regenerative medicine – Drug Discovery Opinion

Blocking Tumour Suppressors May Allow Damage Repair

The ability to grow a replacement tail or limb, present in some species of amphibians such as salamanders and newts, has been lost in vertebrates. Earlier this year, scientists from the Wistar Institute and Washington University showed that mice lacking p21, a downstream target of the tumour suppressor p53, have a greater regenerative capacity than normal mice and now scientists at the Stanford University School of Medicine have shown the importance of other tumour suppression pathways in limiting regeneration in mammalian muscle cells.

Differentiated mammalian muscle cells are not normally able to divide but the team found that mouse myocytes can be induced to re-enter the cell cycle and begin proliferating by blocking the expression of two tumour suppressors, retinoblastoma protein (Rb) and ARF, a protein transcribed from an alternate reading frame of the INK4a locus. ARF is found in birds and mammals but not in lower vertebrates and, interestingly, is expressed at lower than normal levels in mammalian livers – the only organ with some regenerative capacity. When RNA interference was used to temporarily block expression of Rb and Arf in cultured mouse myocytes, the cells lost their differentiated properties, re-entered the cell cycle and began to proliferate. The cells are incorporated into existing muscle fibres when transplanted into mice, but only if Rb function was restored. Without functioning Rb, the new cells proliferated excessively and disrupted the structure of the muscle tissue.

Previous studies had shown that suppression of the Rb gene alone causes newt muscle cells, but not mammalian muscle cells, to re-enter the cell cycle.

Although knocking down tumour suppressor genes has obvious potential dangers, temporary silencing of gene expression may eventually allow regeneration of cardiac or pancreatic tissue if the technique is also successful in other cell types.

The study is published in the journal Cell Stem Cell.

Repairing the Damage

mouse and salamander — Salamander photo: Flickr - brian.gratwicke; Mouse photo: Wikimedia - Ilmari Karonen

Although some species of amphibians – such as salamanders and newts – are able to regenerate lost or damaged tissue, the capacity for regeneration diminished as vertebrates evolved and adult mammals generally have limited regenerative capacity. Over a decade ago, it was discovered that the Murphy Roths Large (MRL) mouse strain was able to regenerate new epidermis, new hair follicles, and new cartilage to repair punch holes in the ears, unlike other strains of mice that close the holes with scar tissue. The MRL mice were later shown to have the ability to repair damaged heart muscle and spinal cord with restoration of normal structure and function and to have some limited capacity for digit re-growth. Scientists have been trying to identify the gene or genes that are responsible for the increased capacity for regeneration in MRL mice and researchers at the Wistar Institute and Washington University have now shown that the p21 gene is involved in regulating the regeneration process.

The team showed that p21, a cell cycle regulator, was found to be consistently inactive in cells from MRL mice. When they looked at p21 knockout mice, they found that, unlike normal mice which heal wounds by forming a scar, mice that lack p21 begin by forming a blastema, a mass of cells capable of rapid growth and de-differentiation which behave more like embryonic stem cells than adult mammalian cells. The p21 knockout mice were able to replace missing or damaged tissue with healthy tissue that showed no signs of scarring.

Since the cyclin-dependent kinase p21 is one of the best characterized downstream targets of the tumour suppressor p53, knockout of p21 might be expected to increase the incidence of cancer and other studies have suggested that p21-deficient mice develop tumours at an earlier age than their wild-type counterparts and are more susceptible to the effects of some carcinogens. Although increased DNA damage was observed in the present study, there was also an increased incidence of apoptosis and no net increase in the incidence of cancer. If MRL mice and p21 knockout mice are a good model for tissue regeneration in humans, temporary inactivation of the p21 gene could eventually be used to speed up wound healing in people.

The study is published in PNAS.

