Basic Study
Copyright ©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Hepatol. Oct 27, 2022; 14(10): 1884-1898
Published online Oct 27, 2022. doi: 10.4254/wjh.v14.i10.1884
Long-term and non-invasive in vivo tracking of DiD dye-labeled human hepatic progenitors in chronic liver disease models
Chaturvedula Tripura, Srinivas Gunda, Sandeep Kumar Vishwakarma, Avinash Raj Thatipalli, Jedy Jose, Mahesh Kumar Jerald, Aleem Ahmed Khan, Gopal Pande
Chaturvedula Tripura, Srinivas Gunda, Avinash Raj Thatipalli, Jedy Jose, Mahesh Kumar Jerald, Gopal Pande, Cell and Stem Cell Biology, CSIR-Centre for Cellular and Molecular Biology, Hyderabad 500007, Telangana, India
Sandeep Kumar Vishwakarma, Aleem Ahmed Khan, Central Laboratory for Stem Cell Research and Translational Medicine, Centre for Liver Research and Diagnostics, Deccan College of Medical Sciences, Hyderabad 500058, Telangana, India
Author contributions: Khan AA, Pande G, and Tripura C were responsible for the study concept, design, and supervision; Thatipalli AR, Vishwakarma SK, Jose J, and Jerald MK performed the experiments; Tripura C, and Gunda S were responsible for data acquisition and analysis; Tripura C also performed data organization and manuscript writing; Tripura C, Vishwakarma SK, Khan AA, and Pande G performed editing and revision of the manuscript draft.
Supported by Department of Science and Technology (DST), Ministry of Science and Technology, Govt. of India and Indian Council of Medical Research (ICMR), New Delhi, Govt. of India Grants to GP, No. GAP-0220 and No. GAP-0383.
Institutional review board statement: The total fetal liver cells (tFLCs) isolation protocol was approved by the Institutional Ethics Committee (IEC) of Deccan College of Medical Sciences (DCMS), Hyderabad.
Institutional animal care and use committee statement: The study was approved by the Institutional Animal Ethics Committee (Animal trial registration number 20/1999/CPCSEA dated 10/03/1999) of CCMB.
Informed consent statement: Informed consent was obtained prior to sample collection, and cell processing was performed according to the ethical guidelines for the use of human cells.
Conflict-of-interest statement: Dr. Tripura reports grants from the Department of Science and Technology (DST, Ministry of Science and Technology, Govt. of India and Indian Council of Medical Research (ICMR), New Delhi, Govt. of India Grants No. GAP-0220 and No. GAP-0383 to GP during the study period.
Data sharing statement: Data will be shared on request.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Corresponding author: Chaturvedula Tripura, PhD, Senior Scientist, Cell and Stem Cell Biology, CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Habsiguda, Hyderabad 500007, Telangana, India.
Received: July 26, 2022
Peer-review started: July 26, 2022
First decision: August 18, 2022
Revised: September 2, 2022
Accepted: October 2, 2022
Article in press: October 2, 2022
Published online: October 27, 2022

Chronic liver diseases (CLD) are the major public health burden due to the continuous increasing rate of global morbidity and mortality. The inherent limitations of organ transplantation have led to the development of stem cell-based therapy as a supportive and promising therapeutic option. However, identifying the fate of transplanted cells in vivo represents a crucial obstacle.


To evaluate the potential applicability of DiD dye as a cell labeling agent for long-term, and non-invasive in vivo tracking of transplanted cells in the liver.


Magnetically sorted, epithelial cell adhesion molecule positive (1 × 106 cells/mL) fetal hepatic progenitor cells were labeled with DiD dye and transplanted into the livers of CLD-severe combined immunodeficiency (SCID) mice. Near-infrared (NIR) imaging was performed for in vivo tracking of the DiD-labeled transplanted cells along with colocalization of hepatic markers for up to 80 d. The existence of human cells within mouse livers was identified using Alu polymerase chain reaction and sequencing.


NIR fluorescence imaging of CLD-SCID mice showed a positive fluorescence signal of DiD at days 7, 15, 30, 45, 60, and 80 post-transplantation. Furthermore, positive staining of cytokeratin, c-Met, and albumin colocalizing with DiD fluorescence clearly demonstrated that the fluorescent signal of hepatic markers emerged from the DiD-labeled transplanted cells. Recovery of liver function was also observed with serum levels of glutamic-oxaloacetic transaminase, glutamate-pyruvate transaminase, and bilirubin. The detection of human-specific Alu sequence from the transplanted mouse livers provided evidence for the survival of transplanted cells at day 80.


DiD-labeling is promising for long-term and non-invasive in vivo cell tracking, and understanding the regenerative mechanisms incurred by the transplanted cells.

Keywords: Chronic liver diseases, Cell transplantation, Cell tracking and imaging, DiD, Hepatic progenitors

Core Tip: Non-invasive tracking of transplanted cells is crucial to understand the homing, distribution, and differentiation into the desired cell types contributing to organ regeneration. Lipophilic fluorescent dye DiD-labeled fetal hepatic progenitor cells (fHPCs) were transplanted into the livers of mice with chronic liver diseases. DiD labeling of cells enabled long-term and non-invasive tracking of transplanted cells in vivo up to 80 d. Immunostaining and colocalization using liver-specific markers with DiD confirmed the persistence of transplanted cells in mice liver post-transplantation. Transplanted fHPCs supported liver function recovery, while identification of the Alu gene sequence revealed survival and engraftment of human cells within the mouse liver.