5th Annual PCTC Mtg _ booklet_Final A

https://translationalcells.org 9 March 2018 Cycle Minzhe Guo Whitsett CCHMC RH06 Online Single Cell Visualization and Analysis of Lung Progenitor Cells Michael Herriges Collaborator: Je

5 TH ANNUAL (VIRTUAL) MEETING

THE UNIVERSITY OF ALABAMA AT BIRMINGHAM

SEPTEMBER 29–30, 2021

https://translationalcells.org 1

Welcome 2

Acknowledgments 3

Overview of the PCTC 4

PCTC Committees 6

Funding Opportunities 7

List of Funded PCTC Awards to Date 8

Keynote Speakers 11

Agenda 13

Breakout Room Assignment of Abstracts 16

Abstracts 19

Chapman (UCSF) Research Hub 01 20

Chou (CHOP) Research Hub 02 25

Daley (BCH) Research Hub 03 30

Snoeck (CUMC) Research Hub 04 34

Thomson (UW-M) Research Hub 05 40

Whitsett (CCHMC) Research Hub 06 46

Zhang (UAB) Research Hub 07 52

U01 Research Hub Principal Investigators 59

Meeting Participants 62

Networking and Collaboration by Registration Institution 74

Index 115

Birmingham Civil Rights Institute (Virtual Visit) 132

Welcome

Dear Friends and Colleagues,

On behalf of the Department of Biomedical

Engineering at the University of Alabama at

Birmingham (UAB), and the Zhang Research Hub

of the NHLBI Progenitor Cell Translational

Consortium (PCTC), we warmly welcome you to

the Fifth Annual PCTC (Virtual) Meeting While

we hoped to host you in person in Birmingham to

explore our campus and the city together, we have

organized this virtual meeting to provide an

accessible forum to promote collaborative

interaction As PCTC investigators, we look

forward to reviewing the past year’s progress and

establishing plans for the future in these truly

historic times

We hope this meeting will provide a stimulating

environment for all attending investigators and

trainees in the fields of heart, lung, and blood

research to foster lively discussions that will

catalyze progress and promote future research

This year we are excited to welcome keynote

speaker Eric Olson, Ph.D., Chair of the

Department of Molecular Biology at the

University of Texas Southwestern Medical

bringing their discoveries to improve public health

The symposium’s day-and-a-half agenda will provide a stimulating environment for all investigators and trainees in the field of heart, lung, and blood, to have lively discussions that will catalyze progress and promote future collaborations This year’s Trainee’s Only Session (TOS) was organized by Asher Kahn- Krell, Danielle Pretorius, Eric Zhang, and Yang Zhou from the University of Alabama, and Matt Brown from the University of Wisconsin at Madison At the TOS, trainees will have the opportunity to present and interact with one another The main meeting session will allow additional trainees and junior investigators to present their research in the Plenary sessions, followed by a Breakout Session, comprising 21 abstracts

We are extremely grateful to Michael Terrin, M.D., MPH, Professor, and his team at the University of Maryland School of Medicine for their tireless efforts in organizing this meeting and making it available virtually We look forward to seeing you online in September and very much hope to see you in person for our

2023 meeting

Best wishes,

https://translationalcells.org 3

The University of Alabama (UAB)

investigators, the National Heart, Lung, and

Blood Institute (NHLBI) Progenitor Cell

Translational Consortium Administrative

Coordinating Center (NHLBI PCACC), and

affiliated consortium investigators, organized

this 5th Annual (Virtual) PCTC Meeting,

under the leadership of Drs Denis Buxton

(NHLBI Program Director of the PCTC) and

Victor Dzau (Chair of the PCTC)

The Consortium extends gratitude to Drs Jay

Zhang, Timothy Kamp, Nenad Bursac, and

their team for their generous support in

organizing the annual meeting

Special acknowledgment also goes to Ms

Andrea Lefever (University of Maryland,

Baltimore), Ms Elizabeth Casher (University

of Maryland, Baltimore), Ms Tina Kramer

(University of Maryland, Baltimore), Dr

Yuji Zhang (University of Maryland,

Baltimore), and Ms Ling Tang (University

of Maryland, Baltimore) for work on meeting logistics

The Consortium further extends appreciation

to Asher Kahn-Krell, Danielle Pretorius, Eric Zhang, and Yang Zhou from the University

of Alabama at Birmingham, for their enthusiastic efforts in organizing and leading the Trainees Only Session (TOS) with guidance from Drs Minzhe Guo, Serine Avagyan, Matthew Brown (co-chairs of the PCTC Skills Development Committee) and from Drs Michael Terrin and Jennifer Albrecht (University of Maryland, Baltimore)

Please direct any questions or concerns that may arise in planning your participation in the 5th Annual (Virtual) PCTC Meeting to the staff of the PCTC Administrative Coordinating Center:

Michael Terrin Contact Principal Investigator mterrin@som.umaryland.edu Jennifer Albrecht Multiple Principal Investigator jalbrecht@som.umaryland.edu Elizabeth Betz Senior Accountant (Awards/Grants) eensbren@som.umaryland.edu

Ling Tang Research Project Coordinator litang@som.umaryland.edu

Yuji Zhang Bioinformatician/Co-Investigator yuzhang@som.umaryland.edu Tina Kramer Research Project Coordinator tkramer@som.umaryland.edu

Overview of the PCTC

Purpose

The goal of the NHLBI Progenitor Cell

Translational Consortium (PCTC) is to

translate advances in progenitor cell biology

towards application to heart, lung, and blood

diseases The initiative will focus on the use of

progenitor cell-based disease models to

understand disease mechanisms and to develop

novel therapies, and the application of

progenitor cell-based therapies for the

treatment of heart, lung, and blood diseases

Research Objectives and Scope

Stem cell research promises major new insights

and therapeutic approaches for human disease

of the heart, lungs, and blood, but critical

barriers to effective translation exist, many of

which entail shared challenges among these

distinct organ systems The NHLBI has

provided significant support for the

development of the progenitor cell biology

field through the NHLBI Progenitor Cell

Biology Consortium (PCBC) and the Lung

Repair and Regeneration Consortium (LRRC)

The PCTC will leverage the advances in

progenitor cell biology made by the PCBC and

LRRC and the broader scientific community

Scientists have made significant progress in

applying bioengineering to heart, lung and

blood diseases, developing organs-on-a-chip

and developing decellularized scaffolds for

tissue replacement The use of patient-specific

induced pluripotent stem cells (iPSCs),

bioengineering and genome editing offers

unique opportunities for developing

In translating advances in progenitor cell biology to treat specific cardiovascular, pulmonary, or hematologic diseases, the selected disease area may involve more than one organ system, e.g., pulmonary arterial hypertension; and other areas of expertise, e.g., large animals, bioengineering, genome editing, transplant immunology, systems biology, bioreactors for scale-up of cell production and product development

