Cfri presentation 2014

Development and

Characterization of Novel 3D

Airway Cell Models

Carolin Boecking, M.D.

University of California, San Francisco

Human Airway Cell Models for CF Research

Focus of our laboratory

is the development and

characterization of in

vitro (cell culture)

models of the

conducting airway

surface epithelium and

submucosal glands for

use in CF research

Central conducting airway

M

S

Human Airway Cell Models for CF Research

Important roles for airway cell models in CF research

include:

Understanding the functions of CFTR and how alterations in

its function lead to disease

Defining the pathophysiology of secondary lung problems in

CF, e.g., infection and mucus hypersecretion

CF drug discovery

In vitro testing of potential CF gene therapies

Understanding the significance of airway gland hyperplasia

and the mechanism of mucus hypersecretion in CF

Improved human airway cell models are needed for:

Facilitating CF drug discovery

Identifying therapeutics for CF treatment of secondary lung

problems

Current 3D Cell Culture Approaches

The third dimension bridges the gap between cell culture and live tissue, Francesco Pampaloni,

Emmanuel G. Reynaud & Ernst H. K. Stelzer, Nature Reviews Molecular Cell Biology 8, 839-845 (October

2007)doi:10.1038/nrm2236

Organotypic explant culture2D cell culture

Cellular speroids Microcarrier cultureRotating wall vessel

Polarized epithelial cell culture

Relevance of Current Project

Subtrates cell cultures especially important for submusosal gland cell cultures

Culture architecture resembles native tissue

Nutrients can access basolaterally and apically

Cell-cell and cell-ECM network

chemical and mechanical signals

mimic in vivo cell physiology

Might prevent loss of tissue-related functions

Airway surface cells

Cell formations exhibit polarization like planar cell model

Maintains architectural integrity and functionality

Relevance for drug discovery: Multitude of cell units

available for High Throughput Screening

Simple Spheroids

Numerous spheroids can be generated

in collagen gels/spinner flasks using

isolated cells obtained by enzymatic

digestion nasal/airway epithelium

Transform into spheroids over about

3 - 5 days

Potentially, each spheroid serves for a

single experiment for measurement of

responses to changing conditions

Phase microscopy of airway spheroids

Model of Spheroid Formation Process

Cell aggregation: Integrin-ECM binding

Delay Period: Accumulation of

E-cadherin

Compact spheres: adherens

junctions formed

Morphology of Airway Epithelial

Spheroid

Spheroid formed from isolated cells--

arrow shows cilia

Developing spheroid from epithelial cell

clusters

ABC

Drawbacks of Simple Spheroids

Differentiated features decline with time in

culture

CFTR expressed but ion transport is not

detectable

Promoting Spheroid Differentiation

Differentiation of airway epithelial spheroids

may be maintained/improved by adding stem

cell growth factors R-spondin 2 and Noggin

Liu et al. (Functional Cftr in crypt epithelium of organotypic

enteroid cultures from murine small intestine) grew

“enteroid” crypts with functional CFTR using R-spondin 1

and Noggin

Stem Cell Growth factor R-spondin2

Roof plate specific spondinexpressed in neural tube

Secreted agonists of Wntpathway

Wnt signals help regulate cell division and specialization throughout the body during development and tissue regeneration.

R- spondin 2 has been linked to lung development

fetal lung buds exclusively produce R-spondin 2

Stem cells: Orphan receptors find a home, Walter Birchmeier

Nature 476, 287–288 (18 August 2011) doi:10.1038/476287a

Stem Cell Growth Factor Noggin

Noggin, a BMP antagonist

BMP4

transcribed in lung mesenchyme

involved in branching of lungs and surface epithelium differentiation

Noggin causes

proximalization of

distal airway

epithelium

Experiment Overview

300.000 cells plated in 300μl gels

Spheroids recovered from gels, re-plated and stem cell factors added

Ciliated epitheliods, can be recovered for further experiments

Method modified from Sato et al., Paneth cells constitute the niche for Lgr5 stem cells

in intestinal crypts. Nature 469: 415-419, 2011.

