Now

Posters on specific applications using Propagenix
technology
!  Poster 1: Using Conditional Reprogramming technology to expand
patient-derived xenografts (PDX) for in vitro profiling and creating master
cell banks
!  Poster 2: Ex vivo expansion and preservation of functionality of human
islet β-cells using Conditional Reprogramming technology
!  Poster 3: A Manufacturing- and Regulatory-Friendly Cell Expansion
Technology Allowing for Trillion-Fold Expansion of Primary Airway
Epithelial Cells
!  Poster 4: A Feeder-Independent and Serum-Free Cell Culture
Technology that Allows for Over Trillion-Fold Expansion of Diverse
Primary Epithelial Cells
Use of conditional reprogramming to develop and characterize cell
cultures from patient-derived xenograft (PDX) models of human
lung and ovarian cancer
Alexandra Borodovsky1, Travis J. McQuiston2, Brian Dougherty1, Ambar Ahmed1, David Whitston1, Daniel Stetson1, Gretchen K. Hubbard2,
Sharon S. Challberg2, Mike Zinda1, Brian A. Pollok2 and Celina M. D’Cruz1
1 AstraZeneca
641
Pharmaceuticals PLC, Oncology Bioscience, Waltham, MA USA, 2 Propagenix Inc, Gaithersburg MD
Abstract
Introduction
• Development of stable PDX cell lines remains a challenge
due to murine stromal outgrowth, lineage commitment and
limited differentiation potential.
Tumor Volume (mm3)
1400
Figure 1. Propagation and immortalization of human adult
epithelial cells3
1000
800
600
Vehicle
400
Savolitinib
0
0
Figure 3. CR-PDX cell lines are amenable to in vitro
chemosensitivity screens. CR-PDX cells were treated with indicated
inhibitors and in vitro drug sensitivity was compared to in vivo response.
Results
CR-PDX models are amenable to chemosensitivity
screens
• Compounds with known in vivo drug response were
selected for chemosensitivity screening, including the BRD4
inhibitor AZD5153, selumetinib (AZD6244, ARRY-142886),
savolitinib (AZD6094, HMP-504, Volitinib) and docetaxel.
• In some cases, in vitro/in vivo discrepancy can exist, similar
to what is observed in conventional cell line xenografts
models.
Table 1. PDX model characteristics
Model
Indication
Pathology
Parental
Mutations
CR-PDX
mutations
Average
doubling
time/day
OV0857F
Ovarian
Serous
Carcinoma
TP53 (R248Q)
TP53 (R248Q)
0.27
KRAS (G12C)
TP53 (R273C
0.37
LG0567F
Lung
KRAS (G12C)
Adenocarcinoma
TP53 (R273C)
HLXF036LN
Lung
Adenocarcinoma TP53 (P152L)
TP53 (P152L)
0.54
HLXF-056
Lung
Adenocarcinoma
KRAS (G12R)
TP53 (K292*)
KRAS (G12R)
TP53 (K292*)
0.16
Conditional reprogramming expands PDX cells and maintains
key mutations
• Conditional reprogramming expands lung and ovarian PDX cells
• Human cells were enriched by through magnetic murine cell
depletion.
• Sequencing analysis identified that the CR-PDX cell lines
maintained mutations of the parental PDX.
140
120
100
80
60
40
20
0
Relative MET mRNA expression
CR-HLXF-036LN
1.6
1.4
1.2
1
0.8
0.6
0.4
Figure 2. CR technology expands PDX explants. Representative
images of PDX cells growing in co-culture with GFP-expressing irradiated
fibroblast cells (arrows). Left: HLXF-036LN, P1 10X, right: OV0857F, P4,
10X.
0
CR-PDX cells can be utilized for gene knockdown studies
• To determine if CR-PDX models can be utilized for genetic
manipulation studies and confirm sensitivity to MET signaling,
siRNA transfection was used to knockdown MET expression
in CR-HLXF-036LN cells. Knockdown efficiency was
confirmed by qPCR.
• MET knockdown significantly reduced viability of CR-HLXF036LN cells in vitro, as compared to controls.
Presented at the AACR Annual Meeting, New Orleans, LA, 17 April, 2016
2
4
7
9
11
14
16
Days
Figure 5. CR-PDX cells form tumors and retain drug sensitivity in vivo
CR-PDX cells form tumors in vivo, retain drug sensitivity
• Approximately 3 weeks following implantation, CR-HLXF-036LN
tumors formed and had similar growth kinetics to the parental
PDX model.
