In recent years, stem cell therapy has become a very promising and advanced scientific research topic. The development of treatment methods has evoked great expectations. This paper is a review focused on the discovery of different stem cells and the potential therapies based on these cells. The genesis of stem cells is followed by laboratory steps of controlled stem cell culturing and derivation. Quality control and teratoma formation assays are important procedures in assessing the properties of the stem cells tested. Derivation methods and the utilization of culturing media are crucial to set proper environmental conditions for controlled differentiation. Among many types of stem tissue applications, the use of graphene scaffolds and the potential of extracellular vesicle-based therapies require attention due to their versatility. The review is summarized by challenges that stem cell therapy must overcome to be accepted worldwide. A wide variety of possibilities makes this cutting edge therapy a turning point in modern medicine, providing hope for untreatable diseases.

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Stem cells: past, present, and future.

Exosomes are extracellular vesicles that contain a specific composition of proteins, lipids, RNA, and DNA. They are derived from endocytic membranes and can transfer signals to recipient cells, thus mediating a novel mechanism of cell-to-cell communication. They are also thought to be involved in cellular waste disposal. Exosomes play significant roles in various biological functions, including the transfer of biomolecules such as RNA, proteins, enzymes, and lipids and the regulation of numerous physiological and pathological processes in various diseases. Because of these properties, they are considered to be promising biomarkers for the diagnosis and prognosis of various diseases and may contribute to the development of minimally invasive diagnostics and next generation therapies. The biocompatible nature of exosomes could enhance the stability and efficacy of imaging probes and therapeutics. Due to their potential use in clinical applications, exosomes have attracted much research attention on their roles in health and disease. To explore the use of exosomes in the biomedical arena, it is essential that the basic molecular mechanisms behind the transport and function of these vesicles are well-understood. Herein, we discuss the history, biogenesis, release, isolation, characterization, and biological functions of exosomes, as well as the factors influencing their biogenesis and their technical and biological challenges. We conclude this review with a discussion on the future perspectives of exosomes.

Review of the Isolation, Characterization, Biological Function, and Multifarious Therapeutic Approaches of Exosomes

National Institute of General Medical Sciences (U.S.) (Center for Integrative Synthetic Biology Grant P50GM098792)

A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes

Long non-coding RNAs (lncRNAs) are involved in the pathology of various tumors, including colorectal cancer (CRC) The crosstalk between carcinoma- associated fibroblasts (CAFs) and cancer cells in the tumor microenvironment promotes tumor development and confers chemoresistance In this study, we further investigated the underlying tumor-promoting roles of CAFs and the molecular mediators involved in these processes Methods: The AOM/DSS-induced colitis-associated cancer (CAC) mouse model was established, and RNA sequencing was performed Small interfering RNA (siRNA) sequences were used to knock down H19 Cell apoptosis was measured by flow cytometry SW480 cells with H19 stably knocked down were used to establish a xenograft model The indicated protein levels in xenograft tumor tissues were confirmed by immunohistochemistry assay, and cell apoptosis was analyzed by TUNEL apoptosis assay RNA-FISH and immunofluorescence assays were performed to assess the expression of H19 in tumor stroma and cancer nests The AldeRed ALDH detection assay was performed to detect intracellular aldehyde dehydrogenase (ALDH) enzyme activity Isolated exosomes were identified by transmission electron microscopy, nanoparticle tracking and Western blotting Results: H19 was highly expressed in the tumor tissues of CAC mice compared with the expression in normal colon tissues The up-regulation of H19 was also confirmed in CRC patient samples at different tumor node metastasis (TNM) stages Moreover, H19 was associated with the stemness of colorectal cancer stem cells (CSCs) in CRC specimens H19 promoted the stemness of CSCs and increased the frequency of tumor-initiating cells RNA-FISH showed higher expression of H19 in tumor stroma than in cancer nests Of note, H19 was enriched in CAF-derived conditioned medium and exosomes, which in turn promoted the stemness of CSCs and the chemoresistance of CRC cells in vitro and in vivo Furthermore, H19 activated the β-catenin pathway via acting as a competing endogenous RNA sponge for miR-141 in CRC, while miR-141 significantly inhibited the stemness of CRC cells Conclusion: CAFs promote the stemness and chemoresistance of CRC by transferring exosomal H19 H19 activated the β-catenin pathway via acting as a competing endogenous RNA sponge for miR-141, while miR-141 inhibited the stemness of CRC cells Our findings indicate that H19 expressed by CAFs of the colorectal tumor stroma contributes to tumor development and chemoresistance

Carcinoma-associated fibroblasts promote the stemness and chemoresistance of colorectal cancer by transferring exosomal lncRNA H19

Exosomal lipid composition and the role of ether lipids and phosphoinositides in exosome biology

Extracellular Vesicles (EVs) are gaining interest as central players in liquid biopsies, with potential applications in diagnosis, prognosis and therapeutic guidance in most pathological conditions. These nanosized particles transmit signals determined by their protein, lipid, nucleic acid and sugar content, and the unique molecular pattern of EVs dictates the type of signal to be transmitted to recipient cells. However, their small sizes and the limited quantities that can usually be obtained from patient-derived samples pose a number of challenges to their isolation, study and characterization. These challenges and some possible options to overcome them are discussed in this review.

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Technical challenges of working with extracellular vesicles

Clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR-associated gene 9 (Cas9) genome editing is set to revolutionize genetic manipulation of pathogens, including kinetoplastids. CRISPR technology provides the opportunity to develop scalable methods for high-throughput production of mutant phenotypes. Here, we report development of a CRISPR-Cas9 toolkit that allows rapid tagging and gene knockout in diverse kinetoplastid species without requiring the user to perform any DNA cloning. We developed a new protocol for single-guide RNA (sgRNA) delivery using PCR-generated DNA templates which are transcribed in vivo by T7 RNA polymerase and an online resource (LeishGEdit.net) for automated primer design. We produced a set of plasmids that allows easy and scalable generation of DNA constructs for transfections in just a few hours. We show how these tools allow knock-in of fluorescent protein tags, modified biotin ligase BirA*, luciferase, HaloTag and small epitope tags, which can be fused to proteins at the N- or C-terminus, for functional studies of proteins and localization screening. These tools enabled generation of null mutants in a single round of transfection in promastigote form Leishmania major, Leishmania mexicana and bloodstream form Trypanosoma brucei; deleted genes were undetectable in non-clonal populations, enabling for the first time rapid and large-scale knockout screens.

/pdf/a-crispr-cas9-high-throughput-genome-editing-toolkit-for-337zvpn6rz.pdf

Laura Makin

Papers

Technical challenges of working with extracellular vesicles

A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids.

cAMP signalling in trypanosomatids: role in pathogenesis and as a drug target.