Day 0 Day 28Day 14

Day 0-14

Day 14-28

+200ng/ml R-spondin2

+25ng/ml Noggin

Ready For Further Studies

Step 1- Airway Epithelial Cells

Embedded in Collagen Gels

Matrigel only Vitrogen only

Mixed matrix: Matrigel/Vitrogen

Step 2- Recovery of Spheroids without

Disrupting Their 3D Orientation

• Spheroids were

recovered from

collagen gels on day

14 using:

• Dispase for

Matrigel

• 0.2%

Collagenase for

Vitrogen

• Spheroids were re-

plated in same gel

Airway epithelial spheroids recovered from

and re-plated in Matrigel

Methods 3- Stem Cell Growth Factors

Added to Medium

After re-plating ALI

medium was

supplemented with

• 125-200 ng/ml

R-spondin 2

• 25 ng/ml Noggin

“Epithelioids”

“Epithelioids”

Cilia

Spheroid Cavity

Epitheliod Architecture Maintained by Growth factors

Control Rspondin-2 and

Noggin added

Immunofluorescence

Protein Quantification

DNA Quantification

Future Studies

EM/ SEM

Histochemical and immunocytochemical stains for

relevant elements of airway surface epithelium,

including CFTR, e-cadherin ciliated cells, basal cells

and mucin genes (MUC5AC, MUC5B)

CFTR function assessed by fluorescence assays,

intracellular microelectrodes and microfluidics

Ciliary beat pattern and beating frequency will be

studied using high-speed video imaging

Established Cell Models of Airway GlandsBrief summary of methods

After epithelium stripped from airway, submucosal tissue sharply dissected

Tissue minced and then dissociated in enzymes--collagenase,hyaluronidase, DNase

Isolated gland aciniplated in collagen coated flask

Near confluent cultures trypsinized onto cell culture inserts

Gland Hyperplasia in Cystic Fibrosis

+IL-13 +IL-17

Control

IL-13 and IL-17 Induce Mucin Production in

Planar Submucosal Gland Cell Cultures

IL-13 and IL-17 Induce Mucin Production in

Planar Submucosal Gland Cell Cultures

• Pink mucicarmine stain

identifies variable

amounts of mucin in

luminal cells

• A. Control cells

• B. Cells exposed to Il-

13 (10 ng/ml)

• C. Cells exposed to IL-17

(10 ng/ml)

MUC5B Stain of Planar Submucosal Gland

Cell Culture Exposed to IL-17

Negative

Control

exposed to IL-

17 (10ng/ml)

Development of Airway Glands Phases of Gland Development (Plopper)

1. Bud formation

2. Outgrowth and branching of buds into cylinders of undifferentiated cells

3. Proliferation of tubules and aciniwith undifferentiated cells distally and differentiation of mucus cells proximally

4. Differentiation of serous cells in proximal tubules. 14 weeks

Wnt3a induces Lef-1 gene expression

and activation of -catenin in airway

submucosal gland buds of mice and is

required for maintenance of bud growth

(Driskell et al, Devel Biol 305:90, 2007.

Adding Wnt/beta catenin enhancers to Submucosal

Gland Cells 3D cultures induced Cilia growth

Contamination from

epithelial cells

Ciliated duct cells

present

Pluripotent “stem cell”

within gland tissue

reprogrammed by

growth factors to exhibit

characteristics of surface

cells

Submucosal Gland Cultures in Gels Day 5

Control HGF HGF & IL-1399% round or oval

1% cylindrical or stellate

60% round or oval

40% cylindrical or stellate

53% round or oval

47% cylindrical or stellate

Submucosal Gland Cells cultured in Gels

initial gland formation in

gels, day 7

Glands cultured with

MammoCult®

Glands grown on mixed

matrices, day 14

Glands grown in Matrigel alone, day 14

IL4 and HGF Enhance Tubular Growth

Control IL4 only IL4+HGF

IL4 and HGF Enhance Tubular Growth

Control IL13 only IL13+HGF

Future studies

Gene expression analysis

Thank You!

Finkbeiner Lab

Walter E. Finkbeiner, M.D., PhD

Lorna Zlock, Staff Research Associate

Cystic Fibrosis Research, Inc.

For all your support