• AZD6094 induced regressions in CR-HLXF-036LN tumors,
similar to the parental PDX response, resulting in >100% tumor
growth inhibition (Figure 3).
Conclusions
• CR technology generates stable explant cell lines from PDX
models
• CR-PDX models retain parental mutations and are amenable
to high throughput chemosensitivity screening and genetic
manipulation
• CR-PDX models form tumors in vivo and maintain drug
response
0.2
Figure 4. CR-PDX cells can be utilized for gene knockdown studies.
CR-HLXF-036LN cells were treated with siRNAs for MET MYC (positive
control ) and non-targeting control (left). Knockdown efficiency was
confirmed by qPCR (right).
• CR cell technology is dependent upon the combination of
feeder cell-derived factors and Rho Kinase (ROCK) inhibitor2.
Supported by
1200
200
• CR technology is a novel cell culture system facilitating the
generation of stable cultures without genetic manipulation.
• The purpose of this study was to identify the potential
applications of CR technology for derivation of PDX cell lines.
CR-HLXF-036LN
1600
Relative Percent Viability
Patient-derived xenografts (PDX) are widely recognized as a more
physiologically relevant preclinical model to standard cell line
xenografts. PDX models faithfully recapitulate the original patient
genetic profile, gene expression patterns and tissue histology.
Despite their benefits, PDX models are limited by their inherent
variability, lower throughput and lack of growth in vitro. The ability
to generate cell lines from PDX models would enable high
throughput chemosensitivity screens, ex vivo genetic manipulation
and the development of novel orthotopic models. Development of
stable PDX cell lines remains a challenge due to murine stromal
outgrowth, lineage commitment and limited differentiation
potential. Conditional reprogramming (CR) cell technology is a
novel cell culture system facilitating the generation of stable
cultures without genetic manipulation. The success of CR cell
technology is dependent upon the combination of feeder cellderived factors and Rho Kinase (ROCK) inhibitor. CR cells,
therefore, represent a new class of progenitor-like cells, distinct
from the phenotype of embryonic stem (ES) cells and induced
pluripotent stem (iPS) cells. The purpose of this study was to
identify the advantages, limitations and potential applications of
CR technology for derivation of PDX explant cell lines. Early
passage human lung and ovarian PDX tumors were cultured in
CR conditions to create stable explant cell lines. Cell lines were
established from 5/8 (63%) PDX tumors and were expanded over
6 months in culture with varying morphologies and growth kinetics.
Due to normal outgrowth of murine stromal cells, early CR
cultures contained mixed populations and required murine
depletion to enrich for human cells. Key oncogenic mutations in a
model of ovarian papillary serous adenocarcinoma were
preserved in the enriched tumor cell population. While purified CR
PDX cell lines were amenable to high throughput chemosensitivity
screens, in vitro chemosensitivity did not consistently predict
response in in vivo murine models. The CR PDX cell lines were
additionally assessed for genetic manipulation and ability to form
tumors in vivo. Collectively, these results demonstrate the
applications of CR technology for the generation of stable explant
cell lines from PDX models for preclinical studies.
References
1. Saebz FR et al. (2014). Conditionally Reprogrammed Normal and
Transformed Mouse Mammary Epithelial Cells Display a
Progenitor-Cell–Like Phenotype. PLOS One. 15;9(5).
2. Palechor-Ceron N et al. (2013). Radiation Induces Diffusible
Feeder Cell Factor(s) That Cooperate with ROCK Inhibitor to
Conditionally Reprogram and Immortalize Epithelial Cells. Am J
Pathol. 183(6):1862-70.
3. Liu X et al. (2012). ROCK Inhibitor and Feeder Cells Induce the
Conditional Reprogramming of Epithelial Cells. Am J Pathol.
180(2):599-607.
Acknowledgements
The authors would like to Theresa Prioia, Evan Barry, Danielle
Greenawalt and Kelsey Biely for their help with this work.
