Koegler, Gesine; Sensken, Sandra; Airey, Judith A; Trapp, Thorsten; Mueschen, Markus; Feldhahn, Niklas; Liedtke, Stefanie; Sorg, Ruediger V; Fischer, Johannes; Rosenbaum, Claudia; Greschat, Susanne; Knipper, Andreas; Bender, Joerg; Degistirici, Oezer; Gao, Jizong; Caplan, Arnold I; Colletti, Evan J; Almeida-Porada, Graca; Mueller, Hans W; Zanjani, Esmail; Wernet, Peter

Journal of Experimental Medicine [J. Exp. Med.]. Vol. 200, no. 2, pp. 123-135. 19 Jul 2004.

Here a new, intrinsically pluripotent, CD45-negative population from human cord blood, termed unrestricted somatic stem cells (USSCs) is described. This rare population grows adherently and can be expanded to 10 super(15) cells without losing pluripotency. In vitro USSCs showed homogeneous differentiation into osteoblasts, chondroblasts, adipocytes, and hematopoietic and neural cells including astrocytes and neurons that express neurofilament, sodium channel protein, and various neurotransmitter phenotypes. Stereotactic implantation of USSCs into intact adult rat brain revealed that human Tau-positive cells persisted for up to 3 mo and showed migratory activity and a typical neuron-like morphology. In vivo differentiation of USSCs along mesodermal and endodermal pathways was demonstrated in animal models. Bony reconstitution was observed after transplantation of USSC-loaded calcium phosphate cylinders in nude rat femurs. Chondrogenesis occurred after transplanting cell-loaded gelfoam sponges into nude mice. Transplantation of USSCs in a noninjury model, the preimmune fetal sheep, resulted in up to 5% human hematopoietic engraftment. More than 20% albumin-producing human parenchymal hepatic cells with absence of cell fusion and substantial numbers of human cardiomyocytes in both atria and ventricles of the sheep heart were detected many months after USSC transplantation. No tumor formation was observed in any of these animals.

Rice, CM; Scolding, NJ*

Lancet [Lancet]. Vol. 364, no. 9429, pp. 193-199. 0-16 Jul 2004.

Much excitement has surrounded recent breakthroughs in embryonic stem-cell research. Of lower profile, but no less exciting, are the advances in the field of adult stem-cell research, and their implications for cell therapy. Clinical experience from use of adult haemopoietic stem cells in haematology will facilitate and hasten transition from laboratory to clinic - indeed, clinical trials using adult human stem cells are already in progress in some disease states, including myocardial ischaemia. Here, with particular reference to neurology, we review processes that might underlie apparent changes in adult cell phenotype. We discuss implications these processes might have for the development of new therapeutic strategies using adult stem cells.

Pochampally, RR; Neville, BT; Schwarz, EJ; Li, MM; Prockop, DJ

Proceedings of the National Academy of Sciences, USA [Proc. Natl. Acad. Sci. USA]. Vol. 101, no. 25, pp. 9282-9285. 22 Jun 2004.

Cell fusion was recently reported to account for the plasticity of adult stem cells in vivo. Adult stem cells, referred to as mesenchymal stem cells or marrow stromal cells, from rat marrow, were infused into 1.5- to 2-day-old chick embryos. After 4 days, the rat cells had expanded 1.3- to 33-fold in one- third of surviving embryos. The cells engrafted into many tissues, and no multinuclear cells were detected. The most common site of engraftment was the heart, apparently because the cells were infused just above the dorsal aorta. Some of the cells in the heart expressed cardiotin, and alpha -heavy-chain myosin. GFP super(+) cells reisolated from the embryos had a rat karyotype. Therefore, the cells engrafted and partially differentiated without evidence of cell fusion.

Dor, Yuval; Brown, Juliana; Martinez, Olga I; Melton, Douglas A

Nature [Nature]. Vol. 429, no. 6987, pp. 41-46. 6 May 2004.

