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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



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)



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



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.



SP cells in rat bone marrow

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Without verapamil With verapamil



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



Chronic

Damage

Acute

Damage

Histopathological and Biochemical Changes in

Acute and Chronic damage to the liver



Chronic Liver failure and Repopulation FISH images

Negative

Control

(Female)

Positive

Control

(Male)

SP Cell

Transplant

Total Bone

marrow

Transplant



Recovery of serum biochemical parameters after

cell therapy



Damaged

Liver

After TBMC

transfer

After SP cell

transfer

Recovery of histopathological parameters after

cell therapy in acute liver damage



Damaged

Liver

After TBMC

transfer

After SP cell

transfer

Recovery of histopathological parameters after

cell therapy in chronic liver damage



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



Phase Contrast Microscopy of Fetal liver cells

Unsorted

cells

EpC AM +

cells

Ep C AM -

cells



Flow cytometric analysis of expression of various markers

In different types of EpCAM+ cells

Group I Group II



Expression of CK 18 and albumin

in EpCAM+ cells

CK18 Albumin



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.

surgeon workshop gp 28-08-10

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