Functional and molecular characterization of human b-cells
propagated using the CR cell technology platform
Joseph R. Blasic1*, Travis McQuiston1, Kelsey Bieley1, Xiaobo Ma2, Lijuan Fan2,Guoling Chen2, Wanxing Cui2
1Propagenix Inc., 2Medstar Georgetown University Hospital, Transplantation Institute
Abstract
Propagenix has licensed Conditional Reprogramming (CR) technology,
developed at Georgetown University, which enables extensive ex vivo
expansion of primary epithelial cells without genetic modification or complex
re-differentiation protocols. The CR-expanded cells remain lineage committed
and can spontaneously revert to their differentiated phenotype when removed
from the expansion media. Here we show that CR technology allows for over
a billion-fold expansion of primary β-cells. Furthermore, these cells continue
to express key β-cell markers (PDX-1, Neuro D1, MafA, and Insulin C-peptide)
and regain expression of glucose sensing machinery (Glut-2 and glucokinase)
after removal from expansion. Expanded cells also have the ability to secrete
insulin in response to glucose stimulation. We are currently investigating the
ability of these expanded cells to restore euglycemia to STZ-treated diabetic
SCID mice.
48-72 hours
96-120 hours
120 hours no EdU control
Expanding islets were labeled for 20-24 hours with Click-iT EdU reagent
(Thermofisher). In this assay, the modified thymidine analogue EdU is efficiently
incorporated into newly synthesized DNA and fluorescently labeled with a bright,
photostable Alexa Fluor dye in a fast, highly-specific click reaction. By 48-72 hours
some of the cells within the islet are beginning to divide, though the majority of the
cells that have migrated out are not yet dividing. By 96-120 hours, many cells are
dividing both within the original islet body as well as those that have migrated out in
monolayer culture.
P L001
M *0 0 1
M *0 0 2
20
M *0 0 3
M *0 0 4
M *0 0 5
10
0
20
40
60
D a y s i n C u lt u r e
Figure 3. Growth curves of islet cultures.
Each curve represents an individual donor. PL001 was obtained from Prodo
Laboratories, while M*001-M*006 were isolated by the Islet Transplantation Program
at MedStar Georgetown University. After initiation most cultures expand for 20-30
population doublings or a 1 million to 1 billion fold increase in cell number. Typical
growth rates are between 0.6-0.7 PD/day. Isolated islets are typically received within
2-5 days after isolation. Overall, β-cell cultures have been established from 11 of 15
primary islet samples we have received.
4 days
Figure 5. Proliferation of insulin-positive cells. Cells at passage 5 were labeled with
Click-iT EdU reagent for 18 hours, removed from co-culture, fixed and stained for the
incorporated proliferation label (green) and counter stained with an antibody against
human insulin C-peptide (red) in solution. Cells were then deposited on glass slides using
a CytoSpin (Shandon) A) Phase contrast overlay with Click-iT EdU staining. B) Human
insulin C-peptide staining with Click-iT EdU label. This culture had a doubling time of 34
hours (0.7 PD/day). Based on this approximately 53% of cells would be expected to be
labeled. Direct counts of labeled cells show that 53.5% of cells are EdU (n=200) and
>90% were insulin positive.
30
0
3 days
10 days
Figure 6. Expression of b-cell
markers. CR-expanded cells were
immunostained for the expression
of key b- and a-cell markers. A-D
were seeded onto chamber slides
and allowed to attach and grow for
72 hours before fixing and staining.
Cells were all positive for PDX-1,
NKX6.1, NEUROD1, and MafA,
important markers of mature bcells. E-F are aggregated cells that
were removed from expansion for
3 days and allowed to attach
overnight to a collage glass slide.
G is a 5mm section of 5 day
differentiated aggregates.
The
expression of Glut2 (F) and
glucokinase
(G)
are
only
detectable after the cells have
been aggregated and removed
from the expansion media.
Human Insulin (mU/1,000 cells
Sequential Glucose Challenge
Figure 1. Example of human islet cell outgrowth.
Human islets plated in CRM with irradiated feeders begin to migrate out of the
islets and expand across the plate. By 10 days post-plating, a large colony
characterized by small tightly packed cells has formed. Colonies continue to expand
and can be passaged and re-plated using standard tissue culture techniques.
9
8
7
6
5
4
3
2
1
0
Sample 1
Conditional Reprogramming (CR) technology is based on our conditional
reprogramming media (CRM) and irradiated feeder cells. CRM combines a
Rho kinase inhibitor in F12-DMEM media with other growth factors. When
mature epithelial cells are introduced into CR culture conditions, they adopt a
tissue-specific progenitor phenotype and become proliferative. Primary
epithelial cells from airway tissues, retina, prostate, breast, intestine, pancreas,
and other tissues replicate for extended periods under CR conditions. On
removal from the CR culture conditions, these cells re-adopt their original
differentiation state. No genetic manipulation is involved in the switch between
the replicative and differentiated phenotypes, and no complex re-differentiation
strategy is required for the cells to return to their normal functional phenotype.
