Research Laboratories at Penn Vet

Penn Vet faculty are engaged in ongoing groundbreaking research. Here are examples of faculty laboratories and the projects being investigated, both at our Philadelphia campus and at New Bolton Center.

Lengner Laboratory

Our lab is broadly interested in the mechanisms by which stem cells acquire and maintain developmental potency. We are also exploring how deregulation of these mechanisms can contribute to oncogenic transformation, tumorigenesis, and tissue regeneration in response to injury, while looking forward to learn how we might manipulate these mechanisms for application in disease modeling and regenerative medicine.

In the mammalian soma, tissue-specific stem cells capable of maintaining the proliferative output necessary for tissue organization and function exist in a state Lengner image 6 copy

of multipotency (the ability to generate any cell type of that particular tissue, in contrast to the pluripotent state embodied by embryonic stem cells capable of generating all cell types of the mammalian organism). In highly proliferative tissues such as the epithelial lining of the intestine, data from our lab and others has begun to establish a model in the stem cell compartment is organized into a hierarchy, with a mostly dormant population of long-live, radio-resistant reserve stem cells at the top of this hierarchy. When activated, these reserve stem cells give rise to a second, highly proliferative, radiosensitive short-term stem cell that bears the daily proliferative burden required to maintain tissue homeostasis.

Our lab is focused on understanding the relationship between these two stem cell populations, the molecular determinants of reserve intestinal stem cell activation, and how deregulation of the reserve intestinal stem cell compartment contributes to disease states such as colorectal cancer or acute gastrointestinal radiation injury.

We have recently identified the Msi family of RNA binding proteins as potent oncoproteins in both hematopoietic and intestinal malignancies. Msi proteins are expressed in putative somatic Lengner image 8 copy

stem cell compartments, are frequently found to be overexpressed in advanced cancers, and are known to govern asymmetric cell division in Drosophila melanogaster (a process thought to maintain the somatic stem cell niche in mammals). Using mouse genetic approaches integrated with human patient data, we have recently demonstrated that MSI2 acts as an intestinal oncogene, driving activation of the mTORC1 complex and uncontrolled stem cell expansion. We are currently pursuing the role of Msi proteins in epithelial stem cell compartments using tissue-specific gene ablation and drug-inducible gene activation. The effects of Msi proteins on stem cell maintenance and oncogenic transformation are being tied to their RNA binding capacity using CLIP-Seq analysis (immunoprecipitation of Msi-interacting RNAs followed by massively parallel sequencing) in order to determine how specific Msi-RNA interactions affect stem cell self-renewal and oncogenic transformation.

While murine genetic systems are the primary tool of the laboratory, we also work to model human genetic gastrointestinal disorders using induced pluripotent stem (iPS) cells generated from patients. Generation of isogenic diseased and disease-allele corrected iPS cell lines using nuclease-mediated homologous recombination followed by directed differentiation into intestinal tissue provides a controlled platform not only for studying the molecular mechanisms underlying

Photos above: Label retaining cells of the intestinal crypts are identified by loading all cells with a Histone H2B protein fused to a green fluorescent protein (left). Several weeks later, only cells that do not divide retain the fluorescent label in their chromatin.

A glimpse of the rare reserve intestinal stem cell (red). This cell is capable of regenerating the entire intestinal lining after injury such as exposure to high doses of radiation.

Research
Publications
People

Lengner image 1 copy

Left: Human induced pluripotent stem cells (iPS) derived from skin cells of a patient with a genetic gastrointestinal disorder followed by genome editing to correct the disease-causing allele. The disease and corrected iPS cultures retain pluripotency assessed by expression of OCT4 and TRA1-6

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Right: Human intestinal organoids were generated from disease and disease-corrected iPS cell lines. Correction of the disease allele restores the ability of these cells to form budding, branched organoids that recapitulate the crypt structure of the small intestinal epithelium

Lengner image 3 copy Above: Histological section of the entirety of the murine small intestine. Immunofluorescence staining reveals proliferative cells within crypts (green), Paneth cells at crypt bases (red), and total cell nuclei (blue).