How tissues generate and maintain the correct number of cells is a fundamental problem in biology. In principle, tissue turnover can occur by the differentiation of stem cells, as is well documented for blood, skin and intestine, or by the duplication of existing differentiated cells. Recent work on adult stem cells has highlighted their potential contribution to organ maintenance and repair. However, the extent to which stem cells actually participate in these processes in vivo is not clear. Here we introduce a method for genetic lineage tracing to determine the contribution of stem cells to a tissue of interest. We focus on pancreatic beta -cells, whose postnatal origins remain controversial. Our analysis shows that pre-existing beta -cells, rather than pluripotent stem cells, are the major source of new beta -cells during adult life and after pancreatectomy in mice. These results suggest that terminally differentiated beta -cells retain a significant proliferative capacity in vivo and cast doubt on the idea that adult stem cells have a significant role in beta -cell replenishment.

Wagers, AJ; Weissman, IL

Cell [Cell]. Vol. 116, no. 5, pp. 639-648. Mar 2004.

Recent years have seen much excitement over the possibility that adult mammalian stem cells may be capable of differentiating across tissue lineage boundaries, and as such may represent novel, accessible, and very versatile effectors of therapeutic tissue regeneration. Yet studies proposing such 'plasticity' of adult somatic stem cells remain controversial, and in general, existing evidence suggests that in vivo such unexpected transformations are exceedingly rare and in some cases can be accounted for by equally unexpected alternative explanations.

Matsuura, K; Nagai, T; Nishigaki, N; Oyama, T; Nishi, J; Wada, H; Sano, M; Toko, H; Akazawa, H; Sato, T; Nakaya, H; Kasanuki, H; Komuro, I

Journal of Biological Chemistry [J. Biol. Chem.]. Vol. 279, no. 12, pp. 11384-11391. 19 Mar 2004.

Although somatic stem cells have been reported to exist in various adult organs, there have been few reports concerning stem cells in the heart. We here demonstrate that Sca-1-positive (Sca-1+) cells in adult hearts have some of the features of stem cells. Sca-1+ cells were isolated from adult murine hearts by a magnetic cell sorting system and cultured on gelatin-coated dishes. A fraction of Sca-1+ cells stuck to the culture dish and proliferated slowly. When treated with oxytocin, Sca-1+ cells expressed genes of cardiac transcription factors and contractile proteins and showed sarcomeric structure and spontaneous beating. Isoproterenol treatment increased the beating rate, which was accompanied by the intracellular Ca super(2+) transients. The cardiac Sca-1+ cells expressed oxytocin receptor mRNA, and the expression was up-regulated after oxytocin treatment. Some of the Sca-1+ cells expressed alkaline phosphatase after osteogenic induction and were stained with Oil-Red O after adipogenic induction. These results suggest that Sca-1+ cells in the adult murine heart have potential as stem cells and may contribute to the regeneration of injured hearts.

Sekiya, I; Larson, BL; Vuoristo, JT; Cui, J-G; Prockop, DJ

Journal of Bone and Mineral Research [J. Bone Miner. Res.]. Vol. 19, no. 2, pp. 256-264. Feb 2004.

We assayed gene expressions during adipogenesis of human MSCs. Microarray assays demonstrated time-dependent increases in expression of 67 genes, including 2 genes for transcription factors that were not previously shown to be expressed during adipogenesis. Introduction: Increased numbers of bone marrow adipocytes have been observed in patients with osteoporosis and aplastic anemia, but the pathological mechanisms remain unknown. Recently, microarray assays for mRNAs were used to follow adipogenic differentiation of the preadipocytic cell line, 3T3-L1, but adipogenic differentiation has not been examined in primary cells from bone marrow. Here we defined the sequence of gene expression during the adipogenesis ex vivo of human cells from bone marrow referred to as either mesenchymal stem cells or marrow stromal cells (MSCs). Materials and Methods: MSCs were plated at extremely low densities to generate single-cell derived colonies, and adipogenic differentiation of the colonies assayed by accumulation of fat vacuoles, time-lapse photomicroscopy, microarrays, and reverse transcriptase-polymerase chain reaction (RT-PCR) assays. Results and Conclusions: About 30% of the colonies differentiated to adipocytes in 14 days and about 60% in 21 days. Cell proliferation was inhibited by approximately 50% in adipogenic medium. The differentiation occurred primarily at the center of the colonies, and a few adipocytes that formed near the periphery migrated toward the centers. RT-PCR assays demonstrated that the differentiation was accompanied by increases in a series of genes previously shown to increase with adipogenic differentiation: peroxisome proliferator activated receptor gamma , CCAAT enhancer-binding protein alpha , acylCoA synthetase, lipoprotein lipase, and fatty acid binding protein 4. We also followed differentiation with microarray assays. Sixty-seven genes increased more than 10-fold at day 1 and 20-fold at day 7, 14, or 21. Many of the genes identified were previously shown to be expressed during adipocytic differentiation. However, others, such as zinc finger E-box binding protein and zinc finger protein 145, were not. This study should serve as a basis for future study to clarify the mechanisms of adipocyte differentiation of MSCs.