The technology has also been employed successfully to propagate cells from
primary tumors, metastatic tumors and patient-derived xenografts
A
E
PDX-1
Insulin C-peptide
F
B
Glut2
NKX6.1
NeuroD1
G
C
NEUROD1
Glucokinase
D
MafA
Conclusions
2mM
Conditional Reprogramming of islet cells.
B
A
Figure 2.Click-iT EdU labeling of expanding cells.
P o p u la tio n D o u b lin g s
Type I diabetes (T1D) is an autoimmune disorder that targets and destroys the
insulin-producing β-cells of the pancreas, leaving patients dependent on
injected insulin to regulate their blood glucose levels. A true cure for T1D
could be achieved by replacing the lost β-cells with new functional insulin
producing cells. To this point, patients transplanted with human cadaver islets
have become insulin-independent for up to 5 years and show control of blood
glucose. The main impediment to this approach is that the supply of donor
islets is inadequate to meet the demand of diabetic patients, as each patient
can require the islets of 2-3 cadaveric donors. An unlimited supply of
functional human β-cells would allow for increased access to this therapy as
well as allow for further study of basic biology and growth characteristics of
these cells to improve and develop other treatment options.
20mM
Sample 2
2mM
20mM
Figure 4. Formation of 3D aggregates for differentiation and functional
characterization.
Cells were expanded in 2D culture with irradiated feeder cells, and then formed into
spherical aggregates by overnight incubation in a low attachment dish on an orbital
shaker at 370C 5% CO2 in CRM. A) Example of aggregated spheres with an
average distribution of 251um+/- 57 (n=250) with aggregates ranging in size from 90
to 400um. B) Results from a sequential glucose stimulated insulin secretion assay.
Aggregates were washed twice in PBS to remove media and incubated in prewarmed 2mM glucose buffer for 1.5 hours in the incubator. Aggregates were
recovered, washed briefly in 2mM glucose buffer and returned to 2mM glucose
buffer for 30 min. Aggregates were recovered and washed as before and then place
in 20mM glucose buffer for 30 min. The cycle was repeated once more, and the
aggregates were recovered, dissociated with trypsin and counted. The buffer from
each 30 min incubation was analyzed in triplicate for human insulin by ELISA
(Mercodia) and normalized to the cell count. Glucose stimulation index is typically
between 1.5 and 2.5 for our preparations.
Propagenix Conditional Reprogramming technology is able to significantly expand
b-cell populations from human islets that can be readily returned to functional
mature state. These cells express many important mature cell markers and
secrete insulin in a controlled manner in response to changing glucose
concentrations. Given these results CR-driven expansion of islet cell populations,
with attendant preservation of cell functionality, may offer a unique approach to
increasing the biomass of functional cell equivalents for allogeneic and/or
autologous transplantation.
For more information visit www.propagenix.com
References
ROCK Inhibitor And Feeder Cells Induce The Conditional Reprogramming Of
Epithelial Cells.
Ory V, Chapman S, Yuan H, Albanese C, Kallakury B, Timofeeva OA, Nealon C, Dakic A,
Simic V, Haddad BR, Rhim JS, Dritschilo A, Riegel A, McBride A, Schlegel R
(2012) Am J Pathol 180(2):599-607.
Radiation Induces Diffusible Feeder Cell Factor(S) That Cooperate With ROCK
Inhibitor To Conditionally Reprogram And Immortalize Epithelial Cells
Palechor-Ceron N, Suprynowicz FA, Upadhyay G, Dakic A, Minas T, Simic V, Johnson M,
Albanese C, Schlegel R, Liu X
(2013) Am J Pathol 183(6): 1862-1870.
A Manufacturing- and Regulatory-Friendly Cell Expansion Technology Allowing for
Trillion-Fold Expansion of Primary Airway Epithelial Cells
Anura Shrivastava, Chengkang (CK) Zhang
Propagenix Inc., Rockville, Maryland, United States
Trillion-fold expansion of primary airway epithelial
cells in EpiX™ medium
Genetic engineering in EpiX™-expanded airway
epithelial cells and single cell cloning
Mucociliary differentiation of EpiX™-expanded cells
upon withdrawal from proliferation
G r o w t h o f t r a n s g e n ic
E x p a n s io n o f p r im a r y t r a c h e o b r o n c h ia l e p it h e lia l c e lls in E p iX
R F P - e x p r e s s in g a ir w a y e p it h e lia l c e lls
m e d iu m
35
H B E C in E p iX
D H B E - C F in E p iX
10 18
10 16
Karyotype @ P4
10 14
30
25
20
15
10
5
0
10 12
0
10
T e s te d fo r K a r y o ty p e
10 10
20
30
40
50
Day1
60
Day6
Day10
D a y s in c u lt u r e
A stable RFP-expressing transgenic cell line is derived by lentivirus transduction and used for single cell
cloning in EpiX™ medium.