Focus areas include the continued development

of patient-specific disease models using progenitor cells and genome editing; the use of progenitor cells (including gene modified progenitors) and their differentiated progeny for cell therapy and tissue engineering; the development of strategies to promote the reparative potentials of endogenous progenitor cells; and expansion of efforts to employ direct reprogramming of cells in vivo to treat disease When needed to inform translational potential and strategies, studies may further elucidate disease mechanisms, e.g., obtaining a better understanding of the principles governing how terminally differentiated cells re-enter the cell cycle to proliferate Modeling and therapeutic development for rare diseases are encouraged For different diseases the starting point on the translational continuum varies, and this influences how far towards clinical implementation investigators can anticipate to progress during a seven year period Projects may vary substantially in their objectives and

https://translationalcells.org 5

specific long term needs for their project to be

translatable, e.g., advice on standard operating

procedures, Good Laboratory Practices, and

interactions with the FDA to develop IND and

IDE applications; investigators will be

expected to obtain regulatory advice early in

the project, consult frequently with their

advisors to ensure that unforeseen regulatory

barriers do not arise, and report on them to

NHLBI

Consortium Structure

The PCTC is a highly interactive and

synergistic Consortium of investigators who

share ideas, data and resources to advance

addition to interactions within a Research Hub, extensive collaboration among Research Hubs

is expected and may include collaborations between investigators at multiple institutions

The Consortium consists of Research Hubs, and an Administrative Coordinating Center (ACC), responsible for enabling collaboration across the consortium through logistical support and a variety of tasks The ACC administers funds to support Research Hub collaborations, translation to clinical applications, and skills development activities The NHLBI works in partnership with the Research Hubs and ACC under cooperative agreements

NHLBI REPRESENTATIVE ASSOCIATE DIRECTOR BASIC AND EARLY TRANSLATIONAL RESEARCH PROGRAM

PCTC Committees STEERING COMMITTEE

▪ Victor Dzau (Chair)

▪ Denis Buxton (Co-chair)

▪ Serine Avagyan (Co-chair)

▪ Matthew Brown (Co-chair)

▪ Minzhe Guo (Co-chair)

https://translationalcells.org 7

Jump Start Awards - $25,000

The PCTC Jump Start Award Program offers up to $25,000/award for new technologies to

overcome translational research challenges; or, preliminary data for a new translational research goal; or inter–hub collaboration

Link: Apply for a PCTC Jump Start Award!

Next submission deadline: March 1, 2022

Clinical Translation Awards (CTAs) - $200,000

The goal of the NHLBI Progenitor Cell Translational Consortium

(PCTC) is to translate advances in progenitor cell biology towards

application to heart, lung, and blood diseases The initiative will

focus on the use of progenitor cell-based disease models to

understand disease mechanisms and to develop novel therapies, and

the application of progenitor cell-based therapies for the treatment

of heart, lung, and blood diseases

Funding amount - $100,000/year for up to two years

Visiting Scholar Awards (VSA) - $25,000

This Request for Proposals provides funding for PCTC Visiting Scholar Awards up to $25,000

for collaboration and exchange of expertise among PCTC Research Hubs

The PCTC Visiting Scholar Award aims to stimulate innovative collaborations among PCTC

Research Hubs along the translational research continuum Applicants will travel to PCTC

Research Hub laboratories to learn new techniques, exchange scientific knowledge or perform

collaborative experiments to advance translational progenitor cell-based therapies for the

treatment or diagnosis of heart, lung, or blood diseases

Link: Apply for a Visiting Scholar Award

List of Funded PCTC Awards to Date Visiting Scholar Awards

Jaymin Kathiriya Chapman (UCSF) RH 01 PCTC Visiting Scholar Award Program

Clinical Translation Awards

Adrianna Vlachos Daley (BCH) RH 03 Designing a Phase I/II Dose Escalation, Toxicity/Efficacy Trial of L-leucine in DBA

Adrianna Vlachos Daley (BCH) RH 03

A Multi-Center, Phase I/II, Dose Escalation Clinical Trial of L-leucine in Patients with Diamond Blackfan Anemia

Paritha Arumugam

Co-PI: Bruce Trapnell Whitsett (CCHMC) RH06

Translating Gene/ Pulmonary Macrophage Transplantation Therapy from Mouse to Man (RFP PCTC 2019 Clinical Translational 01)

Paritha Arumugam

Co-PI: Bruce Trapnell Whitsett (CCHMC) RH06

Translating Gene/ Pulmonary Macrophage Transplantation Therapy from Mouse to Man (RFP PCTC 2021 Clinical Translational 01)

https://translationalcells.org 9

March 2018 Cycle

Minzhe Guo Whitsett (CCHMC) RH06 Online Single Cell Visualization and

Analysis of Lung Progenitor Cells Michael Herriges

Collaborator: Jeffrey

Whitsett

Chapman (USCF) RH01 A Mouse Model for Cell-Based Therapy of

Human Pulmonary ABCA3 Deficiency Jaymin Kathiriya Chapman (USCF) RH01

Small Scale Protein Analysis of Translationally Repressed Alveolar Progenitors

September 2018 Cycle

Matthew Brown Thomson (UW-M) RH05 T Cell Receptor Sequencing of Human

PSC-Graft-Reactive T Cells George Kwong Chapman (UCSF) RH01 Development of Novel Zebrafish Model to Study Lung Development and Disease

March 2019 Cycle

Arunoday Bhan Daley (BCH) RH03 Preventing Premature Activation During In

Vitro Platelet Production

September 2019 Cycle

Modeling chILD caused by ABCA3 Mutations Using Pluripotent Stem Cell Derived AEC2s

Jason Gokey Whitsett (CCHMC) RH06 The Role of MEG3 in Regulating Basal

Progenitor Cell Differentiation Jessie Huang Whitsett (CCHMC) RH06 Single-Cell Analysis of Self-Renewal Mechanisms in Lung Alveolar Progenitors

March 2020 Cycle

Ran Jing Daley (BCH) RH 03 iPSC-derived CAR T cells For Cancer Immunotherapy Claire Burgess Whitsett (CCHMC) RH06 Generation of Human Alveolar Epithelial Type I Cells From Pluripotent Stem Cells

September 2020 Cycle

Christopher Thom Chou (CHOP) RH 02

Defining Tropomyosin 1-Related Mechanisms to Enhance Blood Progenitor Production

Generation of Anterior Foregut Endoderm from Porcine Expended Pluripotent Stem Cells

Liang Ma Chapman (UCSF) RH01 Development of a mouse model of syngeneic airway stem cell transplantation Mingxia Gu Whitsett (CCHMC) RH06 Generation of Vascularized Lung Organoid from iPSCs

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Drs Eric Olson and Robert Langer will discuss the translational aspects of their research and

their discoveries to improve public health

Dr Olson’s lab studies muscle cells as a model for understanding how stem cells adopt specific fates and how programs of cell differentiation and morphogenesis are controlled during development

They have also explored the roles of microRNAs and long coding RNAs in controlling muscle development and disease

non-The long-term goals are to delineate the complete genetic pathways for the formation and

function of each type of muscle cell, and to use this information to devise pharmacologic and

genetic therapies for inherited and acquired muscle diseases in humans

Thursday, September 30, 2021, 10:00 AM, CDT

Presentation: "From Lab Bench to Clinic How to Solve Global Health Challenges by Developing New Therapies Through Innovation and Founding New Companies"

