Fred Levine

Trehalose expression confers desiccation tolerance on human cells

Many organisms that withstand desiccation express the disaccharide trehalose. We have now expressed the otsA and otsB genes of Escherichia coli, which encode trehalose biosynthetic enzymes, in human primary fibroblasts using a recombinant adenovirus vector. Infected cells produced increased amounts of trehalose with increasing multiplicity of infection (MOI). Human primary fibroblasts expressing trehalose could be maintained in the dry state for up to five days. Fourier transform infrared spectroscopy indicated that dry, but viable, human cells contained no detectable water. This study shows that mammalian cells can be engineered to retain viability in the absence of water.

Cardiovascular Response to Large Doses of Intravenous Morphine in Man

Abstract Large doses of intravenous morphine (0.5 to 3.0 mg per kilogram of body weight) were used alone or in combination with inhalation anesthetic agents for anesthesia in over 1100 patients undergoing open-heart surgery. Morphine, 1 mg per kilogram, was administered intravenously to seven subjects with aortic-valve disease and eight without major heart or lung disease. The cardiac subjects had higher control pulse rates and lower control stroke indexes than the normal subjects. In the cardiac but not in the normal subjects, significant increases in cardiac index, stroke index, central venous pressure, and pulmonary-artery pressure, and a significant decrease in systemic vascular resistance, were observed after morphine was administered, suggesting that large doses of morphine may be used with safety in patients with minimal circulatory reserve.

Beta-cell differentiation from nonendocrine epithelial cells of the adult human pancreas.

The nature and even existence of adult pancreatic endocrine stem or progenitor cells is a subject of controversy in the field of beta-cell replacement for diabetes. One place to search for such cells is in the nonendocrine fraction of cells that remain after islet isolation, which consist of a mixture of epithelia and mesenchyme. Culture in G418 resulted in elimination of the mesenchymal cells, leaving a highly purified population of nonendocrine pancreatic epithelial cells (NEPECs). To evaluate their differentiation potential, NEPECs were heritably marked and transplanted under the kidney capsule of immunodeficient mice. When cotransplanted with fetal pancreatic cells, NEPECs were capable of endocrine differentiation. We found no evidence of beta-cell replication or cell fusion that could have explained the appearance of insulin positive cells from a source other than NEPECs. Nonendocrine-to-endocrine differentiation of NEPECs supports the existence of endocrine stem or progenitor cells within the epithelial compartment of the adult human pancreas.

Telomerase Activity Is Sufficient To Allow Transformed Cells To Escape from Crisis

ABSTRACT The introduction of simian virus 40 large T antigen (SVLT) into human primary cells enables them to proliferate beyond their normal replicative life span. In most cases, this temporary escape from senescence eventually ends in a second proliferative block known as “crisis,” during which the cells cease growing or die. Rare immortalization events in which cells escape crisis are frequently correlated with the presence of telomerase activity. We tested the hypothesis that telomerase activation is the critical step in the immortalization process by studying the effects of telomerase activity in two mortal SVLT-Rasval12-transformed human pancreatic cell lines, TRM-6 and βlox5. The telomerase catalytic subunit, hTRT, was introduced into late-passage cells via retroviral gene transfer. Telomerase activity was successfully induced in infected cells, as demonstrated by a telomerase repeat amplification protocol assay. In each of nine independent infections, telomerase-positive cells formed rapidly dividing cell lines while control cells entered crisis. Telomere lengths initially increased, but telomeres were then maintained at their new lengths for at least 20 population doublings. These results demonstrate that telomerase activity is sufficient to enable transformed cells to escape crisis and that telomere elongation in these cells occurs in a tightly regulated manner.

Pancreatic β‐Cell Neogenesis by Direct Conversion from Mature α‐Cells

Because type 1 and type 2 diabetes are characterized by loss of β‐cells, β‐cell regeneration has garnered great interest as an approach to diabetes therapy. Here, we developed a new model of β‐cell regeneration, combining pancreatic duct ligation (PDL) with elimination of pre‐existing β‐cells with alloxan. In this model, in which virtually all β‐cells observed are neogenic, large numbers of β‐cells were generated within 2 weeks. Strikingly, the neogenic β‐cells arose primarily from α‐cells. α‐cell proliferation was prominent following PDL plus alloxan, providing a large pool of precursors, but we found that β‐cells could form from α‐cells by direct conversion with or without intervening cell division. Thus, classical asymmetric division was not a required feature of the process of α‐ to β‐cell conversion. Intermediate cells coexpressing α‐cell‐ and β‐cell‐specific markers appeared within the first week following PDL plus alloxan, declining gradually in number by 2 weeks as β‐cells with a mature phenotype, as defined by lack of glucagon and expression of MafA, became predominant. In summary, these data revealed a novel function of α‐cells as β‐cell progenitors. The high efficiency and rapidity of this process make it attractive for performing the studies required to gain the mechanistic understanding of the process of α‐ to β‐cell conversion that will be required for eventual clinical translation as a therapy for diabetes. STEM CELLS 2010; 28:1630–1638.

Characterization of ataxia telangiectasia fibroblasts with extended life-span through telomerase expression.