10 8
10 6
10
20
30
40
50
60
70
D a y s in c u lt u r e
Karyotype @ P16
Primary human bronchial epithelial cells from a normal donor (HBEC) and a diseased
donor (cystic fibrosis, DHBE-CF) can be expanded in EpiX™ medium to generate biomass
(>1020 cells) far beyond the conventional BEGM (Lonza). The cells retain normal
karyotypes after extended expansion.
M e a n F lu o r e s c e n c e In t e n s it y
K n o c k d o w n R F P e x p r e s s io n b y s iR N A
0
Transient transfection of two
independent siRNAs targeting the
coding region of RFP gene effectively
suppresses RFP protein expression in
the airway epithelial cells, using
Lipofectamine® with standard protocol
provided by ThermoFisher.
150000
50000
0
s iR N A # 1
E x p a n s io n o f a ir w a y e p it h e lia l c e lls in a C D & A C F
s iR N A # 2
Air-liquid-interface
Gene
Name
Description
HBEC in EpiX
ITGA6
Integrin, Alpha 6
0.015
0.22
0.21
ITGB4
Integrin, Beta 4
0.0019
0.034
0.026
KRT14
Keratin 14, Type I
0.00021
1.19
1.21
KRT5
Keratin 5, Type II
0.0028
1.47
1.03
TP63
Tumor Protein P63
0.00057
0.066
0.03
NGFR
Nerve Growth Factor Receptor
0.00011
0.0046
0.0022
LIN28A
Lin-28 Homolog A
0.00031
ND
ND
NANOG
Nanog Homeobox
0.00057
0.00044
ND
POU5F1
POU Class 5 Homeobox 1, OCT4
0.00066
0.00036
0.00039
SOX2
SRY (Sex Determining Region Y)-Box 2
0.00021
0.0026
0.00097
CFTR
Cystic Fibrosis Transmembrane
Conductance Regulator
0.00069
ND
ND
FOXJ1
Forkhead Box J1
0.0043
ND
ND
SCGB1A1
Secretoglobin, Family 1A, Member 1
0.18
ND
ND
SFTPB
Surfactant Protein B
0.082
ND
ND
SFTPD
Surfactant Protein D
0.038
ND
ND
AXIN2
Axin 2
0.0029
ND
ND
P2
p8
Cell Acquisition
Tissue engineering
Serum-free, Feeder-free
>1012-fold expansion
Applications
ND
ND
ND
Prominin 1, CD133
0.085
ND
ND
0.0017
600
ALI on
9605 Medical Center Drive, Suite 325
|
Rockville, MD 20850
W e ll # 1
Day 6
Gene expression levels are examined by qRT-PCR, and expressed as relative
levels to that of actin-B (whose level is set as 1). ND, not detected.
Human primary bronchial epithelial cells from a normal donor are expanded
in EpiX™ medium for more than 8 passages, and examined for the expression
of basal epithelial cell markers. The cells ubiquitously express the
transcription factor TP63. Other basal epithelial cell markers (Integrin α6,
Integrin β4, KRT5, KRT14) are also expressed at high levels. Pluripotent stem
cells or other tissue-specific stem cells markers are not detected.
