Bryon E. Petersen

Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion

Recent studies have demonstrated that transplanted bone marrow cells can turn into unexpected lineages including myocytes, hepatocytes, neurons and many others. A potential problem, however, is that reports discussing such ‘transdifferentiation’ in vivo tend to conclude donor origin of transdifferentiated cells on the basis of the existence of donor-specific genes such as Y-chromosome markers. Here we demonstrate that mouse bone marrow cells can fuse spontaneously with embryonic stem cells in culture in vitro that contains interleukin-3. Moreover, spontaneously fused bone marrow cells can subsequently adopt the phenotype of the recipient cells, which, without detailed genetic analysis, might be interpreted as ‘dedifferentiation’ or transdifferentiation.

In vitro trans-differentiation of adult hepatic stem cells into pancreatic endocrine hormone- producing cells

Although organ-specific stem cells possess plasticity that permit differentiation along new lineages, production of endocrine pancreas and insulin-secreting β cells from adult nonpancreatic stem cells has not been demonstrated. We present evidence that highly purified adult rat hepatic oval “stem” cells, which are capable of differentiation to hepatocytes and bile duct epithelium, can trans-differentiate into pancreatic endocrine hormone-producing cells when cultured in a high-glucose environment. These differentiated cells can self-assemble to form three-dimensional islet cell-like clusters that express pancreatic islet cell differentiation-related transcripts detectable by reverse transcription–PCR/nested PCR (e.g., PDX-1, PAX-4, PAX-6, Nkx2.2 and Nkx6.1, insulin I, insulin II, glucose transporter 2, and glucagon) and islet-specific hormones detectable by immunocytochemistry (e.g., insulin, glucagon, and pancreatic polypeptide). In addition, these cells concomitantly lose expression of the hepatocyte protein Hep-par. When stimulated with glucose, these cells synthesize and secrete insulin, a response enhanced by nicotinamide. In a pilot study, the oval cell-derived islet cell-like clusters displayed the ability to reverse hyperglycemia in a diabetic NOD-scid mouse. These results indicate that primary adult liver stem cells can differentiate in a nonlineage-restricted manner. Trans-differentiation into endocrine pancreas could have significant implications for future therapies of diabetes.

MESENCHYMAL STEM CELLS SPONTANEOUSLY EXPRESS NEURAL PROTEINS IN CULTURE AND ARE NEUROGENIC AFTER TRANSPLANTATION

Reports of neural transdifferentiation of mesenchymal stem cells (MSCs) suggest the possibility that these cells may serve as a source for stem cell–based regenerative medicine to treat neurological disorders. However, some recent studies controvert previous reports of MSC neurogenecity. In the current study, we evaluate the neural differentiation potential of mouse bone marrow–derived MSCs. Surprisingly, we found that MSCs spontaneously express certain neuronal phenotype markers in culture, in the absence of specialized induction reagents. A previously published neural induction protocol that elevates cytoplasmic cyclic AMP does not upregulate neuron‐specific protein expression significantly in MSCs but does significantly increase expression of the astrocyte‐specific glial fibrillary acidic protein. Finally, when grafted into the lateral ventricles of neonatal mouse brain, MSCs migrate extensively and differentiate into olfactory bulb granule cells and periventricular astrocytes, without evidence of cell fusion. These results indicate that MSCs may be “primed” toward a neural fate by the constitutive expression of neuronal antigens and that they seem to respond with an appropriate neural pattern of differentiation when exposed to the environment of the developing brain.

Teratoma Formation Leads to Failure of Treatment for Type I Diabetes Using Embryonic Stem Cell-Derived Insulin-Producing Cells

Embryonic stem (ES) cells have been proposed to be a powerful tool in the study of pancreatic disease, as well as a potential source for cell replacement therapy in the treatment of diabetes. However, data demonstrating the feasibility of using pancreatic islet-like cells differentiated from ES cells remain controversial. In this study we characterized ES cell-derived insulin-expressing cells and assessed their suitability for the treatment of type I diabetes. ES cell-derived insulin-stained cell clusters expressed insulin mRNA and transcription factors associated with pancreatic development. The majority of insulin-positive cells in the clusters also showed immunoreactivity for C-peptide. Insulin was stored in the cytoplasm and released into the culture medium in a glucose-dependent manner. When the cultured cells were transplanted into diabetic mice, they reversed the hyperglycemic state for approximately 3 weeks, but the rescue failed due to immature teratoma formation. Our studies demonstrate that reversal of hyperglycemia by transplantation of ES cell-derived insulin-producing cells is possible. However, the risk of teratoma formation would need to be eliminated before ES cell-based therapies for the treatment of diabetes are considered.

