Epigenetic changes improve differentiation of hepatozyte-like cells from adipose tissue: possible application for cell therapies in surgery

Question Due to donor organ scarcity, researchers nowadays focus on cell transplantation as alternative method to orthotopic liver transplantation. For this purpose, several groups attempt to generate hepatocyte-like cells from various adult stem or precursor cells such as mesenchymal stem cells (MSCs), fat tissue (Ad-) or liver. Aim of this study was to improve hepatic-function and amount of hepatocyte-like cells via epigenetic changes. Methods Human Ad-MSCs were isolated from different patients, with their informed consent according to ethical guidelines of the MRI. For hepatic differentiation several supplement combinations of 5-Azacytidine, FGF-4, Dexamethasone, Nicotinamid, ITS, HGF and EGF were used. The generated hepatocyte-like cells were stained for Glycogen, Glucose-6-phosphatase and neutral lipids. We further investigated glucose and urea metabolism as well as several phase I and II drug metabolizing enzyme activities. Expression of pluripotency-, mesoderm- and endoderm-markers was analyzed by RT-PCR. Results The isolated Ad-MSCs were tested on their pluripotency via Oct3/4, KLF4, Sox2 and c-Myc. After 14 days of differentiation, Ad-MSCs show similar morphological features than primary human hepatocytes and gain the ability to accumulate glycogen and express glucose-6-phophatase. To investigate the metabolic ability Urea- and Glucose metabolism was analyzed. Phase I and II enzyme activities reached levels up to 70% of primary human hepatocytes. Pre-treatment with 5-Azacytidine further increased both metabolic and enzymatic activities of the cells significantly. Conclusion Our work shows, inhibition of DNA-methyltransferase leads to a better hepatic differentiation of Ad-MSCs. Furthermore, a large number of AD-MSC´s can be generated. Hence, these cells may be used for alternative autologous therapies in surgery to bridge liver dys-functions.

Supplements Analysis	FGF4, Nic, Dex, I T S	EGF, FGF4, Nic, Dex, I T S	Aza EGF FGF4 + coll	Aza EGF FGF4 NIC I T S + coll	Aza EGF FGF4 Dex NIC I T S HGF+ coll
Morphology	-	+	++	+++	+++++
Glucosemetabolism	+	-	+++++	++++	++
Lipidakkumulation	-	+	++	++++	+++++
Ureaformation	-	+	+++++	++	++++
Glucoseproduction	+	+	++	++++	+++++
Phase I/II-Enzymactivity	-	+	++	+++++	++++

Literatur: BILIR B.M., GUINETTE D., KARRER F., KUMPE D.A., ET AL.(2000): Hepatocyte transplantation in acute liver failure. Transplant (6), p.32-40; NAJIMI M., KHUU D.N., LYSY P.A., ET AL.(2007): Adult-derived human liver mesenchymal-like cells as a potential progenitor reservoir of hepatocytes? Cell transplantation (16), p.717-728; STROM S., FISHER R.A., THOMPSON M.T., SANYAL A.J., ET AL.(1997): Hepatocyte transplantation as a bridge to orthotropic liver transplantation in terminal liver failure. Transplantation (63), p. 559-569

Epigenetic changes - hepatozyte-like cells - transplantation