Hepatocyte differentiation of mesenchymal stem cells from rat peritoneal adipose tissue in vitro and in vivo.

Mesenchymal tissues harbour stromal cells capable of multilineage differentiation. Here, we demonstrate the isolation of mesenchymal stem cells (MSC) from rat peritoneal adipose tissue capable of osteogenic and adipogenic differentiation. Under in vitro conditions favouring hepatocyte differentiation, these MSC gained characteristic functions of hepatocytes such as the capacity to synthesize urea or store glycogen. Hepatocyte-specific transcripts of dipeptidylpeptidase type IV (CD26), albumin, cytochrome P450 type 1A1 (CYP1A1) and connexin CX32 (CX32) were detected only in differentiated but not undifferentiated cells. Transient transgenic expression of luciferase could be stimulated by cAMP when driven by the hepatocyte-specific promoter of the cytosolic phosphoenolpyruvate carboxykinase (PCK1) gene. Finally, stem cell-derived hepatocytes from wild type (CD26+/+) rats were transplanted into the livers of CD26-deficient animals after lentiviral transduction with the GFP gene under the control of the ubiquitin promoter. GFP-positive cells engrafted in the host liver predominantly in the periportal region of the liver lobule. They continued to express CD26, a prominent feature of differentiated hepatocytes, indicating their topologically and functionally proper integration into the host liver parenchyma. Thus, MSCs from rat peritoneal adipose tissue exhibit the potential to differentiate into hepatocyte-like cells in vitro and in vivo.

Functional integration of hepatocytes derived from human mesenchymal stem cells into mouse livers.

AIMS: At present, clinical success of hepatocyte transplantation as an alternative to whole liver transplantation is hampered by the limited availability of suitable donor organs for the isolation of transplantable hepatocytes. Hence, novel cell sources are required to deliver hepatocytes of adequate quality for clinical use. Mesenchymal stem cells (MSCs) from human bone marrow may have the potential to differentiate into hepatocytes in vitro and in vivo. METHODS: Isolated MSCs were selected by density gradient centrifugation and plastic adherence, differentiated in the presence of human hepatocyte growth medium and transplanted in immunodeficient Pfp/Rag2 mice. RESULTS: Here, we demonstrate that human MSCs gain in vitro the characteristic morphology and function of hepatocytes in response to specified growth factors. Specifically, preconditioned MSCs store glycogen, synthesise urea and feature the active hepatocyte-specific gene promoter of phosphoenolpyruvate carboxykinase (PCK1). After transplantation into livers of immunodeficient mice, preconditioned MSCs engraft predominantly in the periportal portion of the liver lobule. In situ, the cells continue to store glycogen and express PCK1, connexin32, albumin and the human hepatocyte-specific antigen HepPar1, indicating that the transplanted cells retain prominent qualities of hepatocytes after their regional integration. CONCLUSION: MSCs derived from human bone marrow may serve as a novel source for the propagation of hepatocyte-like cells suitable for cell therapy in liver diseases.

Disposition of calcium release units in agarose gel for an optimal propagation of Ca2+ signals.

Clusters of calcium-loaded sarcoplasmic reticulum (SR) vesicles in agarose gel were previously shown to behave as an excitable medium that propagates calcium waves. In a 3D-hexagonal disposition, the distance between neighboring spheres (which may stand for SR vesicles) is constant and the relationship between distance and vesicular protein concentration is expected to be nonlinear. To obtain a distribution of SR vesicles at different protein concentrations as homogeneous as possible, liquid agarose gels were carefully stirred. Electron micrographs, however, did not confirm the expected relationship between inter-SR vesicle distance and vesicular protein concentration. Light micrographs, to the contrary, resulted in a protein concentration-dependent disposition of clusters of SR vesicles, which is described by a linear function. Stable calcium waves in agarose gel occurred at SR vesicle protein concentrations between 7 and 16 g/l. At lower protein concentrations, local calcium oscillations or abortive waves were observed. The velocities of calcium waves were optimum at approximately 12 g/l and amounted to nearly 60 microm/s. The corresponding distance of neighboring calcium release units was calculated to be approximately 4 microm. The results further show that calcium signaling in the described reaction-diffusion system is optimal in a relatively small range of diffusion lengths. A change by +/-2 microm resulted in a reduction of the propagation velocity by 40%. It would appear that 1), the distance between calcium release units (clusters of ryanodine receptors in cells) is a sensitive parameter concerning propagation of Ca2+ signals; and 2), a dysfunction of the reaction-diffusion system in living cells, however, might have a negative effect on the spreading of intracellular calcium signals, thus on the cell's function.