10
13
10
11
10
9
10
7
10
5
TM
m e d iu m
HBEC
D H B E -C F
0 .8
0 .6
0 .4
0 .2
0 .0
10
20
30
40
50
1
2
3
4
5
6
7
8
9
10
Passages
Summary
“Dome-like” formations indicate epithelial cells differentiate to polarized
epithelium with trans-epithelial solute transport capability
W e ll # 2
2
ND
Leucine-Rich Repeat Containing G Protein- 0.00073
Coupled Receptor 5
PROM1
v e r s io n o f E p iX
1 .0
HBEC
A chemically defined, animal-component free version of EpiX™ medium supports
trillion-fold expansion of primary airway epithelial cells
cm )
CD34 Molecule
LGR5
E x p a n s io n o f a ir w a y e p it h e lia l c e lls in a C D & A C F
m e d iu m
D H B E -C F
D a y s in c u lt u r e
TEER (
TP63
CD34
TM
15
0
R e s is t a n c e a c r o s s T r a n s w e ll m e m b r a n e
Genetic engineering
10
C e ll d iv is io n p e r d a y
Human
Lung
EpiX™-expanded airway epithelial cells form an
intact barrier upon withdrawal from proliferation
C a lc u la t e d T o t a l C e ll N u m b e r
v e r s io n o f E p iX
Lung
Frozen cells
Upon withdrawal from proliferation, the EpiX™-expanded airway epithelial cells (the RFPexpressing cell line) readily undergo mucociliary differentiation in two in vitro formats –
bronchosphere (left) and air-liquid interface (middle and right, top-down view)
Performance of a chemically defined, animalcomponent free version of EpiX™ medium
100000
C o n tr o l
EpiX™-expanded airway epithelial cells express
basal cell markers
At-A-Glance
OR
MUC5AC
H B E C in B E G M ( L o n z a )
10 20
acetylated tubulin
10 22
P o p u la t io n d o u b lin g s
The availability of serum-free and feeder-free cell culture
media for human airway epithelial cells has existed for over 20
years. However, these culture systems generally support less
than a million-fold expansion (i.e., less than 20 population
doublings) of normal primary airway epithelial cells before
they succumb to cellular senescence. The limited expansion
potential of conventional media not only greatly restricts the
use of primary airway epithelial cells for research purposes,
but also thwarts the development of cell replacement
regenerative therapy for respiratory diseases. We have
developed a novel serum-free and feeder-free culture
medium (EpiX™ medium) that allows for >1012-fold expansion
of several types of airway epithelial cells without using viral or
oncogenic factors. Airway epithelial cells maintain normal
chromosome karyotype even after more than 40 population
doublings (i.e., trillion-fold expansion) in the EpiX™ medium.
In addition, EpiX™-expanded airway epithelial cells can
differentiate into intact mucociliary epithelia monolayers as
reflected by elevated trans-epithelial electric resistance (TEER)
in air-liquid-interface conditions. Second-generation versions
of EpiX™ medium lacking animal-derived components now
enable us to address attaining sufficient biomass needed for
cell replacement therapy for lung diseases.
C a lc u la t e d T o t a l C e ll N u m b e r
Abstract
400
200
0
ZO-1
0
5
10
15
20
25
30
35
40
D a y s in c u lt u r e
Barrier function are also examined by the expression of tight junction marker
ZO-1 and functional measurement (trans-epithelial electric resistance, TEER)
|
Phone: 240.713.3300
|
www.propagenix.com
We have developed a novel serum-free and feeder-free
culture method (PCT patent pending) that allows for >1012
folds expansion of primary airway epithelial cells in a short
timeframe without using any genetic manipulations. The cells
quickly revert to tissue-specific differentiation states upon
withdrawal from the expansion phase. This method enables
us to address attaining enough biomass for regenerative cell
replacement therapy in the respiratory area. The long runway
and single cell cloning capability supported by the EpiX™
medium opens the door to develop phenotype-relevant
airway models using diverse genetic engineering tools.
A Feeder-Independent And Serum-Free Cell Culture Technology That Allows
For Over Trillion-Fold Expansion Of Various Primary Epithelial Cells
Chengkang (CK) Zhang, Anura Shrivastava
Propagenix Inc., Rockville, Maryland, United States
We have developed a novel serum-free and feeder-free
culture medium (EpiX™ medium) that allows for greater
than 1012-fold expansion of several types of primary
epithelial cells without using viral or oncogenic factors.
Primary epithelial cells maintain normal chromosome
karyotype even after more than 40 population doublings
(i.e., over trillion-fold expansion) in the EpiX™ medium.
The epithelial cells retain basal cells characteristics and low
levels of genes that are associated with stress response and
senescence. Upon exiting the proliferation conditions, the
epithelial cells expanded in EpiX™ medium readily
differentiate into epithelial structures resembling original
tissues.
Furthermore, second-generation versions of EpiX™
medium lacking animal-derived components now provide
us unique manufacturing- and regulatory-friendly solutions,
and enable us to address attaining clinically relevant
biomass needed for cell replacement therapy for various
diseases affecting epithelial tissues.
