pooled-shrna-library-screening-to-identify-factors-that-modulate-drug

Pooled shRNA Library Screening to Identify Factors that Modulate a Drug Resistance Phenotype

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve ...

Thiruvalluvar University

Christian Medical College

Adult bone marrow niche contains rare primitive but highly functional multipotent progenitors (e.g., mesenchymal stem cells; MSCs) capable of differentiating into specific mesenchymal tissues like bone, cartilage, muscle, fat tissues, ligament, dermis, bone marrow stroma, tendon, and other connective tissues. Upon in vivo transplantation, MSCs also secrete a wide range of growth factors, immunomodulatory cytokines, and important bioactive macromolecules including cell-derived exosomes to structure regenerative microenvironments. This protocol describes a mouse model to study bone formation/regeneration from adult mesenchymal stem cells. 

Bone defect repair in mice by mesenchymal stem cells.

The Human mesenchymal stromal/stem cells (MSCs) have been isolated from various tissue sources. Yet, the lack of a distinctive marker for identifying in vivo MSCs in their tissue niche has hampered the MSC's in vivo behavior tracking and compared that to the in vitro expanded cultures. In this review, we present a comprehensive report on MSCs history, isolation from assorted tissue sources, classification, long-term cultures for comprehensively characterized MSCs, immunomodulation, regenerative medical applications, iMSCs as a novel source of patient-specific iPSCs and scaleup strategies for translational applications. We have emphasized on prenatal tissue-derived MSCs and iMSCs derived from hiPSCs as an effective alternative to adult MSCs. We also highlight the urgent requirement to revisit the initial criteria laid down by International Society for Cellular Therapy (ISCT) and propose more stringent criteria to define, identify and exclusively characterize the MSCs derived from various tissue sources using advanced molecular tools; also more international workshops are necessary for delineating unique features of MSCs. Unless the proposed goal is achieved, it is extremely difficult to realize the full potential of MSCs in translational applications. Although numerous patients have been tested with MSCs to date, no immediate adverse outcomes or infusion-related toxicity has been reported, suggesting MSCs infusion to be safe. However, rare adverse event and late complications of the treatment may be detected in large cohorts of patients with long-term follow-up. 

Quest for alternate personalized clinical source of MSCs: Advancing towards hiPSCs derived iMSCs.

Mesenchymal stromal cells (MSCs) and induced pluripotent stem cells (iPSCs) have stimulated much interest in the scientific community and hopes among the general public since their discovery in 1966 due to a variety of potential applications it has in the field of regenerative medicine. Copious amount of literature, as well as long-term animal and human clinical trials, indicates that MSCs can be successfully used for therapeutic purpose without any extreme adversities. MSCs have been isolated from adult and fetal tissues. Recently, MSCs from placenta have generated much inquisitiveness. In this article, we will demonstrate the step-by-step procedure for isolating human placental MSCs from term placenta, reprogramming of placental MSCs into iPSCs using plasmid vectors, evaluation of functional recovery in mice spinal cord injury models, and in vivo tracking of the transplanted cells.

Therapeutic Application of Placental Mesenchymal Stem Cells Reprogrammed Neurospheres in Spinal Cord Injury of SCID.

Mesenchymal stem cells (MSCs) are blossoming as a credible source for regenerative medical applications. The use of fetal MSCs is gaining momentum for therapeutic use. The ease of isolation, enhanced characteristics, and immunomodulation properties renders the utilization of fetal MSCs for numerous clinical applications. In this article, we will demonstrate a step-by-step protocol for isolation of Wharton's jelly MSCs (WJMSCs) from the human umbilical cord matrix, preparation of human platelet lysate, fabricating amniotic membrane scaffold and mice model to study skin regeneration using a combination of MSCs and decellularized amniotic membrane scaffold.

Therapeutic Application of Human Wharton Jelly Mesenchymal Stem Cells in Skin Injury of SCID.

Mesenchymal stem cells (MSCs) are derived from Wharton's jelly tissue of the human umbilical cord. Given appropriate culture conditions, these cells can self-renew and differentiate into multiple cell types across the lineages. Among the properties exhibited by these cells, immunomodulation through secretion of trophic factors has been widely exploited in a broad spectrum of preclinical/clinical regenerative applications. Moreover, the extracellular matrix is found to play a major role apart from niche cells in determining stem cell fate including that of MSCs. Therefore, the currently employed technique of two-dimensional culture expansion can alter the inherent properties of naïve MSCs originally residing within the three-dimensional space. This limitation can be overcome to some extent by using native extracellular matrix scaffold culture system which mimics the in situ microenvironment. In this chapter, we have elucidated the protocol for the preparation of a native extracellular matrix scaffold by decellularization of the MSC sheet and thereof culture expansion and characterization of human Wharton's jelly-derived MSCs.

3D Decellularized Native Extracellular Matrix Scaffold for In Vitro Culture Expansion of Human Wharton's Jelly-Derived Mesenchymal Stem Cells (hWJ MSCs).

This article reports the utilization of Malus domestica for the synthesis of silver nanoparticles (AgNPs) with cytotoxic activity against the Michigan Cancer Foundation-7 (MCF-7) cell line as well as their antibacterial and radical scavenging potential. The biosynthesized AgNPs were confirmed using various analytical characterization techniques. The cytotoxic effect of Malus domestica-AgNPs (M.d-AgNPs) was studied by MTT assay and scavenging efficacy was assessed by DPPH, nitric oxide radical and phosphomolybdate assays. Furthermore, green synthesized nanoparticles were evaluated for their antibacterial activity against multidrug resistant-clinical isolates. M.d-AgNPs were observed to be almost spherical in shape with an average diameter from 50 to 107.3 nm as assessed by TEM and DLS. M.d-AgNPs revealed the dose-dependent antioxidant activity and antimicrobial activity against multidrug-resistant bacterial strain viz. Enterobacter aerogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli. Also, in vitro studies revealed dose-dependent cytotoxic effects of M.d-AgNPs treated MCF-7 cell line. The data strongly suggest that M.d-AgNPs had a potential antioxidant, antimicrobial and cytotoxicity activity.

Green synthesis, characterization and antibacterial activity of silver nanoparticles by Malus domestica and its cytotoxic effect on (MCF-7) cell line.

Spinal cord injury is a devastating condition that is critically challenging and progressive, needing immediate medical attention due to its complex pathophysiology and affecting the social status and economic burden. Stem cell therapy has been the emerging therapeutic trend to treat various diseases for decades. Mesenchymal stem cells pose more advantages over other stem cells in immune-modulation, immune evasiveness, self-renewal, multipotency, etc. Due to various issues in the recent past related to allogenic transplants, ethical concerns in obtaining tissues and adult cells, host immune response, GMP grade production and certification, cell-derived products or cell secretome have proven to be a promising approach and have been implicated in many studies and also in many clinical trials. Utilization of these human MSC-derived exosomes/extracellular vesicles in spinal cord injury has also been demonstrated in many pre-clinical animal models. It is now proven to be therapeutically more efficient and safer than cell therapy. This review focuses on employing human MSC derived EVs for SCI and continues to elucidate the recent advances and emerging EVs trends from other cell types. We discuss biomaterial-based synergistic intervention, mention mimetics and nanovesicles and finally touch upon safety concerns in EV therapy.

Sanjay Kumar

Department of Biotechnology

Thiruvalluvar University

Pooled shRNA Library Screening to Identify Factors that Modulate a Drug Resistance Phenotype