Dr. Shaji R Velayudhan

Career Interests

Awards

• MSc: Marine Biotechnology Goa University (1991-1993).
• PhD: Christian Medical College, Vellore (1995-2001).
• Post Doctoral Research Fellowship:  St Jude Children's Research Hospital, Memphis (2004-2006).
• Post Doctoral Research Fellowship: University of Chicago, Chicago.

Disease Modelling Using Induced Pluripotent Stem Cells (iPSCs):

Our laboratory is involved in disease modeling using iPSCs for understanding the molecular basis of disease pathogenesis and for drug screening on the differentiated cells derived from disease-specific iPSCs. Recently, we generated iPSC lines from the fibroblasts of a patient with Fanconi anemia (FA) by nonintegrative methods, after complementing the defective gene with a normal gene using a doxycycline-inducible lentiviral vector. The iPSCs generated were completely characterized for their pluripotency and, in the absence of doxycycline, these cells showed the cellular phenotypes of FA including cell cycle arrest, lack of FANCD2 monoubiquitination and absence of co-localization of FANCD2 and gamma H2AX. Currently, we are using these iPSC lines to understand the molecular basis of Fanconi Anaemia disease pathogenesis. In addition to FA, using a similar approach, we are creating disease models for Congenital Dyserythropoietic Anaemia (CDA) and Diamond Blackfan Anaemia (DBA).

Fig 1: Establishment of IndC-FANCA-iPSC line that exhibits the FA cellular phenotypes. (A) Schematic of molecular diagnosis of FANCA deficiency and iPSC generation. (B) Morphology of iPSCs grown on feeder layers (upper figure) and feeder-free vitronectin coated plates (lower figure). (C) Immunofluorescence analysis of NANOG, OCT4, SOX2, SSEA-4, TRA-1-81 and TRA-1-60. (D) Real-time PCR analysis of mRNA levels of NANOG, OCT4, SOX2, GDF3 AND DNMT3B, relative to the BC1 control iPSC line (data represented as mean±SD). (E) H&E-staining of teratoma sections showing trilineage differentiation; hc-hyaline cartilage (mesoderm), ne-neuroepithelium (ectoderm) and ce-columnar epithelium (endoderm). (F) Chromosome analysis showing normal male karyotype. (G) Western blot analysis of the cells treated with Hydroxyurea showing the lack of FANCD2-monoubiquitination in the absence of DOX and FANCA expression. L and S indicate the larger monoubiquitinated and the smaller unmodified FANCD2 bands, respectively. (H) Immunofluorescence analysis showing nuclear foci formed by ?H2A.X and FANCD2. The nuclei are stained with DAPI (violet colour). In DOX+ cells, colocalization of the foci formed by ?H2A.X and FANCD2 are seen as yellow spots due to the merging of green spots of ?H2A.X foci and red spots of FANCD2 foci, and in DOX- cells, only green spots of ?H2A.X foci are seen. (I) EdU incorporation based cell cycle analysis showing a spontaneous progressive G2/M arrest in the absence of DOX and FANCA expression for two passages. (J) Alkaline phosphatase staining showing spontaneous progressive exhaustion of iPSCs in the absence of DOX and FANCA expression for two passages.

Induced pluripotent stem cells:

Introduction of four transcription factors Oct-4, Sox2, Klf4 and c-Myc into adult cells can covert them to pluripotent stem cells. These induced pluripotent stem cells (iPSCs) exhibit features characteristic of embryonic stem (ES) cells. These cells have exciting new prospects for biomedical research, drug discovery and for regenerative medicine. Our lab is interested in understanding the mechanism of the reprogramming process and the use of these cells to understand the pathophysiology of human diseases.

A. miPSCs exhibited mouse embryonic stem cell like morphology and high level of expression of alkaline phosphatase (AP). Expression of other pluripotency markers SSEA-1, Oct4 and Nanog were examined by immunofluorescence.

B. miPSCs formed embryoid bodies and could be differentiated into lineages from all three germ layers. Immunostaining for (i) alpha-fetoprotein (AFP; endoderm), smooth muscle actin (SMA; mesoderm) and b3 tubulin (ectoderm) are shown.

Mechanism of globin gene regulation

During human development, three different types of hemoglobins are produced; embryonic, fetal and adult hemoglobins. In this project we are trying to understand the roles of transcription factors and epigenetics in developmental stage specific regulation of globin genes. We are also trying to understand the regulatory sequences in the globin clusters that help in stage specific expression of these genes. We are carrying out ex-vivo differentiation of haematopoietic stem cells to erythroid lineage and studying interaction of proteins with the DNA sequences using chromatin immunoprecipitation followed by microarray (ChIP-chip) to understand the roles of transcription factors and epigenetic changes in the regulation of globin genes.

