Structure-activity relationship of dihydropyridines for rhabdomyosarcoma.

Childhood muscle-related cancer rhabdomyosarcoma is a rare disease with a 50-year unmet clinical need for the patients presented with advanced disease. The rarity of ∼350 cases per year in North America generally diminishes the viability of large-scale, pharmaceutical industry driven drug development efforts for rhabdomyosarcoma. In this study, we performed a large-scale screen of 640,000 compounds to identify the dihydropyridine (DHP) class of anti-hypertensives as a priority compound hit. A structure-activity relationship was uncovered with increasing cell growth inhibition as side chain length increases at the ortho and para positions of the parent DHP molecule. Growth inhibition was consistent across n = 21 rhabdomyosarcoma cell line models. Anti-tumor activity in vitro was paralleled by studies in vivo. The unexpected finding was that the action of DHPs appears to be other than on the DHP receptor (i.e., L-type voltage-gated calcium channel). These findings provide the basis of a medicinal chemistry program to develop dihydropyridine derivatives that retain anti-rhabdomyosarcoma activity without anti-hypertensive effects.

Induction of muscle stem cell quiescence by the secreted niche factor Oncostatin M.

The balance between stem cell quiescence and proliferation in skeletal muscle is tightly controlled, but perturbed in a variety of disease states. Despite progress in identifying activators of stem cell proliferation, the niche factor(s) responsible for quiescence induction remain unclear. Here we report an in vivo imaging-based screen which identifies Oncostatin M (OSM), a member of the interleukin-6 family of cytokines, as a potent inducer of muscle stem cell (MuSC, satellite cell) quiescence. OSM is produced by muscle fibers, induces reversible MuSC cell cycle exit, and maintains stem cell regenerative capacity as judged by serial transplantation. Conditional OSM receptor deletion in satellite cells leads to stem cell depletion and impaired regeneration following injury. These results identify Oncostatin M as a secreted niche factor responsible for quiescence induction, and for the first time establish a direct connection between induction of quiescence, stemness, and transplantation potential in solid organ stem cells.

The microprotein Minion controls cell fusion and muscle formation.

Although recent evidence has pointed to the existence of small open reading frame (smORF)-encoded microproteins in mammals, their function remains to be determined. Skeletal muscle development requires fusion of mononuclear progenitors to form multinucleated myotubes, a critical but poorly understood process. Here we report the identification of Minion (microprotein inducer of fusion), a smORF encoding an essential skeletal muscle specific microprotein. Myogenic progenitors lacking Minion differentiate normally but fail to form syncytial myotubes, and Minion-deficient mice die perinatally and demonstrate a marked reduction in fused muscle fibres. The fusogenic activity of Minion is conserved in the human orthologue, and co-expression of Minion and the transmembrane protein Myomaker is sufficient to induce cellular fusion accompanied by rapid cytoskeletal rearrangement, even in non-muscle cells. These findings establish Minion as a novel microprotein required for muscle development, and define a two-component programme for the induction of mammalian cell fusion. Moreover, these data also significantly expand the known functions of smORF-encoded microproteins.

Transient delivery of modified mRNA encoding TERT rapidly extends telomeres in human cells.

Telomere extension has been proposed as a means to improve cell culture and tissue engineering and to treat disease. However, telomere extension by nonviral, nonintegrating methods remains inefficient. Here we report that delivery of modified mRNA encoding TERT to human fibroblasts and myoblasts increases telomerase activity transiently (24-48 h) and rapidly extends telomeres, after which telomeres resume shortening. Three successive transfections over a 4 d period extended telomeres up to 0.9 kb in a cell type-specific manner in fibroblasts and myoblasts and conferred an additional 28 ± 1.5 and 3.4 ± 0.4 population doublings (PDs), respectively. Proliferative capacity increased in a dose-dependent manner. The second and third transfections had less effect on proliferative capacity than the first, revealing a refractory period. However, the refractory period was transient as a later fourth transfection increased fibroblast proliferative capacity by an additional 15.2 ± 1.1 PDs, similar to the first transfection. Overall, these treatments led to an increase in absolute cell number of more than 10(12)-fold. Notably, unlike immortalized cells, all treated cell populations eventually stopped increasing in number and expressed senescence markers to the same extent as untreated cells. This rapid method of extending telomeres and increasing cell proliferative capacity without risk of insertional mutagenesis should have broad utility in disease modeling, drug screening, and regenerative medicine.

Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein.

A method for non-invasive visualization of genetically labeled cells in animal disease models with micrometer-level resolution would greatly facilitate development of cell-based therapies. Imaging of fluorescent proteins (FPs) using red excitation light in the 'optical window' above 600 nm is one potential method for visualizing implanted cells. However, previous efforts to engineer FPs with peak excitation beyond 600 nm have resulted in undesirable reductions in brightness. Here we report three new red-excitable monomeric FPs obtained by structure-guided mutagenesis of mNeptune. Two of these, mNeptune2 and mNeptune2.5, demonstrate improved maturation and brighter fluorescence than mNeptune, whereas the third, mCardinal, has a red-shifted excitation spectrum without reduction in brightness. We show that mCardinal can be used to non-invasively and longitudinally visualize the differentiation of myoblasts into myocytes in living mice with high anatomical detail.

Rejuvenation of the muscle stem cell population restores strength to injured aged muscles.

The elderly often suffer from progressive muscle weakness and regenerative failure. We demonstrate that muscle regeneration is impaired with aging owing in part to a cell-autonomous functional decline in skeletal muscle stem cells (MuSCs). Two-thirds of MuSCs from aged mice are intrinsically defective relative to MuSCs from young mice, with reduced capacity to repair myofibers and repopulate the stem cell reservoir in vivo following transplantation. This deficiency is correlated with a higher incidence of cells that express senescence markers and is due to elevated activity of the p38α and p38ß mitogen-activated kinase pathway. We show that these limitations cannot be overcome by transplantation into the microenvironment of young recipient muscles. In contrast, subjecting the MuSC population from aged mice to transient inhibition of p38α and p38ß in conjunction with culture on soft hydrogel substrates rapidly expands the residual functional MuSC population from aged mice, rejuvenating its potential for regeneration and serial transplantation as well as strengthening of damaged muscles of aged mice. These findings reveal a synergy between biophysical and biochemical cues that provides a paradigm for a localized autologous muscle stem cell therapy for the elderly.

A critical role for AID in the initiation of reprogramming to induced pluripotent stem cells.

Mechanistic insights into the reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs) are limited, particularly for early acting molecular regulators. Here we use an acute loss of function approach to demonstrate that activation-induced deaminase (AID) activity is necessary for the initiation of reprogramming to iPSCs. While AID is well known for antibody diversification, it has also recently been shown to have a role in active DNA demethylation in reprogramming toward pluripotency and development. These findings suggested a potential role for AID in iPSC generation, yet, iPSC yield from AID-knockout mouse fibroblasts was similar to that of wild-type (WT) fibroblasts. We reasoned that an acute loss of AID function might reveal effects masked by compensatory mechanisms during development, as reported for other proteins. Accordingly, we induced an acute reduction (>50%) in AID levels using 4 different shRNAs and determined that reprogramming to iPSCs was significantly impaired by 79 ± 7%. The deaminase activity of AID was critical, as coexpression of WT but not a catalytic mutant AID rescued reprogramming. Notably, AID was required only during a 72-h time window at the onset of iPSC reprogramming. Our findings show a critical role for AID activity in the initiation of reprogramming to iPSCs.

Transient inactivation of Rb and ARF yields regenerative cells from postmitotic mammalian muscle.

An outstanding biological question is why tissue regeneration in mammals is limited, whereas urodele amphibians and teleost fish regenerate major structures, largely by cell cycle reentry. Upon inactivation of Rb, proliferation of postmitotic urodele skeletal muscle is induced, whereas in mammalian muscle this mechanism does not exist. We postulated that a tumor suppressor present in mammals but absent in regenerative vertebrates, the Ink4a product ARF (alternative reading frame), is a regeneration suppressor. Concomitant inactivation of Arf and Rb led to mammalian muscle cell cycle reentry, loss of differentiation properties, and upregulation of cytokinetic machinery. Single postmitotic myocytes were isolated by laser micro-dissection-catapulting, and transient suppression of Arf and Rb yielded myoblast colonies that retained the ability to differentiate and fuse into myofibers upon transplantation in vivo. These results show that differentiation of mammalian cells is reversed by inactivation of Arf and Rb and support the hypothesis that Arf evolved at the expense of regeneration.

