Mesenchymal Stem Cells or Marrow Stromal Cells (MSCs) have long been viewed as a potent tool for regenerative cell therapy. MSCs are easily accessible from both healthy donor and patient tissue and expandable in vitro on a therapeutic scale without posing significant ethical or procedural problems. MSC based therapies have proven to be effective in preclinical studies for graft versus host disease, stroke, myocardial infarction, pulmonary fibrosis, autoimmune disorders and many other conditions and are currently undergoing clinical trials at a number of centers all over the world. MSCs are also being extensively researched as a therapeutic tool against neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Amyotrophic Lateral Sclerosis (ALS), Huntington's disease (HD) and Multiple Sclerosis (MS). MSCs have been discussed with regard to two aspects in the context of neurodegenerative diseases: their ability to transdifferentiate into neural cells under specific conditions and their neuroprotective and immunomodulatory effects. When transplanted into the brain, MSCs produce neurotrophic and growth factors that protect and induce regeneration of damaged tissue. Additionally, MSCs have also been explored as gene delivery vehicles, for example being genetically engineered to over express glial-derived or brain-derived neurotrophic factor in the brain. Clinical trials involving MSCs are currently underway for MS, ALS, traumatic brain injuries, spinal cord injuries and stroke. In the present review, we explore the potential that MSCs hold with regard to the aforementioned neurodegenerative diseases and the current scenario with reference to the same.

Transcriptional recording by CRISPR spacer acquisition from RNA endows engineered Escherichia coli with synthetic memory, which through Record-seq reveals transcriptome-scale records. Microbial sentinels that traverse the gastrointestinal tract capture a wide range of genes and pathways that describe interactions with the host, including quantitative shifts in the molecular environment that result from alterations in the host diet, induced inflammation, and microbiome complexity. We demonstrate multiplexed recording using barcoded CRISPR arrays, enabling the reconstruction of transcriptional histories of isogenic bacterial strains in vivo. Record-seq therefore provides a scalable, noninvasive platform for interrogating intestinal and microbial physiology throughout the length of the intestine without manipulations to host physiology and can determine how single microbial genetic differences alter the way in which the microbe adapts to the host intestinal environment.

No abstract

It is difficult to elucidate the transcriptional history of a cell using current experimental approaches as they are destructive in nature and therefore only describe a moment in time. Overcoming these limitations, we recently established Record-seq, a technology that enables transcriptional recording by CRISPR spacer acquisition from RNA. The recorded transcriptomes are recovered by SENECA, a method that selectively amplifies expanded CRISPR arrays, followed by deep sequencing. The resulting CRISPR spacers are aligned to the host genome, thereby enabling transcript quantification and associated analyses. Here, we describe the experimental procedures of the Record-seq workflow as well as subsequent data analysis. Beginning with the experimental design, Record-seq data can be obtained and analyzed within 1-2 weeks. CRISPR spacer acquisition from RNAIn order to overcome prior limitations and enable molecular recording and DNA writing on a massive scale we developed Record-seq 17 , an approach that leverages CRISPR spacer acquisition from RNA. This was initially achieved using Cas1 and Cas2 orthologs derived from the type III CRISPR system of the human commensal bacterium Fusicatenibacter saccharivorans. The most important hallmark of the Cas1-Cas2 complex in this species is the naturally occurring fusion of Cas1 to a reverse transcriptase (RT-Cas1). Spacer acquisition from RNA presumably occurs through the binding of RT-Cas1-Cas2 to intracellular singlestranded RNA molecules and subsequent ligation of this RNA into the CRISPR array followed by reverse transcription 14 .Due to the low spacer acquisition frequencies in RNA-adapting CRISPR systems, compared to their DNA adapting counterparts, RNA spacer acquisition had not been used for DNA writing applications before the development of Record-seq. Leveraging overexpression of FsRT-Cas1-Cas2 in an E. coli host and using a newly developed protocol for the selective PCR amplification of expanded CRISPR arrays, Record-seq overcomes this low efficiency limitation and thus enables the direct recording of intracellular RNAs. Utilizing this technology, we demonstrated the recording of stimuli that transiently altered the cellular transcriptome but remained refractory to detection by traditional RNA-seq. Development of the Record-seq protocolHere we describe a detailed protocol for recording transcriptional events into plasmid-borne CRISPR arrays, named Record-seq. During bacterial growth, RNA-derived spacers are integrated into plasmid-encoded CRISPR arrays and reverse transcribed by the RT domain fused to Cas1, thereby achieving permanent storage of transcriptional events in the form of plasmid DNA (Figure 1a). The Record-seq protocol entails extracting this plasmid DNA and retrieving the spacer sequences by means of selective amplification, size selection, and deep sequencing (Figure 1b). Since only a small fraction of CRISPR arrays acquire new spacers during the course of the experiment, Record-seq incorporates a newly developed procedure to selectively amplify newly acqu...

No abstract