Robert Langer, ScD

David H Koch Professor in Biochemistry

Massachusetts Institute of Technology

The Langer group’s work is at the interface of biotechnology

and materials science A major focus is the study and

development of polymers to deliver drugs, particularly

genetically engineered proteins, continuously at controlled rates

and for prolonged periods of time

The laboratory’s interest in drug delivery systems has extended

to situations where drugs may serve a potentially useful purpose

and then cause toxicity In such cases, it would be useful to have

a selective drug or substance removal system Examples include removal of heparin, bilirubin, and cholesterol All of these studies involve reactor design, understanding biomaterials with respect to blood interactions, and modeling of in vivo situations

Finally, the group is developing drugs that specifically inhibit the process of neovascularization but do not interfere with existing blood vessels Neovascularization is critical to the progression

of several diseases, including cancer, retinopathy, rheumatoid arthritis and psoriasis These projects involve biochemical purification and tissue culture studies

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Wednesday, September 29, 2021

08:00 AM, CDT Trainees Only Session

12:15 PM Welcome and Housekeeping Remarks

• Jianyi Zhang, Chair of the Organizing Committee

12:20 PM Introduction to the University of Alabama at Birmingham (UAB) School

of Medicine

• Selwyn M Vickers, Senior Vice President of Medicine,

and Dean, University of Alabama School of Medicine, UAB

• Victor Dzau, President, National Academy of Medicine

12:30 PM Report from the NHLBI

• Denis Buxton, Associate Director, Basic and Early Translational

Research Program, NHLBI

o Progress Reports

o NHLBI Initiatives Related to PCTC

12:35 PM Report from the Administrative Coordinating Center

• Michael Terrin

o Administration

o Web Site

Scientific Program

12:40 PM Snoeck Research Hub 04: Modeling, Pathogenesis and Treatment of

Idiopathic Pulmonary Fibrosis – Moderator: Hans Snoeck

• A Clinically Relevant Model of Acute Respiratory Distress

Syndrome in Human-Sized Swine – Sarah Kaslow

• Regional De-Epithelialization of the Lung in a Rat Model –

Camilla Predella 01:20 PM Keynote Speaker Introduction – Victor Dzau

01:25 PM Gene Therapy for Muscular Dystrophy Syndromes: Eric Olson, Annie

and Willie Nelson Professor in Stem Cell Research, University of Texas Southwestern

02:45 PM Daley Research Hub 03: Stem Cells for Therapeutics Discovery in Genetic

Blood Disorders – Moderator: George Daley

• Role of Transposable Elements in Hematopoiesis and Cancer –

Deepak Jha

03:25 PM Chou Research Hub 02: Improving Transfusion Therapy for Patients with

Sickle Cell Disease with Pluripotent Stem Cell-Derived Red Cells –

Moderator: Stella Chou

• Detection of Rh Antibodies in Patient Plasma Using Customized

Induced Pluripotent Stem Cell-Derived Red Cells – Hyun Hyung

An

04:05 PM Working Group Reports

• Cardiac Working Group – Jianyi Zhang and Timothy Kamp

• Hematopoietic Working Group – Leonard Zon

• Lung Working Group – Jeffrey Whitsett

04:20 PM Trainees Only Session Report

05:30 PM Civil Rights Museum Virtual Visit

Thursday, September 30, 2021

09:55 AM, CDT Keynote Speaker Introduction

• Jeffrey Holmes, Dean, University of Alabama School of

Engineering, UAB

• From Lab Bench to Clinic How to Solve Global Health Challenges by Developing New Therapies Through Innovation and

Founding New Companies: Robert Langer, David H Koch

Professor in Biochemistry, Massachusetts Institute of Technology 11:00 AM Zhang Research Hub 07: Integrated Cellular and Tissue Engineering for

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Vascular Progenitors in Primates – Moderator: James Thomson

• Generation of an Off-the-Shelf Allogeneic Arterial Endothelialized

Small Diameter Vascular Graft – Jue Zhang

12:50 PM Whitsett Research Hub 06: Editing Alveolar Progenitor Cells for

Correction of Monogenic Disease – Moderator: Jeffrey Whitsett

• Endothelial ATF3 Plays a Critical Role in Lung Regeneration –

Terren Niethamer

• Rescue of an Interstitial Lung Disease Related Surfactant

Protein-C Mutation by Inhibition of the Endoplasmic Reticulum

Membrane Complex Activity – Xiaofang Tang

01:30 PM Chapman Research Hub 01: Epithelial Stem/Progenitor Cells as Repair

Agents in Diffuse Alveolar Damage – Moderator: Harold Chapman

• Transplantation of Mouse Pluripotent Stem Cell Derived Distal

Tip-Like Cells into Immunocompetent Mice – Michael Herriges

• Human Alveolar Type 2 Epithelium Transdifferentiates into

Metaplastic KRT5+ Basal Cells – Jaymin Kathiriya

• Victor Dzau, Chair of the PCTC and hosts, Jianyi Zhang, Nenad Bursac and Timothy Kamp

Breakout Room Assignment of Abstracts

RESEARCH HUB

LAST NAME

FIRST NAME ABSTRACT TITLE

Breakout Room #1:

Moderator: Igor Slukvin

ACC Staff - - Lauren Schmand

Chou Research Hub 02 Nations Catriana Modeling Thrombocytopenia Absent Radius

Syndrome Using Induced Pluripotent Stem Cells

Snoeck Research Hub 04 Petrillo Carolina Mitochondrial Dynamics Regulate Interferon

Signaling And Age-Related Changes in hPSCs

Whitsett Research Hub 06 Burgess Claire Generation of Human Alveolar Epithelial Type I

Cells From Pluripotent Stem Cells

Breakout Room #2:

Moderator: Jeffrey Whitsett

ACC Staff - - Andrea Lefever

Snoeck Research Hub 04 Pinezich Meghan Quantitative Analysis Of Cystic Fibrosis Lung

Matrix

Thomson Research Hub 05 Maufort John Identifying Transcriptomic Signatures And

Decomposing Cellular Subpopulations Of Occluded And Patent Cellularized Expanded Polytetrafluoroethylene Arterial Grafts In Rhesus Macaques

Zhang Research Hub 07 Zhou Yang Transcriptomic Comparison Identifies Distinct

Molecular Features in Cardiomyocytes Generated

by Different Reprogramming Approaches

Breakout Room #3:

Moderator: Nenad Bursac

ACC Staff - - Liz Betz

https://translationalcells.org 17

Breakout Room #4:

Moderator: Stella Chou

ACC Staff - - Yuji Zhang

Daley Research Hub 03 Jing Ran Human Pluripotent Stem Cell-Derived T Cells For

Cancer Immunotherapy

Thomson Research Hub 05 Smith Portia Generation of Functional Macrophages from

SIRPα-Knockout Human Pluripotent Stem Cells Confer Superior Anti-Tumor Potential Against CD47+ Solid Tumor Cancers

