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Studies on liver regeneration with murine bonemarrow and human fetal liver cells
Gopal PandeCentre for Cellular and Molecular Biology
Uppal Road Hyderbad 500 007
Workshop of Indian Society of Pediatric Surgeons
CCMB, Hyderabad August 28, 2010
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Acknowledgements
CCMB
M. Subbarao, C V Saritha, M.Gerald MaheshKumar, T
Avinash Raj and G Srinivas
DCMS
Mohammed Aleem and C M Habibullah
Funding: CSIR Network Program on Cell and Tissue Engineering
(CMM002)
Indian Council of Medical Research
Department of Science and Technology (DST) Government
of India (CMM0220)
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Publications
Khan, AA, Parveen N, Vali, MS, Rajendraprasad A, Ravindraprakash, Venkateswarlu J,
Rao P, Pande G, Narusu, ML, Khaja MN, Pramila, Habeeb A and Habibullah CM. (2008)
Treatment of Crigler-Najjar Syndrome Type 1 by Hepatic Progenitor Cell Transplantation: A Simple Procedure for Management of Hyperbilirubinemia.
Transplantation Proceedings 40:1148-1150.
Khan AA, Parveen N, Mahaboob VS, Rajendraprasad A, Ravindraprakash HR, Venkateswarlu J, Rao P, Pande G,
Narusu ML, Khaja MN, Pramila R, Habeeb A, Habibullah CM. (2008) Management of hyperbilirubinemia in
biliary atresia by hepatic progenitor cell transplantation through hepatic arter y: a case report.
Transplant Proc. 40:1153-1155
Mekala, S., Khan, A. A., Nyamath, P. and Habibullah, C.M. Pande, G., (2008) Characterization of
EpC AM positive enriched hepatic progenitors from human fetal liver during second trimester
W orld Jour. Gastero. 14: 5617-5780.
Satyavani R, Fatima A, Sundaram CS, Anabalagan Ch, Saritha CV, Srinivas G, Khan AA,
Habibullah CM and Pande G. (2009) Proteomic Analysis Of The Side Population (SP) Cells FromMurine Bone Marrow. J Proteomics & Bioinformatics 2: 398-407.
Khan A. A., Shaik M V, Parveen N, Rajendraprasad A, Aleem MA, Habeeb M A,
Srinivas G, Avinash Raj T, Santosh K Tiwari, Kumaresan K, Venkateswarlu J,
Pande G.*, Habibullah CM (2010) Human fetal liver derived stem cell transplantation
as supportive modality in the management of end stage decompensated liver
cirrhosis. Cell Transplantation 19:409-418
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Sorting and characterization of SP Cells
Prepare cells in a suspension- bone marrow cells can be flushed
and suspended directly; from other tissues they need to be
treated with appropriate enzymes and then suspended.
Cells at 1-5X106 /ml are stained with 2-5Qg/ml Hoechst33342 at
37ºC for 45 mins and washed.
Cells are analysed/sorted in a dual laser FACS Vantage SE cell
sorter using a UV laser at 355nm (150mW) and a visible laser at
488nm (50mW).
If the cell sorting is done in sterile conditions the sorted cells can
be used for colony in vitro culture and/or animal transplantation.
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SP cells in rat bone marrow
RBMC.001
0 200 400 600 800 1000
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CHST-RED
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Without verapamil With verapamil
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Female Wistar rats were used for the development of chronic liver
injury model by injection of carbon tetra chloride1ml / kg body weight
twice in a week for 5 weeks intraperitonially.
Acute liver failure model is generated by injection of D-galactosamine
hydrochloride (Sigma) 2-3 gm/kg body weight intraperitonially in
female rats .
A known number of SP cells or whole bone marrow cells were injected
in the affected animals intrahepatically. The recovery of liver damage
in the acute and chronic models were assessed using biochemical &
histological investigations.
Induction of liver specific damage
and cell transplantation
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Chronic
Damage
Acute
Damage
Histopathological and Biochemical Changes in
Acute and Chronic damage to the liver
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Chronic Liver failure and Repopulation FISH images
Negative
Control
(Female)
Positive
Control
(Male)
SP Cell
Transplant
Total Bone
marrow
Transplant
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Chronic Liver failure and Repopulation FISH images
Negative
Control
(Female)
Positive
Control
(Male)
SP Cell
Transplant
Total Bone
marrow
Transplant
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Recovery of serum biochemical parameters after
cell therapy
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Damaged
Liver
After TBMC
transfer
After SP cell
transfer
Recovery of histopathological parameters after
cell therapy in acute liver damage
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Damaged
Liver
After TBMC
transfer
After SP cell
transfer
Recovery of histopathological parameters after
cell therapy in chronic liver damage
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CONCLUSIONS
Viable SP cells can be isolated from the bone marrow withor without pre-enrichment of cells by other methods.
SP cells can be isolated in sterile condition for injectionsin syngenic rats and even SCID mice.
Small numbers of SP cells from bone marrow are capable
of repairing large scale of chronic and acute liver damageas determined by serum biochemistry and tissuehistopathology
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Phase Contrast Microscopy of Fetal liver cells
Unsorted
cells
EpC AM +
cells
Ep C AM -
cells
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Flow cytometric analysis of expression of various markers
In different types of EpCAM+ cells
Group I Group II
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Expression of CK 18 and albumin
in EpCAM+ cells
CK18 Albumin
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CONCLUSIONS
EpCAM+ve cells can be isolated from human fetal liversbetween 10-20 weeks of pregnancy.
EpCAM+ve cells show lesser expression of HLA and
hematopoetic markers as compared to EpCAM ±ve cells.
Our marker studies suggest that EpCAM+ve cells can be
used a source of doing cell based therapy of acutely (or
chronically) damaged human liver.