iMyoblasts for ex vivo and in vivo investigations of human myogenesis and disease modeling.

Skeletal muscle myoblasts (iMyoblasts) were generated from human induced pluripotent stem cells (iPSCs) using an efficient and reliable transgene-free induction and stem cell selection protocol. Immunofluorescence, flow cytometry, qPCR, digital RNA expression profiling, and scRNA-Seq studies identify iMyoblasts as a PAX3+/MYOD1+ skeletal myogenic lineage with a fetal-like transcriptome signature, distinct from adult muscle biopsy myoblasts (bMyoblasts) and iPSC-induced muscle progenitors. iMyoblasts can be stably propagated for >12 passages or 30 population doublings while retaining their dual commitment for myotube differentiation and regeneration of reserve cells. iMyoblasts also efficiently xenoengrafted into irradiated and injured mouse muscle where they undergo differentiation and fetal-adult MYH isoform switching, demonstrating their regulatory plasticity for adult muscle maturation in response to signals in the host muscle. Xenograft muscle retains PAX3+ muscle progenitors and can regenerate human muscle in response to secondary injury. As models of disease, iMyoblasts from individuals with Facioscapulohumeral Muscular Dystrophy revealed a previously unknown epigenetic regulatory mechanism controlling developmental expression of the pathological DUX4 gene. iMyoblasts from Limb-Girdle Muscular Dystrophy R7 and R9 and Walker Warburg Syndrome patients modeled their molecular disease pathologies and were responsive to small molecule and gene editing therapeutics. These findings establish the utility of iMyoblasts for ex vivo and in vivo investigations of human myogenesis and disease pathogenesis and for the development of muscle stem cell therapeutics.

Muscular dystrophies are a group of inherited genetic diseases characterised by progressive muscle weakness. They lead to disability or even death, and no cure exists against these conditions. Advances in genome sequencing have identified many mutations that underly muscular dystrophies, opening the door to new therapies that could repair incorrect genes or rebuild damaged muscles. However, testing these ideas requires better ways to recreate human muscular dystrophy in the laboratory. One strategy for modelling muscular dystrophy involves coaxing skin or other cells from an individual into becoming 'induced pluripotent stem cells'; these can then mature to form almost any adult cell in the body, including muscles. However, this approach does not usually create myoblasts, the 'precursor' cells that specifically mature into muscle during development. This limits investigations into how disease-causing mutations impact muscle formation early on. As a response, Guo et al. developed a two-step protocol of muscle maturation followed by stem cell growth selection to isolate and grow 'induced myoblasts' from induced pluripotent stem cells taken from healthy volunteers and muscular dystrophy patients. These induced myoblasts can both make more of themselves and become muscle, allowing Guo et al. to model three different types of muscular dystrophy. These myoblasts also behave as stem cells when grafted inside adult mouse muscles: some formed human muscle tissue while others remained as precursor cells, which could then respond to muscle injury and start repair. The induced myoblasts developed by Guo et al. will enable scientists to investigate the impacts of different mutations on muscle tissue and to better test treatments. They could also be used as part of regenerative medicine therapies, to restore muscle cells in patients.

Dicer cooperates with p53 to suppress DNA damage and skin carcinogenesis in mice.

Dicer is required for the maturation of microRNA, and loss of Dicer and miRNA processing has been found to alter numerous biological events during embryogenesis, including the development of mammalian skin and hair. We have previously examined the role of miRNA biogenesis in mouse embryonic fibroblasts and found that deletion of Dicer induces cell senescence regulated, in part, by the p53 tumor suppressor. Although Dicer and miRNA molecules are thought to have either oncogenic or tumor suppressing roles in various types of cancer, a role for Dicer and miRNAs in skin carcinogenesis has not been established. Here we show that perinatal ablation of Dicer in the skin of mice leads to loss of fur in adult mice, increased epidermal cell proliferation and apoptosis, and the accumulation of widespread DNA damage in epidermal cells. Co-ablation of Dicer and p53 did not alter the timing or extent of fur loss, but greatly reduced survival of Dicer-skin ablated mice, as these mice developed multiple and highly aggressive skin carcinomas. Our results describe a new mouse model for spontaneous basal and squamous cell tumorigenesis. Furthermore, our findings reveal that loss of Dicer in the epidermis induces extensive DNA damage, activation of the DNA damage response and p53-dependent apoptosis, and that Dicer and p53 cooperate to suppress mammalian skin carcinogenesis.

MdmX regulates transformation and chromosomal stability in p53-deficient cells.

The cellular homologues Mdm2 and MdmX play critical roles in regulating the activity of the p53 tumor suppressor in damaged and non-damaged cells and during development in mice. Recently, we have utilized genetically defined primary cells and mice to reveal that endogenous levels of MdmX can also suppress multipolar mitosis and transformation in hyperploid p53-deficient cells and tumorigenesis in p53-deficient mice. These MdmX functions are not shared by Mdm2, and are distinct from the well-established ability of MdmX to complex with and inhibit p53 activity. Here we discuss some of the ramifications of MdmX loss in p53-deficient cells and mice, and we explore further the fate of MdmX/p53-double null embryonic fibroblasts undergoing multi-polar cell division using time-lapse video microscopy. We also discuss the relationship between chromosomal loss, cell proliferation, and the tumorigenic potential of p53-deficient cells lacking MdmX.

MdmX promotes bipolar mitosis to suppress transformation and tumorigenesis in p53-deficient cells and mice.

