Hepatocyte-like cells derived from pancreatic cells for use in a bioartifical liver system and in the identification of glucocorticoid receptor interacting partners

Michael Buchholz

Research output: ThesisDoctoral Thesis

Abstract

Liver transplantation is currently the established treatment for patients with liver failure. Unfortunately there is a shortage of donor livers and therefore a number of patients die while on the waiting list for liver transplantation. Due to the high mortality rates and the increased waiting times for transplantation, there has been great interest in culturing hepatocytes in extracorporeal liver devices (known as bioartificial liver systems (BAL)). BAL systems may provide temporary liver support to bridge the patient prior to transplantation or to allow sufficient time for liver regeneration to occur. Many BAL systems have previously been developed and clinically tested on human patients. Apart from some minor improvement in the clinical status of patients all of the BAL systems failed to promote long-term survival of the patient. One critical point in BAL systems is the origin and functionality of hepatocytes used in the system. Hepatocytes derived from a non-human origin can be immunogenic and therefore not suitable for inclusion in BAL systems. On the other hand, human hepatocytes are in limited supply, as are the whole organs. One alternative to using freshly isolated hepatocytes or transformed cell lines is to generate hepatocyte-like cells from alternative sources. To address this issue we therefore focused on using hepatocytes derived from pancreatic cells, via a process termed transdifferentiation (or sometimes it is referred to as reprogramming) and in general means the conversion of one cell type to another. The model is based on culturing the rat pancreatic cell line AR42J-B13 (B13) with the synthetic glucocorticoid, dexamethasone (Dex). To develop proof-of-concept data for our BAL system we used a biocompatible matrix poly (lactic-co-glycolic acid) (PLGA) as a scaffold for culturing hepatocytes. This approach offers the option of transplanting the BAL system into the patient for the first time. I now provide evidence that the pancreatic B13 cells can be successfully cultured on biodegradable scaffolds of PLGA and transdifferentiated to hepatocyte-like cells with Dex (based on the morphological appearance). We were also interested in identifying novel glucocorticoid receptor interacting partners in the transdifferentiation of pancreatic cells to hepatocyte-like cells. To address this issue I developed B13 cell clones stably expressing functionally N- or C-terminal-
Abstract
V
tagged glucocorticoid receptor. Stable expression of N- or C-terminal tagged glucocorticoid receptor enhanced the sensitivity to Dex. This resulted in increased cell death at higher concentrations of Dex but more efficient conversion of pancreatic cells to hepatocyte-like cells at lower concentrations of Dex compared to normal B13 cells. The B13 cell clones were used to develop a protocol for determination of the early signalling events (interacting partners) in the conversion of pancreatic cells to hepatocytes. The protocol includes gentle protein extraction from the B13 cells followed by purification of the EGFP tagged GR and analysis of co-purified proteins by mass spectrometry. I have now identified 11 potential GR interacting partners, 7 of which are already published and 4 are completely novel. These results suggest that the interacting partners may be involved in the reprogramming of one cell type to another.
LanguageEnglish
QualificationPh.D.
Awarding Institution
  • University of Bath
Supervisors/Advisors
  • Tosh, David, Supervisor
  • Ellis, Marianne, Supervisor
  • Chaudhuri, Julian, Supervisor
Award date29 Feb 2012
StatusUnpublished - 29 Feb 2012

Fingerprint

Glucocorticoid Receptors
Artificial Liver
Hepatocytes
Liver
Dexamethasone
Liver Transplantation
Clone Cells
Transplantation
Cell Transdifferentiation
Transformed Cell Line
Liver Regeneration
Waiting Lists
Liver Failure
Glucocorticoids
Mass Spectrometry
Proteins
Cell Death
Tissue Donors