Reprogramming towards pluripotency requires AID-dependent DNA demethylation.

Reprogramming of somatic cell nuclei to yield induced pluripotent stem (iPS) cells makes possible derivation of patient-specific stem cells for regenerative medicine. However, iPS cell generation is asynchronous and slow (2-3 weeks), the frequency is low (<0.1%), and DNA demethylation constitutes a bottleneck. To determine regulatory mechanisms involved in reprogramming, we generated interspecies heterokaryons (fused mouse embryonic stem (ES) cells and human fibroblasts) that induce reprogramming synchronously, frequently and fast. Here we show that reprogramming towards pluripotency in single heterokaryons is initiated without cell division or DNA replication, rapidly (1 day) and efficiently (70%). Short interfering RNA (siRNA)-mediated knockdown showed that activation-induced cytidine deaminase (AID, also known as AICDA) is required for promoter demethylation and induction of OCT4 (also known as POU5F1) and NANOG gene expression. AID protein bound silent methylated OCT4 and NANOG promoters in fibroblasts, but not active demethylated promoters in ES cells. These data provide new evidence that mammalian AID is required for active DNA demethylation and initiation of nuclear reprogramming towards pluripotency in human somatic cells.

Thymic progenitor homing and lymphocyte homeostasis are linked via S1P-controlled expression of thymic P-selectin/CCL25.

Thymic T cell progenitor (TCP) importation is a periodic, gated event that is dependent on the expression of functional P-selectin ligands on TCPs. Occupancy of intrathymic TCP niches is believed to negatively regulate TCP importation, but the nature of this feedback mechanism is not yet resolved. We show that P-selectin and CCL25 are periodically expressed in the thymus and are essential parts of the thymic gate-keeping mechanism. Periodicity of thymic TCP receptivity and the size of the earliest intrathymic TCP pool were dependent on the presence of functional P-selectin ligand on TCPs. Furthermore, we show that the numbers of peripheral blood lymphocytes directly affected thymic P-selectin expression and TCP receptivity. We identified sphingosine-1-phosphate (S1P) as one feedback signal that could mediate influence of the peripheral lymphocyte pool on thymic TCP receptivity. Our findings suggest a model whereby thymic TCP importation is controlled by both early thymic niche occupancy and the peripheral lymphocyte pool via S1P.

Extensive fusion of haematopoietic cells with Purkinje neurons in response to chronic inflammation.

Transplanted bone marrow-derived cells (BMDCs) have been reported to fuse with cells of diverse tissues, but the extremely low frequency of fusion has led to the view that such events are biologically insignificant. Nonetheless, in mice with a lethal recessive liver disease (tyrosinaemia), transplantation of wild-type BMDCs restored liver function by cell fusion and prevented death, indicating that cell fusion can have beneficial effects. Here we report that chronic inflammation resulting from severe dermatitis or autoimmune encephalitis leads to robust fusion of BMDCs with Purkinje neurons and formation of hundreds of binucleate heterokaryons per cerebellum, a 10-100-fold higher frequency than previously reported. Single haematopoietic stem-cell transplants showed that the fusogenic cell is from the haematopoietic lineage and parabiosis experiments revealed that fusion can occur without irradiation. Transplantation of rat bone marrow into mice led to activation of dormant rat Purkinje neuron-specific genes in BMDC nuclei after fusion with mouse Purkinje neurons, consistent with nuclear reprogramming. The precise neurological role of these heterokaryons awaits elucidation, but their frequency in brain after inflammation is clearly much higher than previously appreciated.