Selected Publications

• Raina K, Joshi G, Modak K, Premkumar C, Priyanka S, Rajesh P, Velayudhan SR,Thummer RP. Generation and characterization of induced pluripotent stem cell line IITGi001-A derived from adult human primary dermal fibroblasts. Stem Cell Res. 2023 Jun 28;71:103159. doi: 10.1016/j.scr.2023.103159.
• Rajamani BM, Illangeswaran RSS, Benjamin ESB, Balakrishnan B, Jebanesan DZP, Das S, Pai AA, Vidhyadharan RT, Mohan A, Karathedath S, Abraham A, Mathews V, Velayudhan SR, Balasubramanian P. Modulating retinoid-X-receptor alpha (RXRA)expression sensitizes chronic myeloid leukemia cells to imatinib in vitro and reduces disease burden in vivo. Front Pharmacol. 2023 May 31;14:1187066.
• Venkatesan V, Christopher AC, Rhiel M, Azhagiri MKK, Babu P, Walavalkar K, Saravanan B, Andrieux G, Rangaraj S, Srinivasan S, Karuppusamy KV, Jacob A, Bagchi A, Pai AA, Nakamura Y, Kurita R, Balasubramanian P, Pai R, Marepally SK, Mohankumar KM, Velayudhan SR, Boerries M, Notani D, Cathomen T, Srivastava A, Thangavel S. Editing the core region in HPFH deletions alters fetal and adult globin expression for the treatment of β-hemoglobinopathies. Mol Ther Nucleic Acids. 2023 Apr 26;32:671-688. doi: 10.1016/j.omtn.2023.04.024. PMID: 37215154; PMCID: PMC10197010.
• Benjamin ES, Babu B, Joshi G, Rajamani BM, Nandy K, Rani S, Anandhan S, PremKumar C, Maddali M, Abraham A, Velayudhan SR*, Balasubramanian P* (*Corresponding Authors) Inhibition of BCR::ABL1 tyrosine kinase activity Aids in the Generation of Stable Chronic Myeloid Leukemia Induced Pluripotent Stem Cells. bioRxiv.
• Illangeswaran RSS, Jebanesan DZP, Sivakumar KK, Vidhyadharan RT, Rajamani BM, Janet NB, David E, Velayudhan SR, Mathews V, Balasubramanian P. Chemotherapeutic drugs elicit stemness and metabolic alteration to mediate acquired drug-resistant phenotype in acute myeloid leukemia cell lines. Leuk Res. 2023 May;128:107054.
• Joshi G, Arthur NBJ, Geetha TS, Datari PVR, Modak K, Roy D, Chaudhury AD,Sundaraganesan P, Priyanka S, Na F, Ramprasad V, Abraham A, Srivastava VM,Srivastava A, Kulkarni UP, George B, Velayudhan SR. Comprehensive laboratory diagnosis of Fanconi anaemia: comparison of cellular and molecular analysis. J Med Genet. 2023 Mar 9:jmedgenet-2022-108714.
• George A, Ravi NS, Prasad K, Panigrahi L, Koikkara S, Rajendiran V, Devaraju N, Paul J, Pai AA, Nakamura Y, Kurita R, Balasubramanian P, Thangavel S, Marepally S, Velayudhan SR, Srivastava A, Mohankumar KM. Efficient and error-free correction of sickle mutation in human erythroid cells using prime editor-2. Front Genome Ed. 2022 Dec 20;4:1085111.
• Dahariya S, Raghuwanshi S, Thamodaran V, Velayudhan SR, Gutti RK. Role of Long Non-Coding RNAs in Human-Induced Pluripotent Stem Cells Derived Megakaryocytes: A p53, HOX Antisense Intergenic RNA Myeloid 1, and miR-125b Interaction Study. J Pharmacol Exp Ther. 2023 Jan;384(1):92-101.
• Bagchi A, Devaraju N, Chambayil K, Rajendiran V, Venkatesan V, Sayed N, Pai AA, Nath A, David E, Nakamura Y, Balasubramanian P, Srivastava A, Thangavel S,Mohankumar KM, Velayudhan SR. Erythroid lineage-specific lentiviral RNAi vectors suitable for molecular functional studies and therapeutic applications. Sci Rep.2022 Aug 18;12(1):14033.
• Das S, Stallon Illangeswaran RS, Ijee S, Kumar S, Velayudhan SR, Balasubramanian P. Pooled shRNA Library Screening to Identify Factors that Modulate a Drug Resistance Phenotype. J Vis Exp. 2022 Jun 17;(184).
• Mahalingam G, Rachamalla HK, Arjunan P, Periyasami Y, M S, Thangavel S, Mohankumar KM, Moorthy M, Velayudhan SR, Srivastava A, Marepally S. Optimization of SARS-CoV-2 Pseudovirion Production in Lentivirus Backbone With a Novel Liposomal System. Front Pharmacol. 2022 Mar 25;13:840727.
• Ravi NS, Wienert B, Wyman SK, Bell HW, George A, Mahalingam G, Vu JT, Prasad K, Bandlamudi BP, Devaraju N, Rajendiran V, Syedbasha N, Pai AA, Nakamura Y,Kurita R, Narayanasamy M, Balasubramanian P, Thangavel S, Marepally S, Velayudhan SR, Srivastava A, DeWitt MA, Crossley M, Corn JE, Mohankumar KM.Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin. Elife. 2022 Feb 11;11:e65421.
• Nath A, Rayabaram J, Ijee S, Bagchi A, Chaudhury AD, Roy D, Chambayil K,Singh J, Nakamura Y, Velayudhan SR. Comprehensive Analysis of microRNAs in Human Adult Erythropoiesis. Cells. 2021 Nov 4;10(11):3018.
• Christopher AC, Venkatesan V, Karuppusamy KV, Srinivasan S, Babu P, Azhagiri MKK, Chambayil K, Bagchi A, Rajendiran V, Ravi NS, Kumar S, Marepally SK, Mohankumar KM, Srivastava A, Velayudhan SR, Thangavel S. Preferential Expansion of Human CD34+CD133+CD90+ Hematopoietic Stem Cells Enhances Gene-Modified Cell Frequency for Gene Therapy. Hum Gene Ther. 2022 Feb;33(3-4):188-201.