Below: Crosslinking, Immunoprecipitation, and massively parallel sequencing of RNA bound to the Msi2 oncoprotein in wildtype small intestinal crypts, in intestinal epithelium ectopically expressing MSI2 (TRE-MSI2), revealing strong interactions between Msi2/MSI2 and the mRNA encoding the tumor suppressor p21.

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Left: Principal component analysis (PCA) of transcriptional profiles of single stem cells. Single cells (spheres) from each of three putative intestinal stem cell populations (red, green, blue) were subjected to expression profiling. Their position on the PCA plot is a representation of their cellular identity.

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Right: The Lengner lab frequently employs Southern Blotting. It enables the unequivocal assessment of specific genetic modifications in stem cells and mice.

Wang N*, Li N*, Yousefi M, Nakauka-Ddamba A, Li F, Parada K, Rao S, Minuesa G, Katz Y, Gregory BD, Kharas MG, Yu Z, Lengner CJ. “Transformation of the intestinal epithelium by the MSI2 RNA binding protein.” Nature Communications, 2015 In Press, PMCID in process. doi:10.1038/ncomms7517 *Authors contributed equally

Park SM, Gönen M, Vu L, Minuesa G, Tivnan P, Barlowe TS, Taggart J, Lu Y, Deering RP, Hacohen N, Figueroa ME, Paietta E, Fernandez HF, Tallman MS, Melnick A, Levine R, Leslie C, Lengner CJ, Kharas MG. “Musashi2 sustains the mixed-lineage leukemia-driven stem cell regulatory program.” J. Clinical Investigation, 2015 Feb 9. pii: 78440. doi: 10.1172/JCI78440.

Li N, Yousefi M, Nakauka-Ddamba A, Jain R, Tobias J, Epstein JA, Jensen S, Lengner CJ. “Single cell analysis of proxy reporter allele-marked epithelial cells establishes intestinal stem cell hierarchy.” Stem Cell Reports, 2014 Nov 11;3(5):876-91. PMCID: PMC4235148

Katz Y, Li F, Lambert NJ, Sokol ES, Tam WL, Cheng AW, Airoldi EM, Lengner CJ, Gupta PB, Yu Z, Jaenisch R, Burge CB. “Musashi proteins are post-transcriptinoal regulators of the epithelial-luminal cell state.” Elife, 2014 Nov 7;3:e03915. doi: 10.7554/eLife.03915. PMID: 25380226. PMCID: In Process

Yildirim O, Hung JH, Cedeno R, Weng Z, Lengner CJ*, Rando OJ*. “A system for
genome-wide histone variant dynamics in ES cells reveals dynamic Macro-H2A replacement at promoters.” PLoS Genetics, 2014 Aug 7;10(8):e1004515, 2014. PMCID: PMC4125097 *Corresponding authors

Park SM, Deering RP, Lu Y, Tivnan P, Lianoglou S, Al-Shahrour F, Ebert BL, Leslie C, Daley GQ, Lengner CJ, Kharas MG. "Msi2 controls self-renewal, lineage restriction and TGF-β signaling in HSCs." Journal of Experimental Medicine, 2014 Jan 13;211(1):71-87. doi: 10.1084/jem.20130736. Epub 2014 Jan 6.

Liu JC, Lengner CJ, Gaur T, Lou Y, Hussain S, Jones MD, Borodic B, Colby JL,
Steinman HA, van Wijnen AJ, Stein JL, Jones SN, Stein GS, Lian JB. “Runx2 protein
expression utilizes the Runx2 P1 promoter to establish osteoprogenitor cell number for normal bone formation." J Biol Chem. 2011 Aug 26;286(34):30057-70.