Origin and distribution of bone marrow-derived cells in the central nervous system in a mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is associated with increased numbers of microglia within the central nervous system (CNS). However, it is unknown whether the microgliosis results from proliferation of CNS resident microglia, or recruitment of bone marrow (BM)-derived microglial precursors. Here we assess the distribution and number of BM-derived cells in spinal cord using transplantation of green fluorescent protein (GFP)-labeled BM cells into myelo-ablated mice over-expressing human mutant superoxide dismutase 1 (mSOD), a murine model of ALS. Transplantation of GFP+ BM did not affect the rate of disease progression in mSOD mice. Mean numbers of microglia and GFP+ cells in spinal cords of control mice were not significantly different from those in asymptomatic mSOD mice and showed no change with animal age. The number of GFP+ cells and microglia (F4/80+ and CD11b+ cells) within the spinal cord of mSOD mice increased compared to age-matched controls at a time when mSOD mice exhibited disease symptoms, continuing up to disease end-stage. Although we observed an increase in the number of GFP+ cells in spinal cords of mSOD mice with disease symptoms, mean numbers of GFP+ F4/80+ cells comprised less than 20% of all F4/80+ cells and did not increase with disease progression. Furthermore, the relative rates of proliferation in CD45+GFP- and CD45+GFP+ cells were comparable. Thus, we demonstrate that the microgliosis present in spinal cord tissue of mSOD mice is primarily due to an expansion of resident microglia and not to the recruitment of microglial precursors from the circulation.

Bone marrow-derived recipient cells in murine transplanted hearts: potential roles and the effect of immunosuppression.

Currently, there is intense debate regarding the origin of reparative cells in injured hearts and vasculature. To determine the contribution of recipient bone marrow (BM)-derived cells to the regeneration of cells in the vasculature of transplanted hearts and to examine the effect of immunosuppression on this phenomenon, we evaluated the fate of green fluorescent protein (GFP)-positive recipient BM cells in non-GFP-expressing cardiac allografts. C57BL/6 BM-GFP chimeric recipients underwent cardiac transplantation. Allografts were immunosuppressed with tacrolimus for 14 or 30 days post-transplantation or were saline treated. Hearts were excised and stained with markers for endothelial cells (EC) or smooth muscle cells (SMC). Colocalization with BM-derived recipient cells was evaluated using confocal microscopy with three-dimensional image analysis. Immunosuppression with tacrolimus did not affect the frequency of recipient BM-derived cell chimerism as EC or SMC phenotypes. A higher frequency of EC chimerism was found at 14 days as compared to 30 days post-transplantation in allograft hearts. BM-derived recipient cells are recruited to areas of donor vascular injury with intercalation of recipient EC and SMC in the setting of ongoing alloimmune recognition of the allograft. Our findings confirm that immunosuppression with tacrolimus does not affect the frequency of recipient BM-derived cell repopulation at an early time point 14 days post-transplantation. EC repopulation by BM-derived recipient cells was found to be an early event in transplanted allograft hearts, which decreased in frequency over time.

Minimal contribution of marrow-derived endothelial precursors to tumor vasculature.

During embryogenesis, vascular and hemopoietic cells originate from a common precursor, the hemangioblast. Recent evidence suggests the existence of endothelial precursors in adult bone marrow cells, but it is unclear whether those precursors have a role in tumor neovascularization. In this report, we demonstrate that murine bone marrow contains endothelial progenitors, which arise from a cell with self-renewing capacity, and can integrate into tumor microvasculature, albeit at a very low frequency. A transgenic double-reporter strategy allowed us to demonstrate definitively that tumor bone marrow-derived endothelial cells arise by transdifferentiation of marrow progenitors rather than by cell fusion. Single cell transplants showed that a common precursor contributes to both the hemopoietic and endothelial lineages, thus demonstrating the presence of an adult hemangioblast. Furthermore, we demonstrate that increased vascular endothelial growth factor (VEGF)-A secretion by tumor cells, as well as activation of VEGF receptor-2 in bone marrow cells does not alter the mobilization and incorporation of marrow-derived endothelial progenitors into tumor vasculature. Finally, in human umbilical cord blood cells, we show that endothelial precursors make up only approximately 1 in 10(7) mononuclear cells but are highly enriched in the CD133+ cell population. By ruling out cell fusion, we clearly demonstrate the existence of an adult hemangioblast, but the differentiation of marrow stem cells toward the endothelial lineage is an extremely rare event. Furthermore, we show that VEGF-A stimulation of hemopoietic cells does not significantly alter this process.

Circulating myogenic progenitors and muscle repair.

