Manipal Academy of Higher Education 6 articles published in JoVE Biology Single-Cell Sorting of Immunophenotyped Mesenchymal Stem Cells from Human Exfoliated Deciduous Teeth Ayona Gupta*1, Risani Mukhopadhyay*1, Himanshi Khandelwal1, Narendra Nala2, Uttara Chakraborty1 1Manipal Institute of Regenerative Medicine, Bengaluru, Manipal Academy of Higher Education, Manipal, 2HIV-AIDS Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR) This protocol describes the use of fluorescence-activated cell sorting of human mesenchymal stem cells using the single-cell sorting method. Specifically, the use of single-cell sorting can achieve 99% purity of the immunophenotyped cells from a heterogeneous population when combined with a multiparametric flow cytometry-based approach. Developmental Biology Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced Pluripotent Stem Cells Sudipta Mahato*1,2, Trupti Agrawal*1,2, Divya Pidishetty*1,2, Savitri Maddileti1, Vinay Kumar Pulimamidi1,3, Indumathi Mariappan1 1Centre for Ocular Regeneration, Prof. Brien Holden Eye Research Centre, LV Prasad Eye Institute, 2Manipal Academy of Higher Education, 3Schepens Eye Research Institute, Massachusetts Eye and Ear, Harvard Medical School This protocol describes an efficient method of differentiating hiPSCs into eye field clusters and generating neuro-retinal organoids using simplified culture conditions involving both adherent and suspension culture systems. Other ocular cell types, such as the RPE and corneal epithelium, can also be isolated from mature eye fields in retinal cultures. Biology Detection and Quantification of Tunneling Nanotubes Using 3D Volume View Images Deepak Kunhi Valappil*1, Abinaya Raghavan*1, Sangeeta Nath1 1Manipal Institute of Regenerative Medicine, Manipal Academy of Higher Education, India Tunneling nanotubes (TNTs) are primarily open-ended F-actin membrane nanotubes that connect neighboring cells, facilitating intercellular communication. The notable characteristic that distinguishes TNTs from other cell protrusions is the hovering nature of the nanotubes between cells. Here, we characterize TNTs by constructing a 3D volume view of confocal z-stack images. Biology CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications Vigneshwaran Venkatesan*1,2, Abisha Crystal Christopher*1,3, Karthik V. Karuppusamy1,2, Prathibha Babu1,2, Manoj Kumar K. Alagiri1,2, Saravanabhavan Thangavel1 1Centre for Stem Cell Research (CSCR), A unit of InStem Bengaluru, Christian Medical College campus, 2Manipal Academy of Higher Education, 3Thiruvalluvar University The present protocol describes an optimized hematopoietic stem and progenitor cell (HSPC) culture procedure for the robust engraftment of gene-edited cells in vivo. Bioengineering A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans Jyoti Dubey1,2,3,4, Sudip Mondal1,5, Sandhya P. Koushika2 1National Centre for Biological Sciences, TIFR, 2Department of Biological Sciences, TIFR, 3InSTEM, 4Manipal Academy of Higher Education, 5Department of Mechanical Engineering, The University of Texas at Austin The protocol describes a simple microfluidic chip design and microfabrication methodology used to grow C. elegans in presence of a continuous food supply for up to 36 h. The growth and imaging device also enables intermittent long-term high-resolution imaging of cellular and sub-cellular processes during development for several days. Developmental Biology Generation of Induced Pluripotent Stem Cells from Turner Syndrome (45XO) Fetal Cells for Downstream Modelling of Neurological Deficits Associated with the Syndrome Nivedha Veerasubramanian*1, Vaishnavi Karthikeyan*1, Sridevi Hegde2, Anandh Dhanushkodi1, Shagufta Parveen1 1Manipal Institute of Regenerative Medicine, Manipal Academy of Higher Education, Manipal, Karnataka, India-576104, 2Department of Medical Genetics, Manipal Hospitals This protocol describes the generation of integration free iPSCs from fetal tissue fibroblasts through delivery of episomal plasmids by nucleofection followed by description of methods used for iPSC characterization and neuronal differentiation.
