Recent developments indicate that the regeneration of beta cell function and mass in patients with diabetes is possible. A regenerative approach may represent an alternative treatment option relative to current diabetes therapies that fail to provide optimal glycemic control. Here we report that the inactivation of GSK3 by small molecule inhibitors or RNA interference stimulates replication of INS-1E rat insulinoma cells. Specific and potent GSK3 inhibitors also alleviate the toxic effects of high concentrations of glucose and the saturated fatty acid palmitate on INS-1E cells. Furthermore, treatment of isolated rat islets with structurally diverse small molecule GSK3 inhibitors increases the rate beta cell replication by 2-3-fold relative to controls. We propose that GSK3 is a regulator of beta cell replication and survival. Moreover, our results suggest that specific inhibitors of GSK3 may have practical applications in beta cell regenerative therapies.

ABSTRACT

Modifications of DNA and chromatin are fundamental for the establishment and maintenance of cell type-specific gene expression patterns that constitute cellular identities. To test whether the developmental potential of fetal brainderived cells that form floating sphere colonies (neurospheres) can be modified by destabilizing their epigenotype, neurosphere cells were treated with chemical compounds that alter the acetylation and methylation patterns of chromatin and DNA. Intravenous infusion of bulk or clonally derived neurosphere cells treated with a combination of trichostatin A (TSA) plus 5-aza-2 0 -deoxycytidine (AzaC) (TSA/AzaC neurosphere cells) yielded long-term, multilineage and transplantable neurospherederived haematopoietic repopulation. Untreated neurosphere cells exhibited no haematopoietic repopulation activity. The neurosphere-derived haematopoietic cells showed a diploid karyotype, indicating that they are unlikely to be products of cell fusion events, a conclusion strengthened by multicolour fluorescence in situ hybridization. Our results indicate that altering the epigenotype of neurosphere cells followed by transplantation enables the generation of neurosphere-derived haematopoietic cells.

IntroductionHematopoietic stem cells (HSCs) are a rare cell type present in the adult bone marrow of mammals that provide the organism with lifelong hematopoiesis. During mammalian embryogenesis, a first transient wave of primitive hematopoiesis originates in the extraembryonic yolk sac. Later, the fetal liver is colonized by HSCs from the aorta gonad mesonephros (AGM) region, which is regarded as the first site of definitive hematopoiesis. Subsequently, HSCs migrate to the bone marrow, which is the hematopoietic active tissue of the adult animal. [1][2][3][4] An in vivo test system for the identification and characterization of human HSCs consists of the intravenous injection of human candidate HSCs into murine nonobese diabetic severe combined immunodeficient hosts and the subsequent evaluation of human hematopoiesis in the recipient mice. 5,6 By means of this assay, human HSCs were shown to be highly enriched within the CD34 ϩ CD38 Ϫ fraction of adult bone marrow and cord blood. 7,8 Previously, we could show that following their injection into day-3.5-old murine blastocysts, murine bone marrow-derived HSCs generate chimeric fetal and adult mice. 9 Now we describe the first step toward an experimental assay system that enables us to analyze the early human hematopoietic system and the developmental potentials of human HSCs. We have injected human cord blood (CB)-derived CD34 ϩ progenitor and CD34 ϩ CD38 Ϫ stem cells into murine blastocysts and could follow the fate of injected human cells during murine embryogenesis and adulthood. Study designCord blood cells were collected from healthy, full-term infants, and sodium citrate was added as an anticoagulant. For CB sampling, approved institutional procedures for obtaining informed consent according to the declaration of Helsinki were observed (Ethics Committee, Medical Faculty, University of Freiburg, Germany); the use of human CB cells for injection into murine blastocysts was approved by the responsible ethics committee (Ethics Committee, Medical Faculty, University of Würzburg, Germany). Low-density cells (less than 1.077 g/mL) were pooled from several CB samples. For CD34 enrichment, CB samples were pretreated with an antihuman Fc antibody (Pharmingen, Franklin Lakes, NJ), incubated with an anti-CD34 antibody conjugated to magnetic beads (Miltenyi Biotec, Auburn, CA) and transferred to an affinity column for positive selection. For sorting, the cells were labeled with anti-CD34-phycoerythrin (clone 581, Coulter Immunotech, Marseille, France) and anti-CD38-fluorescein isothiocyanate (clone T16, Coulter Immunotech), gated for an intermediate forward scatter and low sideward scatter profile, and then sorted for either CD34 ϩ CD38 Ϫ or CD34 ϩ CD38 ϩ . 10 Murine blastocysts were isolated from donor TK Ϫ C57BL/6 or NMRI mice on day 3.5 of gestation, and 70 to 100 human CB CD34 ϩ or CD34 ϩ CD38 Ϫ cells were injected into each blastocyst. Blastocysts were retransferred into foster mothers. All animal experimentation was done in accordance with approved institutional ...

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Stem cell systems represent an effective and powerful approach for tissue development and regeneration of diverse tissue types. Common and defining features of these exceptional cells are the capacity for self-renewal and the potential for differentiation into multiple mature cell types. Recently, surprising new observations have indicated that stem cells isolated from one adult tissue can also give rise to mature cells of other cell lineages, irrespective of classical germ layer designations. This discovery has resulted in quantum leaps in both scientific knowledge and the potential applications of stem cells. The new findings contradict central dogmas of commitment and differentiation of stem and progenitor cells. However, the true potential of somatic stem cells is just emerging and the new findings have to be defined more fully and integrated into a unifying model of stem cell potential and behavior. Here we analyze the developmental potential of hematopoietic stem cells of mouse and man following their injection into the murine preimplantation blastocyst, an environment that allows the development of all cell lineages. In addition, we discuss the emerging lines of evidence of the developmental plasticity of hematopoietic and other somatic stem cells and consider how cellular memory of transcriptional states is established and may be potentially involved in this phenomenon.

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Like many other animals, mammals develop from fertilized oocytes – the ultimate stem cells. As embryogenesis proceeds, most cells lose developmental potential and eventually become restricted to a specific cell lineage. The result is the formation of a complete and structured mature organism with complex organs composed of a great variety of mature, mostly mitotically quiescent effector cells. However, along the way, some exceptional cells, known as somatic stem cells (SSCs) are set aside and maintain a high proliferation and tissue-specific differentiation potential. SSCs, in contrast to embryonic stem (ES) cells, which are able to give rise to all cell types of the body, have been regarded as being more limited in their differentiation potential in the sense that they were thought to be committed exclusively to their tissue of origin. However, recent studies have demonstrated that somatic stem cells from a given tissue can also contribute to heterologous tissues and thus show a broad nontissue restricted differentiation potential. The question arises: how plastic are somatic stem cells? To provide a tentative answer, we describe and review here recent investigations into the developmental potentials of two somatic stem cell types, namely hematopoietic and neural stem cells.