Mdm2 and MdmX are structurally related p53-binding proteins that function as critical negative regulators of p53 activity in embryonic and adult tissue. The overexpression of Mdm2 or MdmX inhibits p53 tumor suppressor functions in vitro, and the amplification of Mdm2 or MdmX is observed in human cancers retaining wild-type p53. We now demonstrate a surprising role for MdmX in suppressing tumorigenesis that is distinct from its oncogenic ability to inhibit p53. The deletion of MdmX induces multipolar mitotic spindle formation and the loss of chromosomes from hyperploid p53-null cells. This reduction in chromosome number, not observed in p53-null cells with Mdm2 deleted, correlates with increased cell proliferation and the spontaneous transformation of MdmX/p53-null mouse embryonic fibroblasts in vitro and with an increased rate of spontaneous tumorigenesis in MdmX/p53-null mice in vivo. These results indicate that MdmX has a p53-independent role in suppressing oncogenic cell transformation, proliferation, and tumorigenesis by promoting centrosome clustering and bipolar mitosis.

Base excision repair sensitizes cells to sulfur mustard and chloroethyl ethyl sulfide.

DNA repair generally functions to improve survival and reduce mutagenesis of cells that have suffered DNA damage. In this study we examine the role of nucleotide excision repair (NER) and base excision repair (BER) in recovery, mutagenesis and DNA repair in response to DNA damage inflicted by the mustard compounds, sulfur mustard (SM) and chloroethyl ethyl sulfide (CEES) in bacteria and mammalian cells. SM and CEES are compared because SM produces cross-links and monoadducts, whereas CEES produces only monoadducts that are similar to those produced by SM, thus allowing the examination of which types of lesions may be responsible for the effects seen. We find that the presence of a functional NER pathway increases survival and reduces mutagenesis, whereas the presence of a functional BER pathway reduces survival, increases mutagenesis, and decreases repair. The deleterious effects of BER appear to be due to an interaction between the DNA glycosylases and the lesions produced by SM and CEES. Possible mechanisms for BER-mediated sensitization by glycosylase action on mustard lesions are discussed.

The role of human alkyladenine glycosylase in cellular resistance to the chloroethylnitrosoureas.

To investigate the possible role of glycosylase action in causing tumor resistance, a full-length, histidine-tagged human alkyladenine glycosylase has been purified from the cloned human gene contained in a pTrc99A vector propagated in a tag alkA mutant Escherichia coli. This human enzyme releases both 3-methyladenine and 7-methylguanine from methylated DNA but in contrast to previous studies of the bacterial AlkA glycosylase, it does not release any adducts from [(3)H]chloroethylnitrosourea-modified DNA. This finding suggests that the alkyladenine DNA glycosylase-dependent resistance to the toxic effects of the chloroethylnitrosoureas reported previously in the literature may occur by a mechanism other than through direct glycosylase action.

Selective protection of non-cancer cells by hypothermia.

BACKGROUND: A serious limitation in cancer treatments is insufficient selectivity of drugs for cancer cells. We have previously demonstrated that, in contrast to p53-deficient cells, cells with wild-type p53 undergo a reversible cell cycle arrest when incubated at 28 degrees C instead of 37 degrees C. Since most of the human tumors are p53-deficient, it suggests that hypothermia may selectively protect normal cells from cytotoxic treatments that primarily target proliferating cells. MATERIALS AND METHODS: We have examined the effect of hypothermia on the survival of wild-type and p53-deficient cells exposed to the anti-tumor drug 5-fluorouracil and compared BrdU incorporation at 28 degrees C and 37 degrees C of normal and tumor cells. RESULTS: p53 wild-type fibroblasts, in contrast to p53-deficient cells, survive much higher doses of 5-fluorouracil when incubated at 28 degrees C than at 37 degrees C. Among tumor cells, the loss of the p53 function coincides with the inability to arrest cell cycle progression at low temperature and with increased sensitivity to prolonged hypothermia as a single modality. CONCLUSION: Hypothermia protects normal cells from cytotoxic treatments and may improve the therapeutic index of chemotherapy by mechanisms based on the differences in cell cycle regulation between normal and tumor cells.

Alkylation resistance of E. coli cells expressing different isoforms of human alkyladenine DNA glycosylase (hAAG).

The alkyladenine DNA glycosylase (AAG) has been cloned from mouse and humans. AAG knock out mouse cells are sensitized to a variety of alkylating and cross-linking agents suggesting AAG is active on a variety of substrates. In humans, two isoforms have been characterized that are generated by alternative splicing and contain either exon 1a or 1b (hAAG1 or hAAG2). In this study, we examine the ability of the both known isoforms of human AAG (hAAG) to contribute to survival of Escherichia coli from treatments with simple alkylating agents and cross-linking alkylating agents. Our results show that hAAG is effective at repairing methyl lesions when expressed in E. coli, but is unable to afford increased resistance to alkylating agents producing larger alkyl lesions such as ethyl lesions or lesions produced by the cross-linking alkylating agents N,N'-bis-chloroethyl-N-nitrosourea (BCNU), N-(2-chloroethyl)-N-nitrosourea (CNU) or mitomycin C. In the case of CNU, expression of hAAG causes increased sensitivity rather than resistance, suggesting deleterious effects of hAAG activity. We also demonstrate that there are no apparent differences between the two isoforms of hAAG when recovery from damage produced by all alkylating agents is tested.