization assays proved that NERCe002-A-3 cells express typical markers of pluripotency and have the ability to form the three germ layers in vivo.

Generation of a human embryonic stem cell line expressing tetrameric Zoanthus sp. green fluorescent protein: NERCe002-A-1.

The human embryonic stem cell (hESC) line NERCe002-A-1 was generated through lentiviral transduction of the original NERCe002-A-1 hESC line with Zoanthus sp. green fluorescent protein (ZsGreen). Cells that expressed ZsGreen showed a >8.6-fold increase in fluorescence intensity compared with that of cells that expressed enhanced green fluorescent protein. The fluorescent hESC line can aid in identification of biological characteristics in vitro and in vivo by tracking cell growth, migration, and differentiation. Characteristic tests confirmed that the NERCe002-A-1 cell line expressed typical markers of pluripotency and had the capability to form the three germ layers in vivo.

[Progress of the application of stem cell therapy for end-stage liver disease].

Many risk factors lead to hypohepatia and hepatic failure, causing people suffering from end-stage liver disease. The conventional treatment for end-stage liver disease is not good enough. Orthotopic liver transplantation is effective. However, the high cost, lack of liver source, immune rejection and other factors limit the large-scale clinical application. Thus, cell therapy is a good option. Studies on common cell sources for the treatment of liver disease and the induction of hepatocytes by embryonic stem cells or pluripotent stem cells have made progress. With the development of stem cell technology, cell transplantation has become a new option, which brings hope to people with end-stage liver diseasetransplantation has become a new option. It brings hope to people with end-stage liver disease.

[Progress in studies on the role of ß-catenin in regulating the self-renewal and pluripotency of embryonic stem cells].

Embryonic stem cells (ESCs) is one of the best cell types for regenerative medicine. It is derived from inner cell mass of the blastocyst stage and characterized by self-renewal and pluripotency, which are regulated by kinds of signal molecules, such as the Wnt/ß-catenin signaling pathway. ß-catenin is a multifunctional protein and plays a key role in Wnt/ß-catenin signaling pathway. ß-catenin involves self-renewal of ESCs and promotes the differentiation of ESCs into three primary germ layers in space and time. Elucidating the mechanisms of ß-catenin in regulating the self-renewal and pluripotency of ESCs will pave the way to use it in research and application.