Cite this

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title = "Hepatocyte-like cells derived from pancreatic cells for use in a bioartifical liver system and in the identification of glucocorticoid receptor interacting partners",
abstract = "Liver transplantation is currently the established treatment for patients with liver failure. Unfortunately there is a shortage of donor livers and therefore a number of patients die while on the waiting list for liver transplantation. Due to the high mortality rates and the increased waiting times for transplantation, there has been great interest in culturing hepatocytes in extracorporeal liver devices (known as bioartificial liver systems (BAL)). BAL systems may provide temporary liver support to bridge the patient prior to transplantation or to allow sufficient time for liver regeneration to occur. Many BAL systems have previously been developed and clinically tested on human patients. Apart from some minor improvement in the clinical status of patients all of the BAL systems failed to promote long-term survival of the patient. One critical point in BAL systems is the origin and functionality of hepatocytes used in the system. Hepatocytes derived from a non-human origin can be immunogenic and therefore not suitable for inclusion in BAL systems. On the other hand, human hepatocytes are in limited supply, as are the whole organs. One alternative to using freshly isolated hepatocytes or transformed cell lines is to generate hepatocyte-like cells from alternative sources. To address this issue we therefore focused on using hepatocytes derived from pancreatic cells, via a process termed transdifferentiation (or sometimes it is referred to as reprogramming) and in general means the conversion of one cell type to another. The model is based on culturing the rat pancreatic cell line AR42J-B13 (B13) with the synthetic glucocorticoid, dexamethasone (Dex). To develop proof-of-concept data for our BAL system we used a biocompatible matrix poly (lactic-co-glycolic acid) (PLGA) as a scaffold for culturing hepatocytes. This approach offers the option of transplanting the BAL system into the patient for the first time. I now provide evidence that the pancreatic B13 cells can be successfully cultured on biodegradable scaffolds of PLGA and transdifferentiated to hepatocyte-like cells with Dex (based on the morphological appearance). We were also interested in identifying novel glucocorticoid receptor interacting partners in the transdifferentiation of pancreatic cells to hepatocyte-like cells. To address this issue I developed B13 cell clones stably expressing functionally N- or C-terminal-AbstractVtagged glucocorticoid receptor. Stable expression of N- or C-terminal tagged glucocorticoid receptor enhanced the sensitivity to Dex. This resulted in increased cell death at higher concentrations of Dex but more efficient conversion of pancreatic cells to hepatocyte-like cells at lower concentrations of Dex compared to normal B13 cells. The B13 cell clones were used to develop a protocol for determination of the early signalling events (interacting partners) in the conversion of pancreatic cells to hepatocytes. The protocol includes gentle protein extraction from the B13 cells followed by purification of the EGFP tagged GR and analysis of co-purified proteins by mass spectrometry. I have now identified 11 potential GR interacting partners, 7 of which are already published and 4 are completely novel. These results suggest that the interacting partners may be involved in the reprogramming of one cell type to another.",
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year = "2012",
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language = "English",
school = "University of Bath",

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TY - THES

T1 - Hepatocyte-like cells derived from pancreatic cells for use in a bioartifical liver system and in the identification of glucocorticoid receptor interacting partners

AU - Buchholz,Michael

PY - 2012/2/29

Y1 - 2012/2/29

N2 - Liver transplantation is currently the established treatment for patients with liver failure. Unfortunately there is a shortage of donor livers and therefore a number of patients die while on the waiting list for liver transplantation. Due to the high mortality rates and the increased waiting times for transplantation, there has been great interest in culturing hepatocytes in extracorporeal liver devices (known as bioartificial liver systems (BAL)). BAL systems may provide temporary liver support to bridge the patient prior to transplantation or to allow sufficient time for liver regeneration to occur. Many BAL systems have previously been developed and clinically tested on human patients. Apart from some minor improvement in the clinical status of patients all of the BAL systems failed to promote long-term survival of the patient. One critical point in BAL systems is the origin and functionality of hepatocytes used in the system. Hepatocytes derived from a non-human origin can be immunogenic and therefore not suitable for inclusion in BAL systems. On the other hand, human hepatocytes are in limited supply, as are the whole organs. One alternative to using freshly isolated hepatocytes or transformed cell lines is to generate hepatocyte-like cells from alternative sources. To address this issue we therefore focused on using hepatocytes derived from pancreatic cells, via a process termed transdifferentiation (or sometimes it is referred to as reprogramming) and in general means the conversion of one cell type to another. The model is based on culturing the rat pancreatic cell line AR42J-B13 (B13) with the synthetic glucocorticoid, dexamethasone (Dex). To develop proof-of-concept data for our BAL system we used a biocompatible matrix poly (lactic-co-glycolic acid) (PLGA) as a scaffold for culturing hepatocytes. This approach offers the option of transplanting the BAL system into the patient for the first time. I now provide evidence that the pancreatic B13 cells can be successfully cultured on biodegradable scaffolds of PLGA and transdifferentiated to hepatocyte-like cells with Dex (based on the morphological appearance). We were also interested in identifying novel glucocorticoid receptor interacting partners in the transdifferentiation of pancreatic cells to hepatocyte-like cells. To address this issue I developed B13 cell clones stably expressing functionally N- or C-terminal-AbstractVtagged glucocorticoid receptor. Stable expression of N- or C-terminal tagged glucocorticoid receptor enhanced the sensitivity to Dex. This resulted in increased cell death at higher concentrations of Dex but more efficient conversion of pancreatic cells to hepatocyte-like cells at lower concentrations of Dex compared to normal B13 cells. The B13 cell clones were used to develop a protocol for determination of the early signalling events (interacting partners) in the conversion of pancreatic cells to hepatocytes. The protocol includes gentle protein extraction from the B13 cells followed by purification of the EGFP tagged GR and analysis of co-purified proteins by mass spectrometry. I have now identified 11 potential GR interacting partners, 7 of which are already published and 4 are completely novel. These results suggest that the interacting partners may be involved in the reprogramming of one cell type to another.