Kharas MG*, Lengner CJ*, Al-Shahrour F, Bullinger L, Ball B, Zaidi S, Morgan K, Tam W, Paktinat M, Okabe R, Gozo M, Scholl C, Frohling S, Fleming M, Ebert BL, Gilliland DG, Jaenisch R, Daley GQ. “Musashi-2 promotes hematopoietic stem cell proliferation and accelerates myeloid leukemogenesis.” Nature Medicine 2010 Aug;16(8):903-8. Epub 2010 Jul 8. *Authors contributed equally (Faculty of 1000 Must Read)

Lengner CJ*, Gimelbrant AA*, Erwin JA, Cheng AW, Guenther MG, Welstead GG, Alagappan R, Frampton GM, Xu P, Muffat J, Santagata S, Powers D, Barrett CB, Young RA, Lee JT, Jaenisch R, Mitalipova M. "Derivation of pre-X inactivation human embryonic stem cells under physiological oxygen concentrations." Cell. 2010 May 28;141(5):872-83. Epub 2010 May 13. (Faculty of 1000 Exceptional)

Lengner CJ. “iPS cell technology and regenerative medicine.” Ann N Y Acad Sci. 2010 Mar;1192(1):38-44

Hanna J*, Saha K*, Pando B, van Zon J, Lengner, CJ, Creyghton MP, van
Oudenaarden A, and Jaenisch R. “Direct reprogramming is a stochastic process amenable to cell division rate dependent and independent acceleration” Nature. 2009 Dec 3;462(7273):595-601. Epub 2009 Nov 8. (Faculty of 1000 Recommended)

Wernig M*, Lengner CJ*, Hanna J, Lodato MA, Steine E, Foreman R, Staerk J, Markoulaki S, and Jaenisch R. “A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types”. Nature Biotechnology. 2008 Aug;26(8):916-24. *Authors contributed equally (Faculty of 1000 Must Read)

Hanna J, Markoulaki S, Schorderet P, Carey BW, Beard C, Wernig M, Creyghton MP, Steine EJ, Cassady JP, Foreman R, Lengner CJ, Dausman JA, Jaenisch R. “Direct
reprogramming of terminally differentiated mature B lymphocytes to pluripotency.” Cell. 2008 Apr 18;133(2):250-64. (Faculty of 1000 Must Read)

Lengner CJ, Welstead GG, Jaenisch R. “The pluripotency regulator Oct4: A role in somatic stem cells?” Cell Cycle. 2008 Jan14; 7(6).

Lengner CJ, Camargo FD, Hochedlinger K, Welstead GG, Zaidi S Gokhale S, Scholer HR, Tomilin A, and Jaenisch R.“Oct4 expression is not required for mouse somatic stem cell self-renewal.” Cell Stem Cell. 2007 Oct; 1(4). (Faculty of 1000 Must Read)

Lengner CJ*, Steinman HA*, Gagnon J, Smith TW, Henderson JE, Kream BE, Stein GS, Lian JB, Jones SN. “Osteoblast differentiation and skeletal development are regulated by Mdm2-p53 signaling.” Journal of Cell Biology. 2006 Mar 13; 172(6): 909-21.

Lengner CJ, Hassan MQ, Serra RW, Lepper C, van Wijnen AJ, Stein JL, Stein GS, Lian JB. “Nkx3.2-mediated repression of Runx2 promotes chondrogenic differentiation.” Journal of Biological Chemistry. 2005 Sep 30; 280(39): 33132-40.

Lengner CJ, Lepper C, van Wijnen AJ, Stein JL, Stein GS, Lian JB. “Primary mouse
embryonic fibroblasts: A model of mesenchymal cartilage formation.” Journal of Cellular Physiology. 2004 Sep; (3): 327-33.

Lengner CJ, Drissi H, Choi JY, van Wijnen AJ, Stein JL, Stein GS, Lian JB. “Activation of the bone-related Runx2/Cbfa1 promoter in mesenchymal condensations and developing chondrocytes of the axial skeleton.” Mechanisms of Development. 2002 Jun; 114(1-2): 167-70.

