John W. Lough, PhD
Department of Cell Biology, Neurobiology & Anatomy
Medical College of Wisconsin
8701 Watertown Plank Road
Milwaukee, WI 53226-0509
Phone: (414) 955-8459
FAX: (414) 955-6517
List of publications
PhD, Washington University, St. Louis, 1975
Postdoctoral, Massachusetts Institute of Technology
Program in Cell and Developmental Biology
Research Area: Cell and molecular biology of cardiac stem cell differentiation
Dr. Lough is Principal Investigator on two separate, independent projects:
1. Several laboratories have shown that human pluripotent human cells can differentiate into contractile cardiomyocytes. For example, Dr. Ana Sepac (formerly associated with this laboratory and that of Prof. Zeljko Bosnjak, MCW) observed, using both embryonic stem cells (ESCs) and induced pluripotent cells (iPSCs), that these can be induced to differentiate into cardiomyocytes, albeit with cell-line-dependent differences as shown in the Video Clips (see below). Hence, the ultimate goal of this laboratory is to develop a scheme to generate cardiomyocytes from pluripotent cells, in a fashion that is rapid, efficient, and reproducible, as well as cell-line-independent.
To fulfill this goal we are supported by a NIH-PPG designed to obtain homogeneous populations of cardiomyogenic cells from pluripotent stem cells, for the purpose of remuscularizing the myocardium following myocardial infarction. Our immediate challenge is to advance this field, which has relied on relatively inefficient cardiomyocyte differentiation occurring in embryoid bodies, to a level where cardiomyocytes can be efficiently and reproducibly produced from pluripotent cells, in universal fashion. Toward this end I am evaluating a two-step in vitro strategy that mimics heart development in vivo. First, Mesp1/Islet1/Nkx2-5-positive precardiac mesoderm is induced in all cells, in monolayer culture, mimicking precardiac events that occur during gastrulation in the embryo. Second, based on my laboratory’s previous observation that only aggregated (not isolated) embryonic precardiac mesoderm cells can terminally differentiate, aggregation is mimicked using nanofibrous scaffolds embedded with selected extracellular matrix proteins (fibronectin and collagen IV), in the presence of medium conditioned by definitive endoderm (DE), which we demonstrated is required to induce terminal cardiomyocyte differentiation in the embryo.
2. The second, longer-standing project addresses the function of Tip60 protein during heart development, as well as in the adult myocardium wherein Tip60 is enriched. For these investigations we globally ablated the Tip60 gene in mice, recently reporting that Tip60 is required for embryonic development to progress beyond the blastocyst stage (Hu et al. 2009). We have subsequently demonstrated a haploinsufficient Tip60 phenotype in the adult heart, wherein either physical or biochemical stressors cause hypertrophy that is accompanied by re-entry of G0 cardiomyocytes into the cell-cycle. These findings support our hypothesis, which is that Tip60 functions to maintain replicative senescence of cardiomyocytes in the adult heart. To extend these findings we have conditionally targeted the Tip60 gene, which should enable enhancement of this phenotype by conditionally and efficiently deleting Tip60 from the adult heart. We expect that Tip60 depletion will induce proliferation of adult cardiomyocytes, and that this condition will enable improved cardiac function following myocardial infarction.
Recent Publications from Project 1:
Rudy-Reil D, Lough J. (2004) Avian precardiac endoderm/mesoderm induces cardiac myocyte differentiation in murine embryonic stem cells. Circ Res. 94:e107-16.
Nelson TJ, Ge ZD, Van Orman J, Barron M, Rudy-Reil D, Hacker TA, Misra R, Duncan SA, Auchampach JA, Lough JW. (2006) Improved cardiac function in infarcted mice after treatment with pluripotent embryonic stem cells. Anat Rec 288:1216-1224.
Li Z, Barron MR, Lough J, Zhao M. (2008) Rapid single-step separation of pluripotent mouse embryonic stem cells from mouse feeder fibroblasts. Stem Cells Dev. 17:383-387.