AB - Liver transplantation is currently the established treatment for patients with liver failure. Unfortunately there is a shortage of donor livers and therefore a number of patients die while on the waiting list for liver transplantation. Due to the high mortality rates and the increased waiting times for transplantation, there has been great interest in culturing hepatocytes in extracorporeal liver devices (known as bioartificial liver systems (BAL)). BAL systems may provide temporary liver support to bridge the patient prior to transplantation or to allow sufficient time for liver regeneration to occur. Many BAL systems have previously been developed and clinically tested on human patients. Apart from some minor improvement in the clinical status of patients all of the BAL systems failed to promote long-term survival of the patient. One critical point in BAL systems is the origin and functionality of hepatocytes used in the system. Hepatocytes derived from a non-human origin can be immunogenic and therefore not suitable for inclusion in BAL systems. On the other hand, human hepatocytes are in limited supply, as are the whole organs. One alternative to using freshly isolated hepatocytes or transformed cell lines is to generate hepatocyte-like cells from alternative sources. To address this issue we therefore focused on using hepatocytes derived from pancreatic cells, via a process termed transdifferentiation (or sometimes it is referred to as reprogramming) and in general means the conversion of one cell type to another. The model is based on culturing the rat pancreatic cell line AR42J-B13 (B13) with the synthetic glucocorticoid, dexamethasone (Dex). To develop proof-of-concept data for our BAL system we used a biocompatible matrix poly (lactic-co-glycolic acid) (PLGA) as a scaffold for culturing hepatocytes. This approach offers the option of transplanting the BAL system into the patient for the first time. I now provide evidence that the pancreatic B13 cells can be successfully cultured on biodegradable scaffolds of PLGA and transdifferentiated to hepatocyte-like cells with Dex (based on the morphological appearance). We were also interested in identifying novel glucocorticoid receptor interacting partners in the transdifferentiation of pancreatic cells to hepatocyte-like cells. To address this issue I developed B13 cell clones stably expressing functionally N- or C-terminal-AbstractVtagged glucocorticoid receptor. Stable expression of N- or C-terminal tagged glucocorticoid receptor enhanced the sensitivity to Dex. This resulted in increased cell death at higher concentrations of Dex but more efficient conversion of pancreatic cells to hepatocyte-like cells at lower concentrations of Dex compared to normal B13 cells. The B13 cell clones were used to develop a protocol for determination of the early signalling events (interacting partners) in the conversion of pancreatic cells to hepatocytes. The protocol includes gentle protein extraction from the B13 cells followed by purification of the EGFP tagged GR and analysis of co-purified proteins by mass spectrometry. I have now identified 11 potential GR interacting partners, 7 of which are already published and 4 are completely novel. These results suggest that the interacting partners may be involved in the reprogramming of one cell type to another.

M3 - Doctoral Thesis

ER -