*Denotes equal author contribution

Meet the Lengner Lab Team
	Christopher Lengner, PhD, Principal Investigator Chris received his B.S. in Biotechnology from the Worcester Polytechnic Institute and his Ph.D. from the University of Massachusetts Medical School. During this time, he became interested in stem cell biology while studying the molecular mechanisms underlying cell fate determination in developing skeletal tissues and osteosarcoma. Chris continued to pursue his interest in stem cell biology as a Ruth L. Kirschstein Postdoctoral Fellow with Rudolf Jaensich at the Whitehead Institute at MIT in Cambridge, MA. At the Whitehead, Chris continued to study somatic stem cells using murine genetic model systems, and also developed an interest in pluripotency of embryonic stem cells and the generation of induced pluripotent stem cells. Upon joining the faculty of the University of Pennsylvania, Chris’ research has focused on somatic stem cells of the intestinal epithelium, how their self-renewal is regulated, and how molecular mechanisms governing self-renewal go wrong in diseases such as cancer. Outside of the lab Chris enjoys hiking, skiing, reading, traveling, and fine American IPAs.
	Stephanie Adams Stephanie is from Germany. She came to Upenn after earning her degree in Biology (Master of Science) and joined the Lengner lab in 2018. Her main focus is generation of transgenic mice for various projects for the Lengner lab and the core. In her free time, she likes to hang out with her family, and she volunteers at the Media Food Bank and at "Kids against Hunger".
	Youngjun Choi Youngjun received his Ph.D. from the Korea University of science and technology. During this time, he researched about differentiation of human pluripotent stem cells into hepatocyte-like cells and finding factors for the maturation of hepatocyte-like cells. After joining the Lengner lab, his research is focused on liver disease modeling in Dyskeratosis congenita patients using patient-derived induced pluripotent stem cells (iPSCs) and genetically corrected iPSCs pair. Outside of the lab, he enjoys watching movies and making some Korean food.
	Zvi Cramer Zvi received his B.Sc. at McGill University where he investigated eIF4A inhibitors as potential therapeutics for cancer cachexia. Zvi is now interested in identifying chromatin factors important in colorectal cancer tumorigenesis using CRISPR/Cas9 Screening in mouse tumoroids. In his spare time Zvi enjoys playing stringed instruments, baking and traveling.
	Nicolette Johnson Nicolette was born and raised in Chicago. She received her B.S.E in Biomedical Engineering from the University of Iowa, where she worked with Dr. Kevin Campbell studying the molecular basis of muscular dystrophy. She joined the Penn MD-PhD program in 2015. In the lab, she is interested in understanding the plasticity of secretory lineage cells of the intestinal epithelium. Outside of lab, she enjoys live music, cycling along the Schuylkill, petting her cat Darwin, and dancing.
	Melissa Kim - CAMB, DSRB Melissa is a PhD student in the Developmental, Stem Cell, and Regenerative Biology graduate group. She is originally from Los Angeles, California, and received her bachelor’s degree from the University of Michigan, Ann Arbor in Neuroscience. While studying at Michigan, she worked in Dr. Ben Allen’s lab as an undergraduate research assistant studying post-injury regeneration of the mammalian olfactory epithelium. After graduating from undergrad, she spent a year working as a post-bac fellow in Dr. Terry Yamaguchi’s lab at the National Cancer Institute in Frederick, MD. There, her research was focused on understanding the role of Wnt signaling in the differentiation of bipotent neuromesodermal progenitors during embryonic development. Aside from lab, she enjoys going out to new restaurants, breweries, and going on weekend trips with friends!