The capability of bone marrow derived cells to contribute to numerous peripheral tissues may hold tremendous promise for the field of regenerative medicine. In the context of skeletal muscle disease in particular, the ability of these cells to reach sites of damage through the circulation would overcome some key limitations of current cell therapy approaches. In muscle however, this non-classical repair process takes place at an exceedingly low frequency and fails to yield any measurable functional improvement. Recent advances regarding the cell types or mechanisms involved in this phenomenon may now provide direction for strategies aimed at increasing its efficiency to therapeutic levels.

Recruitment of adult thymic progenitors is regulated by P-selectin and its ligand PSGL-1.

The molecular mechanisms that direct the migration of early T lymphocyte progenitors to the thymus are unknown. We show here that P-selectin is expressed by thymic endothelium and that lymphoid progenitors in bone marrow and thymus bind P-selectin. Parabiosis, competitive thymus reconstitution and short-term homing assays indicated that P-selectin and its ligand PSGL-1 are functionally important components of the thymic homing process. Accordingly, thymi of mice lacking PSGL-1 contained fewer early thymic progenitors and had increased empty niches for prothymocytes compared with wild-type mice. Furthermore, the number of resident thymic progenitors controls thymic expression of P-selectin, suggesting that regulation of P-selectin expression by a thymic 'niche occupancy sensor' may be used to direct progenitor access.

An alternate core 2 beta1,6-N-acetylglucosaminyltransferase selectively contributes to P-selectin ligand formation in activated CD8 T cells.

Core 2 beta1,6-N-acetylglucosaminyltransferase (C2GlcNAcT) synthesizes essential core 2 O-glycans on selectin ligands, which mediate cell-cell adhesion required for lymphocyte trafficking. Although gene-deletion studies have implicated C2GlcNAcT-I in controlling selectin ligand-mediated cell trafficking, little is known about the role of the two other core 2 isoenzymes, C2GlcNAcT-II and C2GlcNAcT-III. We show that C2GlcNAcT-I-independent P-selectin ligand formation occurs in activated C2GlcNAcT-I(null) CD8 T cells. These CD8 T cells were capable of rolling under shear flow on immobilized P-selectin in a P-selectin glycoprotein ligand 1-dependent manner. RT-PCR analysis identified significant levels of C2GlcNAcT-III RNA, identifying this enzyme as a possible source of core 2 enzyme activity. Up-regulation of P-selectin ligand correlated with altered cell surface binding of the core 2-sensitive mAb 1B11, indicating that CD43 and CD45 are also physiological targets for this alternate C2GlcNAcT enzyme. Furthermore, C2GlcNAcT-I-independent P-selectin ligand induction was observed in an in vivo model. HY(tg) CD8 T cells from C2GlcNAcT-I(null) donors transferred into male recipients expressed P-selectin ligand in response to male Ag, although at reduced levels compared with wild-type HY(tg) CD8 T cells. Our data demonstrate that multiple C2GlcNAcT enzymes can contribute to P-selectin ligand formation and may cooperate with C2GlcNAcT-I in the control of CD8 T cell trafficking.

Contribution of hematopoietic stem cells to skeletal muscle.

Cells from adult bone marrow participate in the regeneration of damaged skeletal myofibers. However, the relationship of these cells with the various hematopoietic and nonhematopoietic cell types found in bone marrow is still unclear. Here we show that the progeny of a single cell can both reconstitute the hematopoietic system and contribute to muscle regeneration. Integration of bone marrow cells into myofibers occurs spontaneously at low frequency and increases with muscle damage. Thus, classically defined single hematopoietic stem cells can give rise to both blood and muscle.

Latest developments and in vivo use of the Tet system: ex vivo and in vivo delivery of tetracycline-regulated genes.

In June this year, the tetracycline-regulated gene expression system (tet system) celebrated its tenth "birthday". In the past ten years a continuous stream of changes made to the tet system's basic components has led to a remarkable improvement in its overall performance. It was not until this year, however, that the full benefits of these improvements became apparent. In particular, usage of the tet system is no longer limited to immortalized cell lines and transgenic animals. In this review, we will describe the obstacles encountered in delivering the tet system's components to primary cells and tissues as well as the methods now used to overcome them. We will also focus on a novel system that is conceptually similar but based on different antibiotic/transcription factor pairs.