Human Induced Pluripotent Stem Cells (hiPSCs) An Innovative Approach for the treatment of Alpha 1 Antitrypsin Deficiency Nelofar Karim*, Kshama Kumari, Manoj Garg and Prakash Baligar Amity Institute of Molecular Medicine And Stem Cell Research (AIMMSCR) Amity University, Noida, Uttar Pradesh, India
Abstract Worldwide millions of patients are affected by liver disease, the one which is rarely diagnosed is an alpha1antitrypsin deficiency (AATD) caused by deficiency of normal human Alpha1 Antitrypsin ( 52kDa glycoprotein ,predominantly produced in the liver and released into the blood) and results into lung alterations like emphysema and bronchiectasis . At present, there are many proven treatments for AATD like Organ transplantation, Bioartificial liver (BAL), Augmentation, Gene therapy, Stem cell therapy, but each having its own limitations such as limited donor, augmentation is expensive and requires repeated administration, insufficient culturing of hepatic cells etc,. Therefore, the existing barriers called for a recent and innovative approach involving the use of Induced pluripotent stem cells (iPSCs). In this proposal, we made hiPSCs, from human foreskin fibroblast cells, which will be differentiated into human hepatic like cells secreting normal a1AT protein and overexpressing the wild type a1AT protein. Thus, the use of these engineered human hepatic like cells will reduce the existing globules which are formed by the misfolded a1AT protein. This approach will not only improve existing diseased liver state but also protects the lung from emphysema. Introduction & Concept Alpha1antitrypsin deficiency (AATD) is inherited in an autosomal recessive manner with codominant expression. In affected patients, circulating levels of mutant A1AT are 15% of the normal protein levels (1). Normal human Alpha1 Antitrypsin (A1AT) is a 52kDa glycoprotein, predominantly produced in the liver and released into the blood (2). There are many variants of the alpha1 antitrypsin. PiMM is the normal gene product whereas PiSS and PiZZ are the most common mutated gene products (3). Alpha1 antitrypsin (AAT), the most potent inhibitor of neutrophil elastase (NE), is coded by the SERPINA1 gene (formerly known as Pi) located on chromosome 14q. The AATD refers to the PiZZ* homozygous mutated variant of the normal human a1AT protein caused by a single basepair substitution encoding a glutamate lysine mutation at position 342 (4 ). Under mutated Image Courtesy : clean eating magazine
state
Hepatic cells secrete mutated A1AT protein (PiZZ)
Methodology Transfected 293T cells
XL blue strain
* Competent cells formation 20x Isolation of plasmid DNAs
40x after 48 hrs
Untransduced human foreskin fibroblast cells
Transformation of XL blue with 3 plasmid DNAs: PSPAX2, STEMCA, PMD2.G
Western blot of Transduced Human foreskin fibroblast cells for the expression of OCT 4 and SOX 2
*
Transfection into 293T cells
Hepatic cells secrete wild type A1AT protein
Released into the blood
Degrades NE and makes it unavailable to act on lungs ( elastin), thus protecting lungs as well
Transduced human foreskin fibroblast cells
Transduction into human foreskin fibroblast cells hiPSCs
20x
40x
At 72hrs
Results
Healthy liver and lungs
Absence of A1AT in the blood leads to availability of neutrophil elastase and degradation of elastin by it in lungs
Results in emphysema and diseased liver state
*
**
Following successful transfection and transduction into respective cell lines, as evident from the confirmatory expression of pluripotent genes ( Oct4 and Sox2) we have made hiPSCs, which can be used to treat AATD.
References
Transformed Colony Culture Plate
20x
40x At 216hrs
Western blot of Transfected 293T cells for the expression of OCT 4 and SOX 2 Untransfected 293T cells
** 20x
** Conclusion
Under normal conditions Not secreted into the blood and accumulates within E.R of hepatocytes leading to globule formation in the liver (liver stress and hepatocytes death)
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40x
* **
* ** transduced/ transfected cells
* un transduced/ untransfected cells
1.Khan, Z. (2016). Pathogenesis of Alpha-1 Antitrypsin Deficiency in the Liver: New Approaches to Old Questions. Journal of Liver Research, Disorders & Therapy, 2(2). 2.Serres, F. D., & Blanco, I. (2014). Role of alpha-1 antitrypsin in human health and disease. Journal of Internal Medicine, 276(4), 311-335. 3.Mitchell, E. L., & Khan, Z. (2017). Liver Disease in Alpha-1 Antitrypsin Deficiency: Current Approaches and Future Directions. Current Pathobiology Reports, 5(3), 243-252 4.Beiko, T. et al (2017). Serum Proteins Associated with Emphysema Progression in Severe Alpha-1 Antitrypsin Deficiency. Chronic Obstructive Pulmonary Diseases: Journal of the COPD Foundation, 4(3), 204-216.
Acknowledgement We are grateful to Dr. Manoj Garg for his immense support and my guide Dr. Prakash Baligar for guiding me and Amity University, Noida.