Zhang Research Hub 07 Kahn-Krell Asher Biomanufacturing of Micro Myocardial Tissue

through Suspension Differentiation with Improved Yields, Purity, and Quality

Breakout Room #5:

Moderator: George Daley

ACC Staff - - Ling Tang

Chapman Research Hub 01 LeSaux Claude

Jourdan Secreted Frizzled Related Protein 2 Contributes to the Transdifferentiation of Human Alveolar

Epithelial Type II Cells

Thomson Research Hub 05 Raja Kalpana SKiM: A Web Interface for Identifying

Literature-Based Discoveries from PubMed

Zhang Research Hub 07 DeLuca Sophia Discovery of Cardiac Mitogens Using a

CRISPR-based High-throughput Approach

Breakout Room #6:

Moderator: Hans Snoeck

ACC Staff - - Jennifer Albrecht

Chapman Research Hub 01 Cohen Max TGFβ-dependent Regulation of sFRP2 Expression

in Fibroblasts In Human Fibrotic Lung Tissue

Chou Research Hub 02

McGrath Kathleen Self-renewing Definitive Erythroblasts Derived

from Human Pluripotent Stem Cells

Whitsett Research Hub 06 Huang Jessie Mechanisms of Self-Renewal and Maturation of

iPSC-Derived Alveolar Epithelial Type 2 Cells

RESEARCH HUB NAME LAST NAME FIRST ABSTRACT TITLE

Breakout Room #7:

Moderator: Hal Chapman

ACC Staff - - Joella Trenchard

Thomson Research Hub 05 D'Souza Saritha MHC-Defined Nonhuman Primate Model for the

Assessment of the Immunogenicity of Derived CD34+ Hematopoietic Progenitors Following Myeloablative HSC Transplantation

iPSC-Zhang Research Hub 07 Zhou Tianhua Transendocardial Injected Human Pluripotent

Stem Cell Derived Cardiac Progenitor Cells Engraft and Differentiate without Associated Tachyarrhythmias in a Swine Ischemic Cardiomyopathy Model

Snoeck Research Hub 04 Pinezich Meghan Ex vivo Lung Repair on Cross-circulation

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Plenary Session and Breakout Session abstracts are organized by Research Hub Within each Research Hub, abstracts are organized alphabetically by last name of the first author Specification for each abstract as being presented in the Plenary Session (P) or Breakout Session (B) are listed

in parentheses following the abstract title The presenter of the abstract is noted with an asterisk beside his/her last name

Chapman (UCSF) Research Hub 01

https://translationalcells.org 21

TGFβ-Dependent Regulation of sFRP2 Expression in Fibroblasts in Human Fibrotic

Lung Tissue (B)

Max Cohen*, Jaymin Kathiriya, Alexis Brumwell, Tsung-Che Ho, Ying Wei,

Claude Jourdan Le Saux, Harold Chapman University of California, San Francisco, CA

TGFβ signaling is important regulator of profibrotic signaling pathways and is increased in Idiopathic Pulmonary Fibrosis In fibroblasts, the TGFβ receptor kinase complex can be inhibited

by trihydroxyphenolic compounds, following modification by the enzyme LOXL2 One such molecule is epigallocatechin gallate (EGCG), a trihydroxyphenolic compound is available over-the-counter as a nutritional supplement purified from green tea We hypothesized that selective inhibition of TGFβ signaling in fibroblasts in patients prior to biopsy would identify genes or pathways that function downstream of TGFβ in fibroblasts as well as the consequences on epithelial cells of altering fibroblast TGFβ signaling EGCG was given to patients with undiagnosed fibrotic lung disease prior to planned surgical lung biopsy, and a small portion of excess tissue was then used for single-cell RNA sequencing Patients who did not take EGCG prior

to biopsy donated fibrotic lung tissue and were used as control samples Comparison of fibroblasts from EGCG-exposed vs control samples identified marked down-regulation of TGFβ signaling,

as the IPA z-score for upstream TGFβ was -4 (p-value 5.8x10-54) and there was strong regulation of genes downstream of TGFβ signaling (e.g., TGFβ1, SNAI2, CTHRC1, multiple collagens) Fibroblast gene expression changes due to EGCG were further examined, and among the most impressive was a decreased in Secreted Frizzled-Related Protein 2 (sFRP2): the transcript expression level decreased by log2FC -1.16 and the percent of cells with detectable expression decreased from 73.3% in the control fibroblasts to 36.4% in the EGCG-exposed fibroblasts IPA analysis of differential gene expression from SFRP2 positive vs negative fibroblasts from control biopsies identified TGFβ as an upstream regulator (z-score 3.991, p-value 1.71E-74) The role for sFRP2 in lung tissue is not well understood In fibrotic lung tissue, fibroblasts that express sFRP2 are present throughout the parenchyma in a scattered distribution, without obvious clustering around the classic histologic features of IPF (such as fibroblast foci or honeycomb cysts), areas of fibrosis, or dysplastic epithelium In summary, sFRP2 is regulated by TGFβ in fibrotic lung

down-fibroblasts and has unknown function Current efforts are focused on characterizing the down-fibroblasts

that express sFRP2, the signaling pathway(s) through which sFRP2 acts on lung cells, and its effects on fibroblasts and epithelia in normal and fibrotic lungs

ABSTRACT #2 Regulation of Type 2 Alveolar Epithelial Cell Reprogramming by Transforming Human Alveolar Type 2 Epithelium Transdifferentiates into Metaplastic KRT5+ Basal Cells (P)

Jaymin Kathiriya*, Chaoqun Wang, Minqi Zhou, Alexis Brumwell, Bruce Wang, Monica Cassandras, Claude Jourdan Le Saux, Kostantinos Alysandratos, Paul Wolters, Michael Matthay,

Darrell Kotton, Harold Chapman, Tien Peng Boston University School of Medicine, Boston, MA

Loss of alveolar type 2 cells (AEC2s) and ectopic appearance of basal cells in the alveoli characterize severe lung injuries such as idiopathic pulmonary fibrosis (IPF) Utilizing 3D organoids, single cell RNAseq, and histologic analyses, we demonstrate th at primary and iPSC-derived human alveolar type 2 cells (hAEC2s), unlike murine AEC2s, transdifferentiate into KRT5+ basal cells in response to fibrotic signaling in the lung mesenchyme in vitro and in vivo Single cell analysis of normal hAEC2s and mesenchymal cells in organoid co-cultures revealed the emergence of pathologic progenitor and niche cell types previously described in IPF BMP and TGF-β1 signaling in the organoids determined the extent of transdifferentiation and preservation

of hAEC2 identity Trajectory analyses of hAEC2-derived organoids and spatial localization of aberrant epithelium in IPF indicated that hAEC2s transdifferentiate into basal cells through alveolar-basal intermediates (ABIs) that are localized based on the severity of injury and proximity

to pathologic CTHRC1+/TGFβ1high mesenchymal niche cells in fibrotic zones Our study indicates that hAEC2-loss and expansion of alveolar metaplastic basal cells in severe human lung injuries are causally connected through a hAEC2-basal cell lineage trajectory driven by aberrant mesenchyme

https://translationalcells.org 23

Transplantation of Mouse Pluripotent Stem Cell Derived Distal Tip-Like Cells into