	Ning Li, MD, Research The goal of his research is to gain an understanding of the molecular mechanisms that govern adult stem cell maintenance and how these contribute to colorectal cancer initiation and progression. His work looking at the function of the Msi family of stem cell-specific RNA binding proteins revealed new tumorigenic pathways underlying gastrointestinal cancer initiation and progression. Moving forward he aims to address whether these novel pathways might be effective points for therapeutic intervention. Looking deeper into the hierarchical structure of the intestinal stem cell compartment, his focus is to combine single cell profiling technology (Single cell RNA-Seq, CyTOF) with mouse genetic markers of the distinct intestinal stem cell compartments in order to dissect the precise pathways that contribute to tumorigenesis and relate this to the cancer cell-of-origin. Ultimately, this knowledge may help to explain tumor heterogeneity and will provide improved murine models of colorectal cancer.
	Hexiang Liu, Master's Candidate Hexiang is a second-year master student in the master of biotechnology program at University of Pennsylvania. She became interested in stem cell technology during her first-year study and joined Dr. Lengner's lab. She is now using CRISPR-HOT method to study the effect of one copy of Apc gene mutation on another copy. Outside of the lab Hexiang likes to stay at home and play with her cat Ray.
	Keara Monaghan Keara received her Bachelor’s in Veterinary and Biomedical Sciences from Penn State University in May 2020. She joined the Lengner Lab in June 2020 where she works as a research assistant specifically managing the labs genetically modified mouse colony. She assists with a variety of projects looking at stem cell potency, colorectal cancer tumorigenesis and the production and use of transgenic mice. She plans to attend veterinary school and has an interest in working in public health, using the ‘One Health’ approach to enhance wildlife and environmental health. Outside of lab Keara loves to exercise, cook, garden and camp.
	Jeeyoon Na Jeeyoon is an undergraduate student (class of 2022) majoring in biology and concentrating in molecular and cell biology. In the Lengner Lab she is studying plasticity of intestinal epithelial cells with Nicolette.
	Joshua Rhoades Josh received his Bachelor's in biology, Masters in Entomology and Bioinformatics Graduate Certificate from the University of Delaware. He has work experience in both agriculture and medical bioinformatics. He is a member of both the Lengner and Blanco labs, as well as the Institute for Biomedical Informatics Bioinformatics Core (BIC). Josh likes traveling, photography, and has a goal to visit all 50 state capitol buildings.
	Kamen Simeonov Kamen completed undergrad at Stanford where he studied colon stem cells in Michael Clarke's lab. After college Kamen moved to San Francisco, where he worked at Genentech studying direct reprogramming of skin cells to liver cells and also initiated an independent epidemiological study of lung cancer incidence. Kamen joined the Penn MD-PhD program in 2014. In the Lengner lab, Kamen is designing methods for dynamically encoding information into DNA to create next-generation lineage tracers, which he is using to study cancer metastasis and stem cell biology. Kamen wishes wet lab work could be in large glass bubbles in the mountains and enjoys fishing and mountain biking.
	Yuhua Tian, Postdoctoral Research Yuhua received her Ph.D. in Biochemistry and Molecular Biology from China Agricultural University. During this period, she mainly studied the function and mechanism of miR-31 in intestinal stem cells and colitis. After joining the Lengner lab, she mainly used organoid as a model to study the function of the Notum in colorectal cancer in vivo and in vitro. In addition, she also does some work on the function of Foxl1 + cells in the initiation and progression of colorectal cancer. Outside of the lab, she enjoy playing with my family, cooking or cleaning up if she has time.

Former Lab Members:

Kim Davidow (Parada), Research Specialist: Kim is currently a medical student at the Jefferson Medical College of Thomas Jefferson University.

Shan Wang, Visiting Scholar: Shan is currently completing her Ph.D. thesis at the College of Biological Sciences, China Agricultural University in Beijing.

Zhengquan Yu, Postdoctoral Felllow: Zhengquan is currently a Principal Investigator at the State Key Laboratories for Agrobiotechnology, College of Biological Sciences, China Agricultural University in Beijing.

Research Laboratories at Penn Vet

Lengner Laboratory

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