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Abstract 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.

Abstract 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. Graphical Abstract

Abstract 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.

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.

Burns not only damage the skin barrier, but also cause a series of inflammatory reactions and oxidative stress states. Among them, elderly patients are prone to suffer severe burns due to degenerative changes of their skin caused by aging factors, such as atrophy and thinning, etc. After burns, the body will continuously release inflammatory factors, resulting in systemic inflammatory response syndrome (SIRS) and oxidative stress, which are related to the poor treatment effect and the poor prognosis of elderly burn patients. It seems to be difficult for conventional treatments to control the disease development of elderly burn patients effectively. Considering the rapidly increasing elderly population, it is priority to understand the pathological process and the mechanisms to formulate more appropriate treatment strategies for elderly burn patients. In recent years, owing to considerable advances in nanotechnology, a variety of nanomaterials have been developed for wound healing and inflammation regulation. Its good biocompatibility, cell proliferation stimulation and antibacterial properties make the clinical treatment strategy more optimized. Concurrently, mesenchymal stem cells (MSCs) have also been used in the burns field and have been proven effective in not only controlling the level of inflammation and regulating the systemic immune balance, but also promoting wound healing and vascularization. Here, this review covers burns classification, the pathological process of elderly burn patients, and the research progress of nanotechnology and MSCs in burns. Eventually, we summarize the advantages and challenges of emerging strategies such as nanotechnology and MSCs in the treatment of elderly burn patients, expecting to promote the clinical transformation.

Stem cell therapy is of great therapeutical potential for anti-aging treatment. Lysates of dental pulp stem cells (DPSC) are rich in numerous cytokines, which can improve the cell growth environment and enhance collagen synthesis in the skin. Frequently, the instability of protein growth factors severely restricts their applications in living systems. In this study, a strategy for delivering the growth factors of DPSC lysate via protein-encapsulated zeolitic imidazolate framework-8 (ZIF-8) nanoparticles was developed. Using ZIF-8 as protein-encapsulated nanoparticles, a ZIF-8@DPSC lysate (ZIF-8@DL) with pH-responsive drug release was produced. ZIF-8@DL demonstrated superior cutaneous uptake, cutaneous penetration, and retention capacity in vivo compared to DPSC lysate. Moreover, ZIF-8@DL demonstrated excellent in vitro and in vivo biocompatibility. Subcutaneous microneedle injection of ZIF-8@DL into a mouse photoaging model resulted in significantly improved epidermal thickness, dermal collagen deposition, and cutaneous inflammation, with no apparent adverse effects. This study provides a safe and efficient method for enhancing the therapeutic application of DPSC lysate. It has the potential to expedite the clinical implementation of lysate-based anti-photoaging therapy.

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.

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.

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.

The human embryonic stem cell (hESC) line NERCe003-A-1 was generated by introducing lentiviral-vector–mediated tetracycline-inducible β-catenin expression into a normal hESC line, NERCe003-A. The resulting cell line can overexpress the β-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.

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.

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.