New Hope for Damaged Hearts

A number of organs, including the heart, have limited regenerative powers, but US scientists have now shown that fully differentiated cardiac muscle cells can be induced to proliferate and regenerate. Writing in the journal Cell, they show that the growth factor neuregulin 1, which plays a role in early development of the heart and nervous system, induces mononucleated, but not binucleated, cardiomyocytes to divide in vitro by acting on the receptor tyrosine kinase, ErbB4. In mice, genetic inactivation of ErbB4 was shown to reduce cardiomyocyte proliferation, whereas increasing ErbB4 expression enhanced proliferation. Following heart attack in adult mice, daily intraperitoneal injection of neuregulin 1 for 12 weeks led to regeneration of the heart muscle and improved function. Unlike the control mice, the treated animals showed reduced signs of heart failure such as left-ventricular dilation and cardiac hypertrophy. If the neuregulin/ErbB4 signalling pathway plays the same role in human heart muscle, stimulating proliferation of differentiated cardiomyocytes by activation of this pathway may provide an alternative to stem cell therapy to regenerate damaged heart muscle in patients with heart failure or children with congenital heart defects. Since ErbB receptor tyrosine kinases and neuregulins have oncogenic potential and may cause proliferation of other tissues, a full safety assessment would be needed before any clinical studies.

Neuregulin 1 has previously been associated with susceptibility to schizophrenia and has also been shown to protect neurones following stroke.

Mending a Broken Heart

Heart disease is a leading cause of death and illness in the developed world and, once damaged, the heart has very limited capacity for regeneration. Following a heart attack, if blood flow is not restored to the heart muscle within 20-40 minutes, the muscle cells (cardiomyocytes) will die. The dead cells are replaced by scar tissue which does not contract or pump as well as healthy heart tissue.

Writing in the journal Nature, Jun Takeuchi and Benoit Bruneauat at the Gladstone Institute of Cardiovascular Disease have now identified a cocktail of three proteins that can turn mouse mesoderm into cardiac muscle cells (cardiomyocytes). Mesoderm is one of the three primary germ cell layers in the very early embryo – the others are the ectoderm and the endoderm – that can differentiate to give a number of tissues such as bone, blood, and muscle, including heart muscle. The three key proteins are the cardiac transcription factors, GATA4 and TBX5, which are believed to be involved in heart development and function, and a cardiac-specific subunit of BAF chromatin-remodelling complexes, Baf60c. Defects in the genes for these proteins have been linked to abnormal development and defects in the heart.

A combination of all three proteins was shown to direct differentiation of mouse mesoderm specifically into cardiac muscle cardiomyocytes that beat rhythmically, just like normal heart cells. Although, so far, only cells from very early mouse embryos have been turned into cardiomyocytes, Takeuchi and Bruneauat hope that their work will help to understand how new cardiomyocytes can be produced for use in regenerative medicine to treat heart disease.

Boost for Stem Cells

Mesenchymal stem cells Mesenchymal stem cells can differentiate into a variety of cells including osteoblasts and chondrocytes A new discovery increases the likelihood that treatments could eventually boost specific subtypes of stem cells, and promote self healing following injury or disease. In response to tissue injury or disease, progenitor cells are mobilised from bone marrow into the tissues and contribute to tissue repair and regeneration. Different subpopulations of progenitor cells are recruited depending on the type and site of disease or tissue injury. Although it is becoming apparent that specific types of progenitor cells could be used to treat a variety of diseases, there are practical and technical difficulties in harvesting, isolation, ex vivo expansion, and delivery of these cells. An alternative strategy would be to directly stimulate the mobilisation of specific populations of stem cells from the bone marrow into the circulation. Scientists at Imperial College, London, have shown that the mobilisation of progenitor cell subsets can be differentially regulated by growth factors that affect their retention in bone marrow and cell-cycle status. Treatment of mice with granulocyte colony-stimulating factor (G-CSF) followed by the CXCR4 antagonist, Mozobil™ (AMD3100), caused maximal mobilisation of hematopoietic stem cells (HPCs) and neutrophils. On the other hand, treatment with vascular endothelial growth factor (VEGF) followed by Mozobil™ maximally stimulated mobilisation of endothelial progenitor cells (EPCs) and stromal progenitor cells (SPCs). By showing that different factors and molecular mechanisms regulate the mobilisation of discrete populations of progenitor cells from the bone marrow, the study has far reaching implications for regenerative medicine. Although it is not yet clear whether such an approach would, for example, speed cardiac repair following myocardial infarction or promote bone healing after a fracture, the ability to selectively mobilise different stem cell populations will enable further research in these areas. The study is published in full in the January 9th issue of Cell Stem Cell.