Epithelial cells remain normal after
extended expansion in the EpiX™ medium
EpiX™-expanded epithelial cells differentiate
into structures resembling original tissues
Expansion of Keratinocytes
TP63
10 25
>1024 fold
HFKn in EpiX
HFKn in KSFM
HEKa in EpiX
HEKa in KSFM
10 20
10 15
>1016 fold
10 10
10 5
10 0
0
10
20
30
40
50
60
70
80
Days in culture
ZO-1
Donor Age
Early Passage
Late Passage
HFKn
2 days
P3 (13.5 PD),
46,XY
P19 (62.0 PD),
46,XY
HBEC
49 yrs
P4 (11.1 PD),
46,XX
P16 (45.1 PD),
46,XX
PrEC
26 yrs
P3 (13.5 PD),
46,XY
P13 (41.1 PD),
46,XY
Tumorigenicity in nude mice
4200
2200
200
Differentiated HFKn (<20 PDs)
Proliferating HFKn (>50 PDs)
Differentiated HFKn (>50 PDs)
Negative control
Positive control
60
40
20
Epithelial tissues
Air-liquid-interface
Genetic engineering
OR
0
7
14
21
28
35
42
49
56
63
70
77
84
Days
HFKn, p19
Primary epithelial cells expanded in EpiX™ medium for more than
40 PDs still retain normal karyotypes. HFKn, neonatal foreskin
keratinocytes; HBEC, human bronchial epithelial cells; PrEC,
prostate epithelial cells. The cells also show homogenous strong
expression of TP63. To evaluate the tumorigenicity of keratinocytes
expanded in EpiX™ medium, 10 x 106 keratinocytes are injected s.c.
into nude mice and monitored for 3 months. No tumor outgrowth
is observed in the samples.
Frozen cells
Tissue engineering
Serum-free, Feeder-free
Applications
>1012-fold expansion
Over trillion-fold expansion of various
primary epithelial cells in the EpiX™ medium
Population
doublings
HFKn/KSFM (p1)
KSFM (p2)
HBEC/BEGM (p1)
EpiX (p2 early)
EpiX (p2, early)
EpiX (p13, mid)
KSFM (p6)
Gene
Name
ITGA6
ITGB4
KRT14
KRT5
TP63
NGFR
EpiX (p23, late)
Description
Integrin, Alpha 6
Integrin, Beta 4
Keratin 14, Type I
Keratin 5, Type II
Tumor Protein P63
Nerve Growth Factor Receptor
LIN28A
NANOG
POU5F1
SOX2
Lin-28 Homolog A
Nanog Homeobox
POU Class 5 Homeobox 1
SRY(Sex Determining Region Y)-Box 2
Gene
Name
AKT1
ATM
CDKN2A
GADD45A
Description
Protein Kinase B
ATM Serine/Threonine Kinase
p16, INK4A
Growth arrest and DNA-damageinducible, alpha
GLB1
Galactosidase, beta 1
PLAU
Plasminogen activator, urokinase
SERPINE1 Plasminogen activator inhibitor 1
1000
Stratum Basale
0
0
5
10
15
Days in culture
EpiX™-expanded keratinocytes (RFP-expressing cell line) form tight
barrier upon differentiation, and readily differentiate into stratified
structure on air-liquid-interface
HBEC in EpiX
P2
P8
0.22
0.21
0.034
0.026
1.19
1.21
1.47
1.03
0.066
0.03
0.0046 0.0022
ND
0.0004
0.0003
0.002
Bronchosphere
acetylated tubulin
ND
ND
0.0003
0.001
P2
0.17
0.031
2.53
1.52
0.092
0.0018
0.0001
ND
0.0004
ND
HFKn in EpiX
P13
P23
0.21
0.39
0.027
0.018
2.19
2.63
1.53
1.66
0.047
0.033
0.0009
0.00009
ND
ND
0.0005
ND
0.0001
0.0001
0.001
ND
HFK in KSFM
P2
P6
0.009
0.22
0.007
0.041
0.002
0.04
0.01
0.07
P2
0.003
0.002
0.001
0.002
0.004
0.02
0.04
0.005
0.001
0.002
HFKn in EpiX
P13
P23
0.003
0.003
0.004
0.002
0.006
0.004
0.003
0.008
Performance of a chemically defined, animalcomponent free version of EpiX™ medium
Expansion of airway epithelial cells in a CD&ACF
version of EpiXTM medium
10 15
DHBE-CF
HBEC
10 13
10 11
10 9
10 7
10 5
0
10
20
30
40
Source
Medium
Fold expansion
achieved (in days)
Neonatal
Gibco
KSFM
122,295
(34 days)
16.9
HFKn
Neonatal
Gibco
EpiX
> 3 x 1021
(95 days)
71.4
HFKn
Neonatal
In-house
isolation
EpiX
> 1 x 1026
(70 days)
87.1
HEKa
Adult
Gibco
KSFM
2,896
(35 days)
11.5
HEKa
Adult
Gibco
EpiX
> 6 x 1016
(79 days)
55.9
HMEC
Adult
Lonza
MEGM
244,589
(39 days)
17.9
HMEC
Adult
Lonza
EpiX
> 1 x 1013
(63 days)
43.8
PrEC
Adult
Lonza
PrGM
4,390
(42 days)
12.1
PrEC
Adult
Lonza
EpiX
> 7 x 1012
(57 days)
42.8
HBEC
Adult
Lonza
BEGM
2,097,152
(37 days)
21.1
50
HBEC
Adult
Lonza
EpiX
> 2 x 1014
(65 days)
47.7
40
DHBE-CF Adult
Lonza
(cystic fibrosis)
EpiX
> 5 x 1015
(69 days)
52.2
30
Day 1
Expansion of neonatal keratinocytes in a CD&ACF
version of EpiXTM medium
10 18
10 16
10 14
0.02
0.07
0.13
0.004
0.002
0.002
0.005
0.001
0.003
Day 6
Day 10
Stable RFP-expressing transgenic cell lines are derived by lentivirus transduction
and used for single cell cloning in EpiX™ medium.
