An optimized protocol for the preparation of blood immune cells for transmission electron microscopy.

Transmission electron microscopy (TEM) is a powerful technique that enables visualization of structural details inside cells. Prior to TEM imaging, biological samples must undergo several preparation steps that are optimized according to the sample type. Currently, there are limited protocols for the preparation of blood samples for TEM imaging. Here, we provide a detailed step-by-step method for preparing blood samples for TEM imaging. This protocol enables robust visualization of the ultrastructures of blood immune cells. In addition, we describe the typical cellular features that can be used to distinguish between different immune cells in the blood, such as neutrophils, eosinophils, monocytes, and lymphocytes. This protocol is useful for studying ultrastructural changes in blood immune cells under various physiological and disease conditions.

Coating Materials for Neural Stem/Progenitor Cell Culture and Differentiation.

Neural stem/progenitor cells (NSPCs) have a potential to treat various neurological diseases, such as Parkinson's Disease, Alzheimer's Disease, and Spinal Cord Injury. However, the limitation of NSPC sources and the difficulty to maintain their stemness or to differentiate them into specific therapeutic cells are the main hurdles for clinical research and application. Thus, for obtaining a therapeutically relevant number of NSPCs in vitro, it is important to understand factors regulating their behaviors and to establish a protocol for stable NSPC proliferation and differentiation. Coating materials for cell culture, such as Matrigel, laminin, collagen, and other coating materials, can significantly affect NSPC characteristics. This article provides a review of coating materials for NSPC culturing in both two dimensions and three dimensions, and their functions in NSPC proliferation and differentiation, and presents a useful guide to select coating materials for researchers.

An overview on small molecule-induced differentiation of mesenchymal stem cells into beta cells for diabetic therapy.

The field of regenerative medicine provides enormous opportunities for generating beta cells from different stem cell sources for cellular therapy. Even though insulin-secreting cells can be generated from a variety of stem cell types like pluripotent stem cells and embryonic stem cells, the ideal functional cells should be generated from patients' own cells and expanded to considerable levels by non-integrative culture techniques. In terms of the ease of isolation, plasticity, and clinical translation to generate autologous cells, mesenchymal stem cell stands superior. Furthermore, small molecules offer a great advantage in terms of generating functional beta cells from stem cells. Research suggests that most of the mesenchymal stem cell-based protocols to generate pancreatic beta cells have small molecules in their cocktail. However, most of the protocols generate cells that mimic the characteristics of human beta cells, thereby generating "beta cell-like cells" as opposed to mature beta cells. Diabetic therapy becomes feasible only when there are robust, functional, and safe cells for replacing the damaged or lost beta cells. In this review, we discuss the current protocols used to generate beta cells from mesenchymal cells, with emphasis on small molecule-mediated conversion into insulin-producing beta cell-like cells. Our data and the data presented from the references within this review would suggest that although mesenchymal stem cells are an attractive cell type for cell therapy they are not readily converted into functional mature beta cells.

A direct and non-invasive method for kidney delivery of therapeutics in mice.

Kidney is a vital organ that maintains the homeostasis in terms of acid-balance, toxin filtration and blood pressure control. Kidney malfunction can be fatal and the renal research administers testing pharmaceutical agents or stem cells in rodents to study their therapeutic efficacy. However, targeted delivery of agents into mice kidney is strenuous and may require laparotomy. Here we present a direct delivery method for cell transplantation or drug injection into the mice kidney. The method is simple and can be performed non-invasively with avoidance of surgical intervention on the animals. Nevertheless, this method serves as an efficient method for in vivo drug delivery or engraftment studies for renal research. •Direct delivery into the kidney.•Non-invasive method.

Facial vein injection of human cells in severe combined immunodeficiency (SCID) neonatal mice.

Intravenous injection is a standard procedure for delivering human stem cells and therapeutic agents. Currently, genetically modified severe combined immunodeficiency (SCID) mice are used for engraftment studies using human cells. SCID neonates have better integration and survivability of human cells compared to adult SCID mice, as their immune system will not be developed in the first few days after birth. However, intravenous injections in neonates are difficult. This protocol describes a reliable and reproducible method for injecting cells into the facial vein of P3/P4 (3 or 4 days post-birth) SCID neonates to study their engraftment. The injection was safe and well tolerated by the pups. Post-injection analysis revealed the distribution of tagged cells in different organs. Results suggest that this new method can serve as a pre-analysis for transplantation studies using human stem cells before in vivo animal model testing.

Urine-derived cells for human cell therapy.

Desirable cells for human cell therapy would be ones that can be generated by simple isolation and culture techniques using a donor sample obtained by non-invasive methods. To date, the different donor-specific cells that can be isolated from blood, skin, and hair require invasive methods for sample isolation and incorporate complex and costly reagents to culture. These cells also take considerable time for their in-vitro isolation and expansion. Previous studies suggest that donor-derived cells, namely urine stem cells and renal cells, may be isolated from human urine samples using a cost-effective and simple method of isolation, incorporating not such complex reagents. Moreover, the isolated cells, particularly urine stem cells, are superior to conventional stem cell sources in terms of favourable gene profile and inherent multipotent potential. Transdifferentiation or differentiation of human urine-derived cells can generate desirable cells for regenerative therapy. In this review, we intended to discuss the characteristics and therapeutic applications of urine-derived cells for human cell therapy. Conclusively, with detailed study and optimisation, urine-derived cells have a prospective future to generate functional lineage-specific cells for patients from a clinical translation point of view.

Small Molecules for Neural Stem Cell Induction.

Generation of induced pluripotent stem cells (iPSCs) from other somatic cells has provided great hopes for transplantation therapies. However, these cells still cannot be used for clinical application due to the low reprogramming and differentiation efficiency beside the risk of mutagenesis and tumor formation. Compared to iPSCs, induced neural stem cells (iNSCs) are easier to terminally differentiate into neural cells and safe; thus, iNSCs hold more opportunities than iPSCs to treat neural diseases. On the other hand, recent studies have showed that small molecules (SMs) can dramatically improve the efficiency of reprogramming and SMs alone can even convert one kind of somatic cells into another, which is much safer and more effective than transcription factor-based methods. In this study, we provide a review of SMs that are generally used in recent neural stem cell induction studies, and discuss the main mechanisms and pathways of each SM.