ADULT BONE MARROW-DERIVED CELLS TRANS-DIFFERENTIATING INTO INSULIN-PRODUCING CELLS FOR THE TREATMENT OF TYPE I DIABETES

Recent findings suggest that bone marrow (BM) cells have the capacity to differentiate into a variety of cell types including endocrine cells of the pancreas. We report that BM derived cells, when cultured under defined conditions, were induced to trans-differentiate into insulin-producing cells. Furthermore, these insulin-producing cells formed aggregates that, upon transplantation into mice, acquired architecture similar to islets of Langerhans. These aggregates showed endocrine gene expression for insulin (I and II), glucagon, somatostatin and pancreatic polypeptide. Immunohistochemistry also confirmed that these aggregates were positive for insulin, somatostatin, pancreatic polypeptide and C-peptide. Also, Western and ELISA analysis demonstrated expression of proinsulin and/or secretion of active insulin upon glucose challenge. Subcapsular renal transplantation of these aggregates into hyperglycemic mice lowered circulating blood glucose levels and maintained comparatively normal glucose levels for up to 90 days post-transplantation. Graft removal resulted in rapid relapse and death in experimental animals. In addition, electron microscopy revealed these aggregates had acquired ultrastructure typically associated with mature beta (β) cells. These results demonstrate that adult BM cells are capable of trans-differentiating into a pancreatic lineage in vitro and may represent a pool of cells for the treatment of diabetes mellitus.

SDF-1α/CXCR4: A Mechanism for Hepatic Oval Cell Activation and Bone Marrow Stem Cell Recruitment to the Injured Liver of Rats

Stromal derived factor-1 alpha (SDF-1alpha) and its receptor CXCR4 have been shown to play a role in the systematic movement of hematopoietic stem cells (HSC) in the fetal and adult stages of hematopoiesis. Under certain physiological conditions liver oval cells can participate in the regeneration of the liver. We have shown that a percentage of oval cells are of hematopoietic origin. Others have shown that bone marrow derived stem cells can participate in liver regeneration as well. In this study we examined the role of SDF-1alpha and its receptor CXCR4 as a possible mechanism for oval cell activation in oval cell aided liver regeneration. In massive liver injury models where oval cell repair is involved hepatocytes up-regulate the expression of SDF-1alpha, a potent chemoattractant for hematopoietic cells. However, when moderate liver injury occurs, proliferation of resident hepatocytes repairs the injury. Under these conditions SDF-1alpha expression is not up-regulated and oval cells are not activated in the liver. In addition, we show that oval cells express CXCR4, the only known receptor for SDF-1alpha. Lastly, in vitro chemotaxis assays demonstrated that oval cells migrate along a SDF-1alpha gradient which suggests that the SDF-1alpha/CXCR4 interaction is a mechanism by which the oval cell compartment could be activated and possibly recruit a second wave of bone marrow stem cells to the injured liver. In conclusion, these experiments begin to shed light on a possible mechanism, which may someday lead to a better understanding of the hepatic and hematopoietic interaction in oval cell aided liver regeneration.

Method for the decellularization of intact rat liver

We have developed a method for the decellularization of whole rat livers by perfusion with increasing concentrations of detergents. This procedure resulted in an intact, decellularized organ with an intact liver capsule. These decellularized organs were analyzed by immunohistochemistry, and retained an appropriate distribution of extracellular matrix components. The laminin basement membranes of the liver vasculature also remain intact. These acellular vessel remnants were strong enough to be cannulated; providing a convenient means for the delivery of cells to areas deep within the decellularized organ. Cannulation of the extrahepatic vessel remnants allow for media to be circulated through the decellularized organ. These decellularized livers provide a natural matrix for research in the fields of bio-artificial livers and liver engineering.