Immunocompetent Mice (P)

Michael Herriges*, Jonathan Lindstrom-Vautrin, Carlos Villacorta-Martin, Darrell Kotton

Boston University School of Medicine, Boston, MA

Recent work suggests that mouse Sox9+ embryonic lung epithelial tip cells cultured in vitro can engraft and differentiate in injured immunocompromised mouse lungs, provid ing a potential method for cell-based therapy of lung injury However, the use of primary embryonic donor cells and immunocompromised recipients severely limits the clinical applicability of this approach Directed differentiation of pluripotent stem cells (PSCs) offers a promising alternative source of donor cells which can overcome these limitations Here we describe an important step toward human PSC-based lung cell therapy with the development of a protocol for the directed differentiation of murine PSCs into distal tip-like cells The resulting cells express distal tip markers, maintain low levels of mature alveolar markers, and can be expanded in culture, similar

to cultured primary embryonic tip cells These mPSC-derived tip-like cells can then be transplanted into syngeneic and immunocompetent injured mouse lungs, where they gave rise to persistent AT1-like and AT2-like cells, as characterized by immunohistochemistry and single cell sequencing Together this work provides the first evidence of successful transplantation of PSC-derived cells into immunocompetent recipients Further characterization of this model system will provide important information on the function of these donor-derived cells and the development

of PSC-derived cell therapy of human pulmonary diseases

ABSTRACT #4 Secreted Frizzled Related Protein 2 Contributes to the Transdifferentiation of Human

Alveolar Epithelial Type II Cells (B)

Claude Jourdan Le Saux*, Tsung-Che Ho, Alexis Brumwell, Jaymin Kathiriya, Max Cohen,

Harold Chapman University of California, San Francisco, CA

Human alveolar epithelial type II cells (hAEC2) are known to serve as alveolar stem cells Recent studies have demonstrated that activated human mesenchymal cells (HMC) secrete factors that promote hAEC2 to transdifferentiate into functional, metaplastic alveolar KRT5+ basal cells during severe lung injury It is well known that MRC5, a fetal lung fibroblast cell line, maintains hAEC2 in culture In contrast, co-culture of hAEC2 isolated by flow cytometry using HTII-280 antibody with activated HMC or irradiated MRC5 led to a dramatic loss of SFTPC expression accompanying with a basal cell transcriptome by Day 14 as shown by RNAseq and histological staining Bulk RNA sequence analysis of activated HMC or irradiated MRC5 compared to non-irradiated non-activated MRC5 indicate a significant upregulation of genes encoding secreted growth factors and cytokines including secreted frizzled related protein 2 (sFRP2) sFRP2 is upregulated in IPF lungs and it is a known regulator of WNT and BMP pathway We hypothesized that sFRP2 plays a significant role in metaplastic transdifferentiation of hAEC2s Using hAEC2 co-cultures with MRC5 treated with recombinant sFRP2 from 10 ng/ml to 60ng/ml produced increasing percentage of KRT5+organoids associated with loss of SFTPC In contrast, siRNA-mediated silencing of sFRP2 in activated HMC or irradiated MRC5 prevented the loss of SFTPC expression and attenuated KRT5 expression in organoids In our hAECII 2D-model, sFRP2 did not regulate BMP4-dependent phosphorylation of Smad1/5/8 Interestingly, transdifferentiation is promoted by TGF-beta1, also a strong inducer of sFRP2 mRNA, suggesting sFRP2 is acting through previously unknown interactions with TGF-beta1 signaling In conclusion, utilizing 3D organoids, bulk RNAseq, and 2D cell culture system, we demonstrate that activated HMC secrete sFRP2 that is critically required for subsequent transdifferentiation of hAEC2s into metaplastic alveolar basal cells

https://translationalcells.org 25

Chou (CHOP) Research Hub 02

ABSTRACT #5 Detection of Rh Antibodies in Patient Plasma Using Customized Induced Pluripotent Stem

Cell-Derived Red Cells (P)

Hyun Hyung An*, Alyssa Gagne, Jean Ann Maguire, Giulia Pavani,Paul Gadue,Deborah

French, Connie Westhoff, Stella ChouChildren’s Hospital of Philadelphia, Philadelphia, PA

Background: Rh alloimmunization remains a major challenge for patients with sickle cell disease Antibody identification is hampered by the lack of appropriate reagent red cells, especially those that can identify antibodies against high prevalence or low prevalence Rh antigens We used human induced pluripotent stem cells (iPSCs) with the goal of producing renewable red cell reagents to aid complex antibody identification

Methods: We generated a panel of iPSCs including Rh null, D , hrS-, hrB-, V+VS+, Goa+, or

DAK+ For the Rh null line, we used CRISPR/Cas9 to disrupt RHCE in D neg iPSCs For D ,

RHD was inserted into the AAVS1 safe harbor locus of an Rh null iPSC line using ZFN resulting

in a line that expresses RhD but no RhCE iPSCs with uncommon variants were reprogrammed

from RH genotyped donors or engineered via ZFN similar to the generation of the D line

iPSC-derived red blood cells (iRBCs) were generated via iPSC hematopoietic differentiation and subsequent erythroid specific culture iRBCs were ficin treated and tested with patient plasma with previously identified Rh antibodies using gel agglutination

Results: Rh null iRBCs showed complete absence of cell surface Rh protein by flow, while ZFN targeted iRBCs (D , Goa+, DAK+) showed Rh expression Using standard Rh typing reagents (Ortho), Rh null iRBCs showed no agglutination with all 5 common Rh antibodies, D iRBCs

showed agglutination with anti-D only, and Rh variant iRBCs agglutinated as predicted by RH

genotype D iRBCs agglutinated against patient plasma containing anti-D while Rh null iRBCs showed no agglutination Rh null, D , and hrS- iRBCs did not agglutinate against patient plasma containing anti-hrS, while hrS+ iRBCs showed strong agglutination iRBCs expressing the low prevalence antigens VVS and Goa showed strong agglutination with plasma containing antibodies directed against these antigens

https://translationalcells.org 27

Self-Renewing Definitive Erythroblasts Derived from Human Pluripotent Stem Cells (B)

Kathleen McGrath*, Anne Koniski, Jayme Olsen, James Palis University of Rochester Medical Center, Rochester, NY