Alonso, L; Fuchs, E

Genes & Development [Genes Dev.]. Vol. 17, no. 10, pp. 1189-1200. 15 May 2003.

Although it seems unlikely that adult stem cells harvested from one tissue might be reprogrammed to take on the characteristics of a different cell type, several tantalizing results have encouraged researchers to consider this possibility. Before scientists can begin to define the limits of adult stem cell plasticity, they need to understand the signals that instruct multipotent cells to self-renew and differentiate within the lineages of their resident tissues. Skin is an excellent model system in which to explore these fundamental mechanisms, because skin keratinocytes are easily accessible and are one of the few adult stem cell types that can be maintained and propagated in vitro. These cells have already been engrafted long term to replace damaged epidermis on burn patients. Now skin biologists have begun to identify some of the key steps involved in generating a functional tissue from multipotent stem cells.

Lee, H-S; Crane, GG; Merok, JR; Tunstead, JR; Hatch, NL; Panchalingam, K; Powers, MJ; Griffith, LG; Sherley, JL*

Biotechnology and Bioengineering [Biotechnol. Bioeng.]. Vol. 83, no. 7, pp. 760-771. 2003.

Adult stem cells have potential use for several biomedical applications, including cell replacement therapy, gene therapy, and tissue engineering. However, such applications have been limited due to difficulties encountered in expanding functional adult stem cells. We have developed a new approach to the problem of adult stem cell expansion based on the suppression of asymmetric cell kinetics (SACK). We postulated that asymmetric cell kinetics, required for adult stem cell function, were a major barrier to their expansion in culture. As such, conversion of adult stem cells from asymmetric cell kinetics to symmetric cell kinetics would promote their exponential expansion and longterm propagation in culture. The purine nucleoside xanthosine (Xs), which promotes guanine ribonucleotide biosynthesis, can be used to reversibly convert cells from asymmetric cell kinetics to symmetric cell kinetics. We used Xs supplementation to derive clonal epithelial cell lines from adult rat liver that have properties of adult hepatic stem cells. The properties of two Xs-derived cell lines, Lig-8 and Lig-13, are described in detail and compared to properties of adult rat hepatic cell lines derived without Xs supplementation. The Xs-derived cell lines exhibit Xs-dependent asymmetric cell kinetics and Xs-dependent expression of mature hepatic differentiation markers. Interestingly, Lig-8 cells produce progeny with properties consistent with hepatocyte differentiation, while Lig-13 progeny cells have properties consistent with bile duct epithelium differentiation. A stable adult cholangiocyte stem cell line has not been previously described. Consistent with the principles of their derivation, the SACK-derived hepatic cell lines exhibit neither senescence nor tumorigenic properties, and their differentiation properties are stable after longterm culture. These characteristics of SACK-derived stem cell lines underscore asymmetric cell kinetics as an essential adult stem cell property with potential to be the basis for a general approach to expansion and propagation of diverse adult stem cells.

Ramalho-Santos, M; Yoon, S; Matsuzaki, Y; Mulligan, RC; Melton, DA*

Science (Washington) [Science (Wash.)]. Vol. 298, no. 5593, pp. 597-600. 18 Oct 2002.

The transcriptional profiles of mouse embryonic, neural, and hematopoietic stem cells were compared to define a genetic program for stem cells. A total of 216 genes are enriched in all three types of stem cells, and several of these genes are clustered in the genome. When compared to differentiated cell types, stem cells express a significantly higher number of genes (represented by expressed sequence tags) whose functions are unknown. Embryonic and neural stem cells have many similarities at the transcriptional level. These results provide a foundation for a more detailed understanding of stem cell biology.

Avots, A; Harder, F; Schmittwolf, C; Petrovic, S; Mueller, AM

Immunological Reviews [Immunol. Rev.]. Vol. 187, pp. 9-21. Sep 2002.

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.