Van Orman JR, Weihrauch D, Warltier DC, Lough J (2009) Myocardial interstitial fluid inhibits proliferation and cardiomyocyte differentiation in pluripotent embryonic stem cells. Am J Physiol Heart Circ Physiol. 297:H1369-1376.
Si-Tayeb K, Noto FK, Sepac A, Sedlic F, Bosnjak ZJ, Lough JW, Duncan SA. (2010) Generation of human induced pluripotent stem cells by simple transient transfection of plasmid DNA encoding reprogramming factors. BMC Developmental Biology 10:81.
Sepac A, Sedlic F, Si-Tayeb K, Lough J, Duncan SA, Bienengraeber M, Park F, Kim J, Bosnjak ZJ. (2010) Isoflurane preconditioning elicits competent endogenous mechanisms of protection from oxidative stress in cardiomyocytes derived from human embryonic stem cells. Anesthesiology 113:906-916.
Zhao M, Barron MR, Li Z, Koprowski S, Hall CL, Lough J. (2010) Making Stem Cells Infarct Avid. Cell Transplant. 19:245-250.
Van Orman JR, Si-Tayeb K, Duncan SA, Lough J. (2011) Induction of Cardiomyogenesis in Human Embryonic Stem Cells (hESCs) by hESC-derived Definitive Endoderm. Stem Cells Dev. Jun 1. [Epub ahead of print] PubMed PMID: 21627569.
Recent Publications from Project 2:
Kim, M-S, Merlo X, Wilson C, Lough J. (2006) Co-activation of atrial natriuretic factor promoter by Tip60 and serum response factor. J. Biol. Chem. 281:15082-15089.
Gorrini C, Squatrito M, Wark L, Martinato F, Sardella D, Bennett S, Marchesi F, Scanziani E, Mai S, Lough J, Amati B. (2007) Tip60 is a haplo-insufficient tumour suppressor required for an oncogene-induced DNA damage response. Nature 448:1063-1067.
Hu Y, Fisher JB, Koprowski S, McAllister D, Kim MS, Lough J. (2009) Homozygous disruption of the Tip60 gene causes early embryonic lethality. Dev. Dynamics 238:2912-2921.
Gehrking KM, Andresen JM, Duvick L, Lough J, Zoghbi HY, Orr HT. (2011) Partial Loss of Tip60 Slows Mid-Stage Neurodegeneration in a Spinocerebellar Ataxia Type 1 (SCA1) Mouse Model. Hum. Mol. Genet. 20:2204-2212.
Fisher JB, Kim M-S, Blinka S, Ge Z-D, Wan T, Duris C, Christian D, Twaroski K, North P, Auchampach J, Lough J. (2011) Stress-Induced Cell-Cycle Activation in Tip60 Haploinsufficient Adult Cardiomyocytes. Submitted to PLoS One.
The video clips below, which show rhythmic contractions in cultures generated from human pluripotent stem cells, are further described in
Sepac et al. (Cell Transplant. 2012 Aug 2.)
Video Clip 1. H1 hESC cardiomyocytes form widespread areas of contractile cells. This video recorded on post-induction day 40 shows randomly selected areas within a 6 cm diameter culture dish that contains widespread areas of rhythmically contracting cellular masses; these areas comprised almost the entire area of the dish.
Video Clip 2. Contracting clusters of H9 hESC cardiomyocytes occur in selected areas. This video recorded on post-induction day 40 shows a representative region of a 6 cm culture dish containing differentiating H9 cells that exhibited spontaneous and rhythmical contractions. Unlike the H1 ESCs shown in Video 1, these areas of contractility occurred only in selected regions.
Video Clip 3. Cardiomyocytes induced from human iPSC C2a cells occur in small beating clusters. Recorded on induction day 40, this video shows a representative region containing spontaneously and rhythmically beating cells. Similar to H9 ESCs, these areas of contractility do not span the entire dish.
Video Clip 4. Areas of beating human iPSC C6a cardiomyocytes occur in restricted foci. Spontaneous and rhythmic beating was observed only in confined foci of the dish, which contains iPSC C6a cardiomyocytes. This video was made on induction day 40.