Eleven million units of red blood cells (RBCs) are transfused yearly in the US Cultured RBCs could serve as a supplemental source to better type and treat alloimmunized patients requiring chronic transfusion therapy The limited proliferative capacity of erythroid precursors is a major obstacle to generating sufficient numbers of RBCs We previously determined that overexpression

of Bmi-1, a member of the polycomb repressive complex 1, is both necessary and sufficient to drive the extensive ex vivo self-renewal of adult murine marrow-derived erythroblasts We have

subsequently determined that overexpression of BMI-1 can induce the extensive self-renewal of adult human peripheral blood mononuclear-derived immature erythroblasts that can be subsequently matured in vitro Since induced pluripotent stem cells (iPSCs) can serve as a genetically-modifiable and a renewable source of blood cells, we sought to derive self-renewing erythroid precursors from human iPSC Differentiation of human iPSCs, which recapitulates yolk sac hematopoiesis, yields mixtures of primitive erythroid cells, expressing high levels of embryonic epsilon-globin, and fetal-type definitive erythroid cells, expressing high levels of gamma-globin Definitive erythroblasts more completely express adult blood type antigens, so we characterized the cell surface phenotype of emerging definitive hematopoietic progenitors by monitoring their ability to generate definitive erythroid cells in culture, as assayed by RNA flow cytometry Lentiviral-driven overexpression of BMI-1 in isolated populations of human iPSC-derived definitive hematopoietic progenitors resulted in over 105-fold expansion of self-renewing erythroblasts when cultured in EPO, SCF, and Dexamethasone These cells expressed predominantly gamma-globin transcripts, consistent with a fetal definitive erythroid identity Self-renewing erythroblasts can be stored frozen and when transitioned into maturation media they terminally mature to late-stage erythroblasts Expanding the self-renewal capacity of definitive erythroid cells from genetically modified human iPSCs will help to pave the way for the production

of sufficient numbers of cultured RBCs for blood typing with reagent RBCs, creation of improved

in vitro modeling of RBC-intrinsic disorders, and ultimately transfusion therapy

ABSTRACT #7 Modeling Thrombocytopenia Absent Radius Syndrome Using Induced Pluripotent

by mutations in the RBM8A gene that lead to reduced RBM8A mRNA expression and significantly lower levels of Y14, the protein encoded by RBM8A, in TAR patient platelets Since Y14 has no

known roles in megakaryopoiesis, this study aims to understand how the deficiency in this protein leads to the MK phenotype seen in TAR patients One mechanism by which Y14 may affect megakaryopoiesis is through its role as a core factor in the exon-junction complex (EJC) The EJC

is important for RNA splicing, and depletion in EJC core proteins has been shown to cause several

alternative splicing (AS) changes in genes regulating apoptosis and cell cycle In vivo, MKs

differentiate from hematopoietic progenitor cells (HPCs) and undergo a complex maturation phase before they are ready to manufacture platelets Here, MKs endure several cycles of replication without division, greatly increase RNA synthesis, and must evade apoptosis throughout this stressful process Because MK maturation diverges from canonical cell cycle progression and RNA processing, it is likely that MKs depend upon the faithful, efficient splicing of proteins involved in these processes for proper maturation Therefore, we hypothesize that reduced Y14 expression in TAR syndrome alters AS in MKs, and that this change in splicing contributes to the

MK phenotype seen in TAR patients To test this hypothesis, we have generated two TAR patient induced pluripotent stem cell (iPSC) lines as well as isogenic corrected lines that contain a CD43-

promoter-driven RBM8A construct in the AAVS1 locus to restore expression in HPCs and MKs

We can successfully differentiate these lines into HPCs and MKs that will be assessed for maturation using flow cytometry for MK-specific markers or functionality using agonist responsiveness and proplatelet formation assays We will then purify these MKs and perform

RNA-seq to determine changes in gene expression as a result of RBM8A/Y14 deficiency The

results of these experiments will expand our understanding of how AS of relevant genes may affect

https://translationalcells.org 29

Investigating the Role of KLF1 in Primitive and Definitive Erythropoiesis (B)

Giulia Pavani*, Joshua Klein, Catriana Nations, Hyun Hyung An, Stella Chou,

Deborah French, Paul GadueChildren's Hospital of Philadelphia, Philadelphia, PA

During development, erythroid cells are produced through at least two hematopoietic waves (primitive and definitive), which generate erythroblasts with different functional characteristics Kruppel-like Factor 1 (KLF1) is a master transcription factor that regulates erythroid cell specification and terminal differentiation Increased expression of KLF1 in fetal liver vs yolk sac suggests a differential requirement of this transcription factor in primitive and definitive erythropoiesis Although extensively studied in mice, the role of KLF1 in human development has not been fully elucidated, with only one case of complete loss-of-function described Induced pluripotent stem cell (iPSC) can help us model processes that would otherwise be impossible to

study in vivo, such as lethal or embryonic phenotypes Upon differentiation, iPSCs can generate

hematopoietic progenitor cells (HPC) and erythroblasts from primitive and definitive programs,

thus allowing in vitro modeling of erythropoiesis and the study of KLF1 through development

Using CRISPR/Cas9 and base editors, we successfully generated isogenic cell lines heterozygous

or homozygous for a known pathogenic missense mutation in the zinc finger domain (L300P) and

a homozygous presumed null frameshift mutation (KO) All KLF1 edited lines were able to generate primitive HPC with similar yields Expression of hematopoietic markers was comparable among the different conditions; however, we observed an increased frequency of megakaryocyte progenitors in KLF1 mutant cells (~2-fold) We further tested the differentiation potential of these HPCs in a colony-forming assay; erythroid colonies were completely absent in KLF1 KO and KLF1L300P/L300P lines, while KLF1L300P/wt showed a 2.8-fold reduction compared to WT To study the effect of KLF1 mutations on erythropoiesis, we differentiated primitive HPCs into erythroblasts KLF1 KO and KLF1L300P/L300P HPCs failed to commit to the erythroid lineage, showing limited cell expansion, lack of hemoglobinization, and no expression of red cell markers Heterozygosity for KLF1 L300P mutation reduced cell proliferation and slowed erythroid commitment, however erythroid CD235a+ cells reached WT levels at the end of the differentiation (>90%) While some red cell antigens such as Kell and CD47 were expressed at normal levels in KLF1L300P/wt erythroblasts, we observed reduced expression of CD44, recapitulating the phenotype

of heterozygous carriers of KLF1 mutations Similar findings were observed in an additional iPSC line harboring another zinc finger mutation (KLF1H329R/wt), further validating our model RNA-seq analysis in WT and edited erythroblasts is currently underway and will elucidate qualitative and quantitative effects of KLF1 mutations on primitive erythropoiesis Future experiments on definitive erythroblasts derived from WT and edited lines will determine differential ontogenetic targets of this transcription factor and possibly reveal novel roles of KLF1 during development

Daley (BCH) Research Hub 03

https://translationalcells.org 31

Role of Transposable Elements in Hematopoiesis and Cancer (P)

Deepak Jha*, Mohamad Najia, Cheng Zhang, Benoit Laurent, Caroline Kubaczka, Bjoern

Chapuy, Arianna Markel, Vivian Morris, Yu-Chung Huang, Michael Morse, Matthew

Marunde, Anup Vaidya, Zachary Gillespie, Sarah Howard, Trista North, Michael-Christopher