Breast Cancer Resistance Protein (BCRP/ABCG2) Is Expressed by Progenitor Cells/Reactive Ductules and Hepatocytes and Its Expression Pattern Is Influenced by Disease Etiology and Species Type: Possible Functional Consequences

Breast cancer resistance protein (BCRP/ABCG2) is an ATP-binding cassette transport protein that is expressed in several organs including the liver. Previous studies have shown that ABC transport proteins play an important pathophysiological role in several liver diseases. However, to date, expression pattern and possible role of BCRP in human liver diseases and animal models have not been studied in detail. Here we investigated the expression pattern of BCRP in normal liver, chronic parenchymal and biliary human liver diseases, and parallel in different rat models of liver diseases. Expression was studied by immunohistochemistry and additionally by RT-PCR analysis in Thy-1-positive rat oval cells. Bile ducts, hepatic progenitor cells, reactive bile ductules, and blood vessel endothelium were immunoreactive for BCRP in normal liver and all types of human liver diseases and in rat models. BCRP was expressed by the canalicular membrane of hepatocytes in normal and diseased human liver, but never in rat liver. Remarkably, there was also expression of BCRP at the basolateral pole of human hepatocytes, and this was most pronounced in chronic biliary diseases. In conclusion, BCRP positivity in the progenitor cells/reactive ductules could contribute to the resistance of these cells to cytotoxic agents and xenotoxins. Basolateral hepatocytic expression in chronic biliary diseases may be an adaptive mechanism to pump bile constituents back into the sinusoidal blood. Strong differences between human and rat liver must be taken into account in future studies with animal models.

An Overview of Stem Cell Research and Regulatory Issues

Stem cells are noted for their ability to self-renew and differentiate into a variety of cell types. Some stem cells, described as totipotent cells, have tremendous capacity to self-renew and differentiate. Embryonic stem cells have pluripotent capacity, able to form tissues of all 3 germ layers but unable to form an entire live being. Research with embryonic stem cells has enabled investigators to make substantial gains in developmental biology, therapeutic tissue engineering, and reproductive cloning. However, with these remarkable opportunities many ethical challenges arise, which are largely based on concerns for safety, efficacy, resource allocation, and methods of harvesting stem cells. Discussing the moral and legal status of the human embryo is critical to the debate on stem cell ethics. Religious perspectives and political events leading to regulation of stem cell research are presented and discussed, with special attention directed toward the use of embryonic stem cells for therapeutic and reproductive cloning. Adult stem cells were previously thought to have a restricted capacity to differentiate; however, several reports have described their plasticity potential. Furthermore, there have been close ties between the behavior of stem cells and cancer cells. True eradication of cancer will require a deeper understanding of stem cell biology. This article was written to inform medical scientists and practicing clinicians across the spectrum of medical education about the research and regulatory issues affecting the future of stem cell therapy.

Monocrotaline, an alternative to retrorsine-based hepatocyte transplantation in rodents.

Retrorsine has been used extensively to inhibit proliferation of resident hepatocytes in various transplantation models. Here we report a successful alternative to currently unavailable retrorsine that can be used in cellular transplantation models. Based on structural and molecular similarities, we investigate the use of monocrotaline (MCT) in cell transplantation studies in rodents. In this study, MCT was given to rats intraperitoneally in two injections 2 weeks apart. Two weeks after the final injection, a partial hepatectomy followed by splenic hepatocyte transplantation was performed. The results indicate that MCT, at two doses of 30 mg/kg, highly enhances liver repopulation by donor hepatocytes following partial hepatectomy and produces 15.3 ± 4.9% liver repopulation within the first 6 weeks following transplantation. Additionally, we tested the effectiveness of MCT in a murine model. Using two injections of 50 mg/kg each, given 2 weeks apart, hepatocyte proliferation in the native liver was inhibited and subsequent oval cell transplants engrafted at 18 ± 21.3% after 16 weeks posttransplantation. In conclusion, MCT can be used as an effective selective pressure for donor hepatocytes in cell transplantation to the liver in rodents.