Growth of transgenic
RFP-expressing epithelial cells
HBEC/nRFP
PrEC/nRFP
HFKn/nRFP
20
10
0
0
10
20
30
40
50
60
70
50
Days in culture
A chemically defined, animal-component free version of EpiX™
medium supports over trillion-fold expansion of primary airway
epithelial cells (top) and neonatal foreskin keratinocytes (bottom)
10 12
10 10
10 8
10 6
10 4
10 2
10 0
0
10
20
30
40
50
60
80
Days in culture
Transient transfections of several siRNAs targeting the coding region
of RFP gene effectively suppress RFP protein expression in
transgenic epithelial cells, using Lipofectamine® with standard
protocol provided by ThermoFisher.
70
Days in culture
Genetic engineering in EpiX™-expanded
epithelial cells and single cell cloning
Donor
Age
HFKn
MUC5AC
EpiX™-expanded bronchial epithelial cells (RFP-expressing cell line)
readily undergo mucociliary differentiation in two in vitro formats –
bronchosphere (left) and air-liquid-interface (top-down view)
EpiX (p8, late)
Gene expression levels are expressed as relative to actin-B (whose level is set as
1). ND, not detected.
Cell
Type
Primary epithelial cells derived from various tissues are expanded in
conventional culture media or in EpiX™ medium. The epithelial cells
acquired from commercial suppliers generally reach <20 population
doublings in the recommended media. The same cells could be
expanded for more than 40 population doublings in the EpiX™
medium. Notably, keratinocytes isolated in EpiX™ medium reach
more PDs in a significant shorter timeframe.
Stratum granulosum
Stratum spinosum
2000
Gene expression signatures of epithelial cells
expanded in EpiX™ medium
Low levels of genes associated with stress response and senescence
Cell Acquisition
Stratum Corneum
3000
0
EpiX™-expanded epithelial cells maintain basal cell characteristics
At-A-Glance
“Dome”-like structure
Barrier function of HFKn
4000
cm2)
Cells
TEER (
Abstract
Feeder-independent and serum-free cell culture media
for primary epithelial cells have been available for over 20
years. However, these culture systems generally support
less than a million-fold expansion (i.e., less than 20
population doublings) of primary epithelial cells before
they succumb to stress-associated cellular senescence
during in vitro expansion. The limited expansion potential
of conventional media not only greatly restricts the use of
primary epithelial cells for research purposes, but also
thwarts the development of cell replacement regenerative
therapy utilizing the potentials of primary epithelial cells.
Summary
We have developed a novel feeder-independent and
serum-free culture method (patent pending) that allows for
>1012-fold expansion of various primary epithelial cells in a
short timeframe without using any genetic manipulations.
The cells quickly revert to tissue-specific differentiation
states upon withdrawal from the expansion phase, and
form epithelial structures that bear resemblance to the
original tissues. This method enables us to generate
clinically relevant biomass for regenerative cell replacement
therapies targeting numerous diseases affecting epithelial
tissues. The long runway and single cell cloning capabilities
supported by the EpiX™ medium also open the door to
develop phenotype-relevant in vitro epithelial cell biology
models and precision diagnostics using diverse genetic
engineering tools.
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