Keogh, Yang Shi, Hu Li, Margaret Shipp, George Daley

Boston Children’s Hospital, Boston, MA

Tissue differentiation depends on transcriptional regulatory programs mediated by signaling pathways that impinge on transcription factors (TFs) and epigenetic modifiers During hematopoiesis, lineage-specific TFs interact with a variety of cis-regulatory elements (CREs) including enhancers, insulators, and promoters Transposable elements (TEs) are sometimes co-opted as CREs, most commonly as enhancers, but their role in directing hematopoietic cell fates remains unclear Here we employ a modified activity-by-contact (ABC) model to catalog enhancer- gene associations throughout the human hematopoietic hierarchy, and find that the enhancers and the genes they regulate recapitulate known biology and are enriched in hematopoietic pathways and lineage- specific TF motifs Moreover, these enhancers are enriched for TEs in hematopoietic cells, and co-expressed with TE regulatory machinery including histone H3K9 methyltransferases such as SETDB1 and SUV39H1, KAP1/TRIM28, DNA methyltransferases, and the TASOR complex Normal lymphoid cells including B, T and NK cells showed high expression of TEs and low expression of TE-repressive machinery, a pattern that was reversed in a common B-cell lymphoma, which showed widespread repression of TEs Genetic and chemical modulation of H3K9 demethylases in B-cell lymphoma led to de- repression of TEs, activation of cGAS/STING-mediated inflammatory cell death and reduced growth in tumor xenografts We discovered a similar repression of TEs in acute myeloid leukemia patient samples, and found that modulating a writer and eraser of H3K9 methylation induced cancer cell death In summary, we describe a novel role of TEs in human hematopoiesis and identify modulators of H3K9 methylation as vulnerable targets in two common hematopoietic malignancies

ABSTRACT #10 Human Pluripotent Stem Cell-Derived T Cells for Cancer Immunotherapy (B)

Ran Jing*, Areum Han, Mohamad Najia, Caroline Kubaczka, Deepak Jha, Trista North,

George Daley Harvard Medical School, Boston, MA

CAR T cell therapy has shown major therapeutic effects and therefore holds great promise for the cure of hematopoietic cancers However, the broad application of this breakthrough anti-cancer strategy has been impeded by the limited source of T cells IPSCs represent an ideal source for scalable manufacture of off-the-shelf products for cell therapy Although T cells can be

differentiated from iPSCs in vitro, the process is inefficient, and the function and gene expression

profile of iPSC-derived T cells are not identical to those of peripheral blood T cells Previous work from our lab has identified a cocktail of transcription factors that help to drive lymphoid fate from iPSC sources We therefore hypothesize that this new knowledge would allow efficient generation

of mature, functional T cells from iPSC-derived hematopoietic stem and progenitor cells (HSPCs).Human iPSCs were first differentiated into CD34+CD45+ HSPCs that can be stably propagated in vitro by virtue of conditional immortalization with a distinct cocktail of 5 doxycycline-inducible

transcription factors (HOXA9, ERG, RORA, SOX4, and MYB) These progenitor cells were then

differentiated to T cells by culturing on tissue culture plates coated with Notch ligands After 5 weeks of differentiation, CD4/CD8 single positive T cells that express CD3 and TCRαβ were detected To further evaluate the effector function of these iPSC-derived T cells, we transduced iPSC-T cells with anti-CD19 Chimeric antigen receptors (CARs) and exposed these cells to CD19-expressing tumor cells, and the cytotoxic response was determined by detecting the specific lysis

of tumor cells Our results suggest that iPSC-derived T cells are capable of robust antigen-specific killing of tumor cells and display an efficacy similar to PBMC T cells In conclusion, we have developed a new platform that allows us to more efficiently generate functional HSPCs and T cells from iPSCs Such an approach will be compatible with commercial-scale production and broad distribution, and therefore will significantly enable CAR T cell-based immunotherapy

https://translationalcells.org 33

GCSF Induced Mobilization of Hematopoietic Stem and Progenitor Cells from the

Embryonic Hematopoietic Niche (B)

Ji Wook Kim*, Samuel Wattrus, Leonard Zon Boston Children’s Hospital and Harvard Medical School, Boston, MA

Hematopoietic stem cell transplantation requires a collection of hematopoietic stem cells from patients or stem cell donors GCSF is widely used in the clinic to mobilize hematopoietic stem and progenitor cells (HSPCs) from the adult bone marrow niche into circulation, allowing a collection

of HSPCs from blood Many interactions between HSPCs and adult niche are also found in the embryonic hematopoietic niche To study if GCSF can mobilize HSPCs from the embryonic hematopoietic niche, we expressed GCSF under the control of heat-inducible promoter in zebrafish embryos GCSF was induced with heat-shock at three days post-fertilization when HSPCs reside

in the caudal hematopoietic tissue (CHT), the embryonic hematopoietic niche of zebrafish Live

imaging Tg(runx:mCherry) reporter line of HSPCs 24 hours post GCSF induction demonstrated

that circulating HSPCs increase in response to GCSF expression Whole -mount in situ hybridization of c-myb displayed HSPCs that prematurely colonize the kidney, the adult hematopoietic niche of zebrafish, in 73% (n = 41/56) of embryos when GCSF is expressed The number of EdU positive proliferating HSPCs in the CHT did not change significantly in response

to GCSF, demonstrating that HSPC proliferation is not affected Together, these data suggest that HSPCs leave the embryonic hematopoietic niche in response to GCSF, enter circulation, and colonize the adult hematopoietic niche Expansion and activation of neutrophil is one of the mechanisms how GCSF mobilizes HSPCs from the bone marrow To verify whether neutrophil is necessary for GCSF induced mobilization from the CHT, we ablated neutrophils with a nitroreductase based system in embryos Regardless of the neutrophil ablation, we observed an increase in circulating HSPCs and premature colonization of the kidney 24 hours post GCSF expression in 77% (n = 36/47) of embryos In conclusion, these results demonstrate that GCSF is sufficient to mobilize HSPCs from the embryonic hematopoietic niche in a neutrophil-independent manner

Snoeck (CUMC) Research Hub 04

https://translationalcells.org 35

A Clinically Relevant Model of Acute Respiratory Distress Syndrome in

Human-Sized Swine (P)

Sarah Kaslow*, Jonathan Reimer, Meghan Pinezich, Maria Hudock, Panpan Chen, Camilla

Predella, Kenmond Fung, Jay Leb, Carrie Ruzal-Shapir, Charles Marboe, Matthew Bacchetta,

N Valerio Dorrello, Gordana Vunjak-Novakovic Columbia University, New York, NY

Purpose: Acute respiratory distress syndrome (ARDS) has a mortality of 30-50% yet treatment strategies have not markedly changed since the ARDSNet trial in 2000 Here, we describe a model

of inducing ARDS in swine by epithelial and endothelial insults for the study of new treatments Methods: Yorkshire swine (n=9) were intubated and mechanically ventilated Gastric contents were delivered bronchscopically into the trachea and mainstem bronchi to simulate aspiration

injury to the lung epithelium, followed by central intravenous infusion of Escherichia coli-derived

lipopolysaccharides (LPS) Once PaO2/FiO2 (P/F) ratio decreased to <150, defined as ARDS 0hr, swine received standard ARDS treatment The experimental endpoints included animal expiration, 24-48 hours after ARDS induction, or P/F >300 on minimal ventilator settings on two consecutive hourly measurements Chest radiographs were scored using the Radiographic Assessment of Lung Edema (RALE) by two radiologists Histologic samples were collected at experimental endpoint and scored for injury severity by a pulmonary pathologist