• Singh G, Manian KV, Premkumar C, Srivastava A, Daniel D, Velayudhan SR.Derivation of Clinical-Grade Induced Pluripotent Stem Cell Lines from Erythroid Progenitor Cells in Xenofree Conditions. Methods Mol Biol. 2022;2454:775-789.
• Thamodaran V, Rani S, Velayudhan SR. Gene Editing in Human Induced Pluripotent Stem Cells Using Doxycycline-Inducible CRISPR-Cas9 System. Methods Mol Biol. 2022;2454:755-773.
• Bagchi A, Nath A, Thamodaran V, Ijee S, Palani D, Rajendiran V, Venkatesan V, Datari P, Pai AA, Janet NB, Balasubramanian P, Nakamura Y, Srivastava A,Mohankumar KM, Thangavel S, Velayudhan SR. Direct Generation of Immortalized Erythroid Progenitor Cell Lines from Peripheral Blood Mononuclear Cells. Cells. 2021 Mar 1;10(3):523.
• Benjamin ESB, Ravindra N, Rajamani BM, Anandan S, Kausalya B, Veldore V, Mathews V, Velayudhan SR, Balasubramanian P. BCR-ABL1 kinase domain mutation analysis by next-generation sequencing detected additional mutations in chronic myeloid leukemia patients with suboptimal response to imatinib. Leuk Lymphoma. 2021 Jun;62(6):1528-1531.
• Ravindra N, Athiyarath R, S E, S S, Kulkarni U, N A F, Korula A, Shaji RV, George B, Edison ES. Novel frameshift variant (c.409dupG) in SLC25A38 is a common cause of congenital sideroblastic anaemia in the Indian subcontinent. J Clin Pathol. 2021 Mar;74(3):157-162.
• Arunachalam AK, Suresh H, Edison ES, Korula A, Aboobacker FN, George B, Shaji RV, Mathews V, Balasubramanian P. Screening of genetic variants in ELANE mutation negative congenital neutropenia by next generation sequencing. J Clin Pathol. 2020 Jun;73(6):322-327.
• Sullivan S, Stacey GN, Akazawa C, Aoyama N, Baptista R, Bedford P, Bennaceur Griscelli A, Chandra A, Elwood N, Girard M, Kawamata S, Hanatani T, Latsis T, Lin S, Ludwig TE, Malygina T, Mack A, Mountford JC, Noggle S, Pereira LV, Price J, Sheldon M, Srivastava A, Stachelscheid H, Velayudhan SR, Ward NJ, Turner ML, Barry J, Song J. Quality control guidelines for clinical-grade human induced pluripotent stem cell lines. Regen Med. 2018 Oct;13(7):859-866.
• Manian KV, Bharathan SP, Maddali M, Srivastava VM, Srivastava A, Velayudhan SR. Generation of an integration-free iPSC line (CSCRi005-A) from erythroid progenitor cells of a healthy Indian male individual. Stem Cell Res. 2018 May;29:148-151.
• Fouzia NA, Edison ES, Lakshmi KM, Korula A, Velayudhan SR, Balasubramanian P, Abraham A, Viswabandya A, George B, Mathews V, Srivastava A. Long-term outcome of mixed chimerism after stem cell transplantation for thalassemia major conditioned with busulfan and cyclophosphamide. Bone Marrow Transplant. 2018 Feb;53(2):169-174.
• Karathedath S, Rajamani BM, Musheer Aalam SM, Abraham A, Varatharajan S, Krishnamurthy P, Mathews V, Velayudhan SR, Balasubramanian P. Role of NF-E2 related factor 2 (Nrf2) on chemotherapy resistance in acute myeloid leukemia (AML) and the effect of pharmacological inhibition of Nrf2. PLoS One. 2017 May 15;12(5):e0177227.
• Bharathan SP, Nandy K, Palani D, Janet A NB, Natarajan K, George B, Srivastava A, Velayudhan SR. Generation of an induced pluripotent stem cell line that mimics the disease phenotypes from a patient with Fanconi anemia by conditional complementation. Stem Cell Res. 2017 Apr;20:54-57.
• Dharmalingam P, Marrapu B, Voshavar C, Nadella R, Rangasami VK, Shaji RV, Abbas S, Prasad RBN, Kaki SS, Marepally S. An anti-oxidant, α-lipoic acid conjugated oleoyl-sn-phosphatidylcholineas a helper lipid in cationic liposomal formulations. Colloids Surf B Biointerfaces. 2017 Apr 1;152:133-142.
• Bharathan SP, Manian KV, Aalam SM, Palani D, Deshpande PA, Pratheesh MD, Srivastava A, Velayudhan SR. Systematic evaluation of markers used for the identification of human induced pluripotent stem cells. Biol Open. 2017 Jan 15;6(1):100-108.
• Srivastava A, Shaji RV. Cure for thalassemia major - from allogeneic hematopoietic stem cell transplantation to gene therapy. Haematologica. 2017 Feb;102(2):214-223.
• Aalam SM, Manian KV, Bharathan SP, Mayuranathan T, Velayudhan SR. Identification of Stable OCT4+NANOG- State in Somatic Cell Reprogramming. Cell Reprogram. 2016 Nov;18(6):367-368.
• Kamath MS, Pradhan S, Edison ES, Velayudhan SR, Antonisamy B, Karthikeyan M, Mangalaraj AM, Kunjummen A, George K. Chorionic villous sampling through transvaginal ultrasound approach: A retrospective analysis of 1138 cases. J Obstet Gynaecol Res. 2016 Oct;42(10):1229-1235.

