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.

Repair spinal cord injury with a versatile anti-oxidant and neural regenerative nanoplatform.

Spinal cord injury (SCI) often results in motor and sensory deficits, or even paralysis. Due to the role of the cascade reaction, the effect of excessive reactive oxygen species (ROS) in the early and middle stages of SCI severely damage neurons, and most antioxidants cannot consistently eliminate ROS at non-toxic doses, which leads to a huge compromise in antioxidant treatment of SCI. Selenium nanoparticles (SeNPs) have excellent ROS scavenging bioactivity, but the toxicity control problem limits the therapeutic window. Here, we propose a synergistic therapeutic strategy of SeNPs encapsulated by ZIF-8 (SeNPs@ZIF-8) to obtain synergistic ROS scavenging activity. Three different spatial structures of SeNPs@ZIF-8 were synthesized and coated with ferrostatin-1, a ferroptosis inhibitor (FSZ NPs), to achieve enhanced anti-oxidant and anti-ferroptosis activity without toxicity. FSZ NPs promoted the maintenance of mitochondrial homeostasis, thereby regulating the expression of inflammatory factors and promoting the polarization of macrophages into M2 phenotype. In addition, the FSZ NPs presented strong abilities to promote neuronal maturation and axon growth through activating the WNT4-dependent pathways, while prevented glial scar formation. The current study demonstrates the powerful and versatile bioactive functions of FSZ NPs for SCI treatment and offers inspiration for other neural injury diseases.

Human dental pulp stem cells mitigate the neuropathology and cognitive decline via AKT-GSK3ß-Nrf2 pathways in Alzheimer's disease.

Oxidative stress is increasingly recognized as a major contributor to the pathophysiology of Alzheimer's disease (AD), particularly in the early stages of the disease. The multiplicity advantages of stem cell transplantation make it fascinating therapeutic strategy for many neurodegenerative diseases. We herein demonstrated that human dental pulp stem cells (hDPSCs) mediated oxidative stress improvement and neuroreparative effects in in vitro AD models, playing critical roles in regulating the polarization of hyperreactive microglia cells and the recovery of damaged neurons. Importantly, these therapeutic effects were reflected in 10-month-old 3xTg-AD mice after a single transplantation of hDPSCs, with the treated mice showing significant improvement in cognitive function and neuropathological features. Mechanistically, antioxidant and neuroprotective effects, as well as cognitive enhancements elicited by hDPSCs, were at least partially mediated by Nrf2 nuclear accumulation and downstream antioxidant enzymes expression through the activation of the AKT-GSK3ß-Nrf2 signaling pathway. In conclusion, our findings corroborated the neuroprotective capacity of hDPSCs to reshape the neuropathological microenvironment in both in vitro and in vivo AD models, which may be a tremendous potential therapeutic candidate for Alzheimer's disease.

Application of dental pulp stem cells for bone regeneration.

Bone defects resulting from severe trauma, tumors, inflammation, and other factors are increasingly prevalent. Stem cell-based therapies have emerged as a promising alternative. Dental pulp stem cells (DPSCs), sourced from dental pulp, have garnered significant attention owing to their ready accessibility and minimal collection-associated risks. Ongoing investigations into DPSCs have revealed their potential to undergo osteogenic differentiation and their capacity to secrete a diverse array of ontogenetic components, such as extracellular vesicles and cell lysates. This comprehensive review article aims to provide an in-depth analysis of DPSCs and their secretory components, emphasizing extraction techniques and utilization while elucidating the intricate mechanisms governing bone regeneration. Furthermore, we explore the merits and demerits of cell and cell-free therapeutic modalities, as well as discuss the potential prospects, opportunities, and inherent challenges associated with DPSC therapy and cell-free therapies in the context of bone regeneration.

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.

Alzheimer's disease: Pathophysiology and dental pulp stem cells therapeutic prospects.

Alzheimer's disease (AD) is a destructive neurodegenerative disease with the progressive dysfunction, structural disorders and decreased numbers of neurons in the brain, which leads to long-term memory impairment and cognitive decline. There is a growing consensus that the development of AD has several molecular mechanisms similar to those of other neurodegenerative diseases, including excessive accumulation of misfolded proteins and neurotoxic substances produced by hyperactivated microglia. Nonetheless, there is currently a lack of effective drug candidates to delay or prevent the progression of the disease. Based on the excellent regenerative and reparative capabilities of stem cells, the application of them to repair or replace injured neurons carries enormous promise. Dental pulp stem cells (DPSCs), originated from ectomesenchyme of the cranial neural crest, hold a remarkable potential for neuronal differentiation, and additionally express a variety of neurotrophic factors that contribute to a protective effect on injured neuronal cells. Notably, DPSCs can also express immunoregulatory factors to control neuroinflammation and potentiate the regeneration and recovery of injured neurons. These extraordinary features along with accessibility make DPSCs an attractive source of postnatal stem cells for the regeneration of neurons or protection of existing neural circuitry in the neurodegenerative diseases. The present reviews the latest research advance in the pathophysiology of AD and elaborate the neurodifferentiation and neuroprotective properties of DPSCs as well as their application prospects in AD.

A descriptive study of random forest algorithm for predicting COVID-19 patients outcome.

BACKGROUND: The outbreak of coronavirus disease 2019 (COVID-19) that occurred in Wuhan, China, has become a global public health threat. It is necessary to identify indicators that can be used as optimal predictors for clinical outcomes of COVID-19 patients. METHODS: The clinical information from 126 patients diagnosed with COVID-19 were collected from Wuhan Fourth Hospital. Specific clinical characteristics, laboratory findings, treatments and clinical outcomes were analyzed from patients hospitalized for treatment from 1 February to 15 March 2020, and subsequently died or were discharged. A random forest (RF) algorithm was used to predict the prognoses of COVID-19 patients and identify the optimal diagnostic predictors for patients' clinical prognoses. RESULTS: Seven of the 126 patients were excluded for losing endpoints, 103 of the remaining 119 patients were discharged (alive) and 16 died in the hospital. A synthetic minority over-sampling technique (SMOTE) was used to correct the imbalanced distribution of clinical patients. Recursive feature elimination (RFE) was used to select the optimal subset for analysis. Eleven clinical parameters, Myo, CD8, age, LDH, LMR, CD45, Th/Ts, dyspnea, NLR, D-Dimer and CK were chosen with AUC approximately 0.9905. The RF algorithm was built to predict the prognoses of COVID-19 patients based on the best subset, and the area under the ROC curve (AUC) of the test data was 100%. Moreover, two optimal clinical risk predictors, lactate dehydrogenase (LDH) and Myoglobin (Myo), were selected based on the Gini index. The univariable logistic analysis revealed a substantial increase in the risk for in-hospital mortality when Myo was higher than 80 ng/ml (OR = 7.54, 95% CI [3.42-16.63]) and LDH was higher than 500 U/L (OR = 4.90, 95% CI [2.13-11.25]). CONCLUSION: We applied an RF algorithm to predict the mortality of COVID-19 patients with high accuracy and identified LDH higher than 500 U/L and Myo higher than 80 ng/ml to be potential risk factors for the prognoses of COVID-19 patients in the early stage of the disease.