Results: All swine (n=9) developed moderate-to-severe ARDS per the Berlin criteria with an average minimum P/F = 79±17 Two swine survived less than 6 hours and the remaining swine (n=7) remained with a P/F <300 for 6 to 48 hours Chest radiographs showed significantly worsening lung edema at ARDS 0hr (corresponding to a total RALE score of 21.2±6.7 ; Figure)

relative to baseline (10.5±6.7, p=0.005) Histopathologic analysis (n=6) demonstrated neutrophil

infiltration of the alveoli, alveolar edema, and mild-to-moderate alveolar membrane thickening without hyaline membranes

Conclusion: Dual injury to the lungs in human-sized swine induced a reproducible, severe, and prolonged lung dysfunction consistent with moderate-to-severe ARDS clinically, radiographically, and histopathologically This model provides a valuable research platform to better understand the pathophysiology of ARDS and test novel

therapeutics

quiescence Here we show that deletion of Mfn2 causes an ‘aged’ HSPC phenotype in young mice,

with relative expansion of the stem and progenitor cell compartment in both medullary and medullary compartments, with exacerbated loss of lymphoid potential during aging RNAseq combined with single cell-RNAseq experiments revealed striking up-regulation of Interferon

extra-stimulated genes in HSCs as well as whole bone marrow (BM) and spleen cells from Mfn2 -/- out (KO) mice compared to wild-type (WT) Detectable IFN was furthermore found in the serum

knock-of Mfn2 -/- mice, albeit its source is unclear Moreover, Mfn2 does not functionally interact with Mitochondrial activator of viral signaling (Mavs), a central mediator of innate immunity signaling associated with mitochondria Instead, double deletion of Mfn2 and Stat1 or the type I interferon receptor fully rescued the effect of Mfn2 deletion on HSPC cycling Finally, aged Mfn2-deleted

BM has decreased hematopoietic regenerative function compared to WT with a full rescue by

deleting Stat1 together with Mfn2 Furthermore, Mfn2-deleted young HSCs sorted from BM or

spleen have lower reconstitution capacity, confirming a stem cell impairment

Our data indicate that MFN2 is required to dampen tonic IFN signaling and production and may play a role in the prevention of age-related changes in the hematopoietic system, including age-associated hematopoietic malignancies We are currently searching for the source of this tonic IFN signaling Taken together, these findings suggest that manipulating mitochondrial dynamics might

https://translationalcells.org 37

Quantitative Analysis of Cystic Fibrosis Lung Matrix (B)

Meghan Pinezich*, Manuel Tamargo, Sharon Fleischer, Jonathan Reimer, Sarah Kaslow, Maria Hudock, Ahmed Hozain, Charles Marboe, Matthew Bacchetta, John O’Neill, N Valerio

Dorrello, Gordana Vunjak-NovakovicColumbia University, New York, NY

Background: Cystic fibrosis (CF) leads to progressive decline in lung function throughout life, necessitating lung transplantation at a median age of 29.5 years Previous studies have revealed that elevated levels of proteolytic enzymes found in sputum of CF patients correlate with worsening lung function We hypothesized that end-stage CF is characterized by degradation of key lung extracellular matrix (ECM) components Methods: Lung tissue was obtained at the time

of transplantation from patients with end-stage CF (n=8) and from uninjured regions of donor lungs to serve as controls (n=3) Structure and composition of CF ECM was assessed using

histology, electron microscopy, liquid chromatography mass spectrometry (LC-MS), and gene

ontology (GO) analysis Results: Histological analysis of CF specimens revealed septal

degradation, fragmented matrix fibers, and proteinaceous fluid-filled airways and alveoli (Fig 1A)

On scanning electron microscopy (SEM), perturbed parenchymal ultrastructure and alveolar architecture was observed in CF, with wall thickening, obstruction, and collapse of the alveoli Transmission electron microscopy (TEM) enabled visualization of basement membrane dysregulation, alveolar collapse, and fragmented matrix fibers in CF (Fig 1B) LC-MS was used

to quantify changes in matrix proteins Collagens (including collagen IV, V, VI, XI, XXII, XVIII, XXI, XXVI) were significantly downregulated in CF compared to normal tissue Key basement membrane constituents, including multiple isoforms of laminin (laminin 2, 4, 5; |log2FC| > 1, p < 0.05) and nidogen 1 (log2FC = -1.31, p = 0.03) which provide structural support and promote cell adhesion, were similarly downregulated in CF Elastin, which confers lung mechanical properties, was decreased in CF compared to healthy lung (log2FC = -1.6, p = 0.026), similar to elastin loss observed in emphysema (Fig 1C)

Conclusions: Degradation of the distal lung ECM

accompanies functional decline of the lung and may

contribute to lung inflammation in end-stage cystic

fibrosis Future studies should evaluate the effects

of ECM alterations to cell phenotype, and

determine if ECM damage can be reversed

Figure 1 Comparison of cystic fibrosis and normal lung ECM

Structural comparison of CF and normal lung with (A)

histology, and (B) electron microscopy (C) Analysis of ECM

composition with LC-MS

ABSTRACT #15

Ex Vivo Lung Repair on Cross-Circulation (B)

Meghan Pinezich*, Ahmed Hozain, John O’Neill, Brandon Guenthart, Yuliya Tipograf, Jonathan Reimer, Sarah Kaslow, Mavis Liu, Katherine Cunningham, Rachel Donocoff, Jinho Kim, Ya-

Wen Chen, Hans Snoeck, Matthew Bacchetta, Gordana Vunjak-Novakovic

Columbia University, New York, NY Purpose: Despite medical and surgical advances in recent years, the majority of donor lungs are not suitable for transplantation Ex vivo studies have potential to support and functionally recover donor lungs to a level acceptable for transplantation Our objective was to develop methodologies

to assess function and deliver therapeutic cargo to donor lungs supported by cross-circulation Methods: A cross-circulation bioreactor was designed to provide normothermic support of extracorporeal donor lungs (Fig 1A) During cross-circulation support, lungs were assessed through real-time diagnostic imaging including video bronchoscopy and thermal imaging (Fig 1B), and therapeutic intervention was achieved through delivery of exogenous surfactant and lung cell populations

Results: Cross-circulation enabled functional maintenance of the lungs for up to 4 days, confirmed

by efficient gas exchange and lung compliance Airway integrity was confirmed by visualization

with video bronchoscopy, and appropriate lung perfusion and ventilation were confirmed using thermal imaging Targeted delivery of exogenous surfactant and therapeutic cells was achieved via bronchoscope, and cells were visualized through trans-pleural imaging following delivery Delivered cells remained viable throughout the duration of cross-circulation support (Fig 1C, D)

Figure 1 Cell delivery in extracorporeal lungs on circulation support (A) Schematic diagram of circulation support circuit: (B) Operating room setup with real-time diagnostic and therapeutic capabilities, TC,