Pre-clinical lentiviral gene therapy

Dr. Alok Srivastava, Centre for Stem Cell Research, CMC, Vellore.

Disease modelling of haematological disorders using iPSCs

Dr. Biju George, Department of Haematology, CMC, Vellore.

Dr. Poonkuzhali Balasubramanian, Department of Haematology, CMC, Vellore.

• Kannan V Manian

  PhD Student (2009-2017)

  Current position: Post Doctoral Fellow

  Ocular Genomics Institute, Harvard Medical School

  Boston, Massachusetts, USA.

• Janakiram Rayabaram

  PhD Student (2009-2015)

  Current position: Scientist - I, Aurigene Discovery Technologies Limited, Bangalore.

• Nancy Beryl Janette

  PhD Student (2009-2015)

  Current position: Lecturer, Department of Haematology, Christian Medical College, Vellore.

• Syed Mohammed Musheer Aalam

  PhD Student (2009-2016)

  Current position: Post doctoral research fellow, Mayo clinic, Rochester.

• Thiyagaraj M

  PhD Student (2009-2016)

  Current position: Post doctoral research fellow, St Jude Children's research hospital, Memphis.

• Sumitha Prameela Bharathan

  PhD Student (2009-2016)

  Current position:Post Doctoral Research Fellow

  Ophthalmology, Children’s Hospital Los Angeles

  Los Angeles, California, USA.

• Aneesha Nath

  PhD Student (2014-2020)

  Current position: Post Doctoral Fellow

  Department of Pediatrics, University of Florida.

• Vasanth Thamodaran

  Post-Doctoral Research (2017-2020)

  Current position: Senior Scientist

  Tata Institute for Genetics and Society

  NCBS, Bengaluru, India.

• Abhirup Bagchi

  PhD Student (2017-2023)

  Current position: Post-Doctoral Research

  Department of Paediatrics

  Perelman School of Medicine

  University of Pennsylvania, US

"Contents will be updated soon."