Dr. Pramod K. Upadhyay, Emeritus Scientist

Summary of Research:

Engineering hepatocytes for supporting acute liver failure

India is placed at intermediate endemic level in hepatitis B. Hepatitis B virus is reported to be responsible for 70% of chronic hepatitis cases and 80% of cirrhosis of liver cases. About 60% of those cases with hepatocellular carcinoma are HBV marker positive. Thus it would be clinically relevant in Indian scenario if a cell based therapeutic option is available for treatment of end stage liver disease cause due to hepatitis B virus.

To address these clinical situations, we developed a strategy to generate hepatocyte-like cells (Neo-Hepatocytes; abbreviated as NeoHeps) from monocyte derived from peripheral blood of HBsAg-NAT positive human peripheral blood samples. To generate NeoHeps; non-activated monocytes were sorted from the human peripheral blood by magnetic assisted cell sorting techniques. The monocytes were then cultured in IMDM basal media containing IL3, MCSF and 2-ME supplemented with 0.5% ESC serum for 6 days to generate Reprogrammed Monocytes (RM) at Day 6. The RM were then cultured in IMDM basal media containing epithelial growth factor (EGF), hepatocyte growth factor (HGF) and fibroblast growth factor-4 (FGF4) with progressive increase in serum concentration from 2% (at day 6) to 5% (at Day8) upto 10% from day 13 to day 21 of the culture. 

Both Monocytes and RM were unable to demonstrate P450 activity in them. The NeoHeps derived from both healthy and HBsAg-NAT positive bloods have the potential to secrete human serum albumin in their respective culture supernatant like primary human hepatocytes. 

To evaluate the stability of NeoHeps in vivo, partial (1/3rd) hepatectomy of SCID mouse was performed and NeoHeps were injected near the spleen of the hepatectomized mouse. 10th day post transplantation, blood was collected from the mouse and serum was isolated. The liver of the mouse was excised out, there was no visible tumours found in the liver lobes of the recipient mouse and cryo-sectioning of the liver was performed to check the homing and engraftment of transplanted NeoHeps. Human serum albumin was detected in the serum of the recipient mouse and the immuno-cytochemistry of the liver tissue section of the recipient mouse stained positive for human albumin and connexin32.

Tissue engineered vascular graft 

We are developing a novel tissue engineered vascular graft which is immune friendly as well as long lasting as compared to the presently available grafts. 

In this approach, the peripheral blood monocytes are reprogrammed to stem cell like cell followed by its re-differentiation into endothelial –like cells and vascular smooth muscle like-cells. These cells express prominent endothelial and smooth muscle cell markers as well as show functional activity. 

The re-differentiated vascular cells are then uniformly seeded onto a decellularized xenogenic vessel matrix using a bioreactor setup such that endothelial cells form the luminal layer while smooth muscle cells form the outer layer of the graft.

Monocytes derived retinal cells

The project involves two steps; in vitro differentiation of PBMC derived monocytes into retinal cells and their further phenotypic and functional characterization by transplantation into a newly developed NOD.SCID-CBA/J mouse model for retinal degeneration. The underlying aim of the project is to discover new options in curing of retinal degeneration diseases like retinitis pigmentosa which unfortunately remain incurable till date. 

Immunization by the aerogenic route

We have developed a methodology to encapsulate live Mycobacterium (MIP and BCG) as a dry powder inhale-able formulation which remains viable for 4 months at 37°C. A non invasive procedure was developed to deliver this formulation to small animals.  We have established by a number of in vitro and in vivo experiments that this inhale-able vaccine of live Mycobacterium is more immunogenic as compared to the aerosol of bacilli and it provide better protection in mice when challenged with H37Rv. The alginate coated DPA of BCG/MIP is a very promising alternative for present day vaccination for Tuberculosis; it does not require the cold chain for transportation and storage, it provide better protection than conventional intradermal or liquid aerosol and its delivery does not require the needle and a syringe.  

Local hyperthermia enhanced immunization

Hyperthermia enhanced transdermal (HET) immunization is a novel needle free immunization strategy employing application of antigen along with mild local hyperthermia (42°C) to intact skin resulting in detectable antigen specific Ig in serum. We have developed a tools for HET immunization and have shown that mice immunized by tetanus toxoid using HET route exhibited protection from challenge with a lethal dose of tetanus toxin.