Ataxia-telangiectasia (A-T) is an autosomal recessive disease characterized by progressive cerebellar degeneration, immunodeficiencies, genomic instability and gonadal atrophy. A-T patients are hypersensitive to ionizing radiation and have an elevated cancer risk. Cells derived from A-T patients require higher levels of serum factors, exhibit cytoskeletal defects and undergo premature senescence in culture. We show here that expression of the catalytic subunit of telomerase (hTERT) in primary A-T patient fibroblasts can rescue the premature senescence phenotype. Ectopic expression of hTERT does not rescue the radiosensitivity or the telomere fusions in A-T fibroblasts. The hTERT+AT cells also retain the characteristic defects in cell-cycle checkpoints, and show increased chromosome damage before and after ionizing radiation. Although A-T patients have an increased susceptibility to cancer, the expression of hTERT in A-T fibroblasts does not stimulate malignant transformation. These immortalized A-T cells provide a more stable cell system to investigate the molecular mechanisms underlying the cellular phenotypes of Ataxia-telangiectasia.

Towards gene therapy of diabetes mellitus

A definitive treatment for diabetes mellitus will be one that maintains a normal blood glucose concentration in the face of fluctuating dietary intake. To accomplish this, there must be mechanisms to sense the amount of blood glucose coupled to rapid release of the right amount of insulin. While mechanical devices to accomplish this are being developed, ultimately the best approach is likely to be based on genetic modification of cells. These could be pancreatic beta-cells, which are the only cells that produce insulin, or other cells that are involved in the pathogenesis of diabetes. Although definitive treatment of diabetes using genetically modified cells is a long-term goal, much progress is being made. This review discusses various approaches to modifying cells genetically, both in vitro and in vivo, for the treatment of diabetes.

Isolation and characterization of a cell line from the epithelial cells of the human fetal pancreas

Pancreatic cell lines are useful for basic studies of pancreatic biology and for possible application to cell transplantation therapies for diabetes. A retroviral vector expressing simian virus 40 (SV40) T antigen and H-rasval12 was used to infect a monolayer culture of epithelial cells from an 18-wk human fetal pancreas. Infected cells gave rise to a clonal epithelial cell line, designated TRM-1. This cell line expresses epithelial markers as well as gult2 and small amounts of insulin and glucagon. TRM-1 is the first cell line to be generated from the human fetal pancreas and also the first cell line derived directly from the fetal pancreas of any species. The approach that we have used to develop TRM-1 should be applicable to isolating cell lines from other stages of human pancreatic development.

NeuroD1 in the endocrine pancreas: Localization and dual function as an activator and repressor

The basic helix–loop–helix transcription factor NeuroD1 regulates cell fate in the nervous system but previously has not been considered to function similarly in the endocrine pancreas due to its reported expression in all islet cell types in the newborn mouse. Because we found that NeuroD1 potently represses somatostatin expression in vitro, its pattern of expression was examined in both strains of mice in which lacZ has been introduced into the NeuroD1 locus by homologous recombination. Analysis of adult transgenic mice revealed that NeuroD1 is predominantly expressed in β‐cells and either absent or expressed below the limit of lacZ detection in mature α‐, δ‐, or PP cells. Consistent with a previous report, NeuroD1 colocalizes with glucagon as well as insulin in immature islets of the newborn mouse. However, no colocalization of NeuroD1with somatostatin was detected in the newborn. In vitro, ectopic expression of NeuroD1 in TRM‐6/PDX‐1, a human pancreatic δ‐cell line, resulted in potent repression of somatostatin concomitant with induction of the β‐cell hormones insulin and islet amyloid polypeptide. Additionally, NeuroD1 induced expression of Nkx2.2, a transcription factor expressed in β‐ but not δ‐cells. Transfection studies using insulin and somatostatin promoters confirm the ability of NeuroD1 to act as both a transcriptional repressor and activator in the same cell, suggesting a more complex role for NeuroD1 in the establishment and/or maintenance of mature endocrine cells than has been recognized previously. Developmental Dynamics 233:946–953, 2005.

Telomere‐independent cellular senescence in human fetal cardiomyocytes

Fetal cardiomyocytes have been proposed as a potential source of cell‐based therapy for heart failure. This study examined cellular senescence in cultured human fetal ventricular cardiomyocytes (HFCs). HFCs were isolated and identified by immunocytochemistry and RT‐PCR. Cells were found to senesce after 20–25 population doublings, as determined by growth arrest, morphological changes and senescence‐associated β‐galactosidase activity. Using the telomeric repeat amplification protocol assay, telomerase activity was undetectable in primary HFCs. Cells were transduced to express the human reverse transcriptase subunit (hTERT) of telomerase. This resulted in greatly increased telomerase activity, but no significant lifespan extension. Analysis of telomere length in primary HFCs revealed that the senescent phenotype was not accompanied by telomere shortening. Telomeres in hTERT‐positive cells were elongated in comparison with primary cells, and elongation was retained in senescent cells. Levels of the tumor suppressor protein p16INK4A increased in all senescent cells whether telomerase‐positive or ‐negative. Senescence was accompanied by a decline in transcript levels of the polycomb gene Bmi‐1, Ets1 and Ets2 transcription factors, and Id1, Id2 and Id3 helix–loop–helix proteins, suggesting roles for these genes in maintenance of cardiomyocyte proliferative capacity. In addition to offering novel insights into the behavior of human fetal cardiomyocytes in culture, these findings have implications for the development of a cell‐based therapy for cardiac injury using primary fetal heart tissue.