104--Lecture

Transplantation
1. Rejection and GvHD
2. Stem cell transplantation

Saints Cosmas and Damian performing a miraculous
transplantation
 Transplantation
The act of transferring
cells, tissues, or organs
from one site to another.

used to replace tissue or

organs that have failed on
functional operation
 The history of transplantation

Cadaveric transplants: cornea transplantation,1906

Alexis Carrel (1908): first transplantation study,
Cat and kidney transplantation: maintained urine output
up to 25 days
Russian surgeon (1935): first human kidney
transpantation,
failed due to blood type not match induced hyperacute
rejection (antibody mediated)
The first successes organ transplantation
Dr. Joseph E. Murray (1954): at Peter Bent Brigham
Hospital in Boston, USA
Live donor: Kidney transplantation between identical twins
(Herrick brothers: Ronald and Richard )
Dr. Joseph Murray was awarded the 1990 Nobel Prize in
Medicine
 Types of transplants
Autograft
Self tissue in the same individual

Burn wound

Isograft
Between genetically identical

individuals
Inbred strain of mice, monozygotic

genetically indentical twins

Allograft
Between genetically different

members of the same species

Two inbred strain

Human A and human B

transplantation
Xenograft
Between different species---pig to

human
Xenotransplantation
Donor:
nonhuman primates
(chimpanzees, baboons)
Pigs
The first xenotransplant:
1964, chimpanzee kidney
transplant
Liver, heart, BM
Immune rejection is quite
vigorous
Xenozoonoses is fatal for
human: chimpanzees HIV-I,
II virus, herpesvirus B
 Current Problems
Lack / short transplanted organs
In USA, 116,000 patients are waited for an organ
transplantation (2012, December)
Over 75% of organ transplantation are required a kidney
Cadaveric transplant (80%) and live transplantation
(20%)
Living donation: kidney, BM, performed between relatives
The development of artifical mechanical organs
Cloning and tissue engineering
Xenotransplantation: pig
Immune rejection problems
Chronic rejection
Immune suppression drugs
Toxicity
incidence of cancer or infection
Process:
3-7 days: re-
vascularized transplant,
leukocytes infiltrated
7-10 days: healing
and more cellular
infiltration
10-14 days:
thrombosis and necrosis
 Rejection is an immunological phenomenon
Exhibits the Specificity and Memory

Alloantigen specific T cells can be

transferred to a second recipient , then
induces a second-set allograft rejection

Adoptive transfer experiment

(Mitchison, 1950s)
 Mechanisms involved in graft rejection

Alloantigen specific T cells can be transferred

to allograft rejection
Lymphoid cells are played a major role.
Major alloantigen is MHC
Minor antigens:
H-Y system
Non-classical MHC molecules
ABO, Rh(rhesus) antigen ------ etc.
 Immune response involved in graft
rejection
Regulated by cell-mediated response
Delayed type hypersensitive: CD4+ T cells
Cell-mediated cytotoxicity: CD8 T cells
Major cells are T cells: recognized and
reacted against graft 1/1001/1000: T to
APC
CD4+
CD8+
Both CD4+ and CD8+ T cells are important
Immune response is initiated by T cell recognition

Infected cell
Antigen-
Microbe presenting
cell

1 A fragment of
foreign protein
Antigen (antigen) inside the Antigen
fragment cell associates with fragment
an MHC molecule
1
and is transported 1
to the cell surface.
Class I MHC Class II MHC
molecule 2 molecule
2 T cell
2 The combination of
T cell receptor
MHC molecule and
receptor antigen is recognized
by a T cell, alerting it
to the infection.

Helper T cell
(a) Cytotoxic T cell (b)
 MHC gene

MHC gene is encoded by a region of multiple

loci
Each locus has many alleles : Is polygenic
All genes are located on same chromosome
Human: chromosome 6HLA complex
HLA-I: 626 alleles for HLA-B; 470 alleles for
DRB; 349 alleles for HLA-A
Mouse: chromosome 17---H-2 complex
 Recognition of non-self MHC molecule as a
foreign antigen is a major determinant of graft
rejection

Alloantigen: antigens that differ between

members of the same species.
Major transplantation antigen: MHC
Minor transplantation antigen
Transplanted antigen is under genetic control.
Alloreactive response: immune response against
such alloantigens
In our body, the frequency of T cells specific for
any non-self MHC molecule is relatively high.
 The recognition of alloreactive T cells

Most recognize the MHC antigens by the directly

pathways: recipient T cells recognize allogeneic MHC
antigen on donor APC,
is more important in acute rejection

The indirect responses: recipient T cells recognize

recipient MHC molecules + donor MHC peptides
presented by recipient APC
Is important in chronic rejection
Chronic rejection of organ transplants is caused by a type III hypersensitivity
reaction and is result from an indirect allorecognition pathway
 The process of graft rejection
Sensitization stage
Host CD4+ and CD8+ T cells recognize the
alloantigens of graft
Host dendritic cells or APC
Donor APC---- passenger leukocytes (DCs are major cells), it
activates Tc cells
Host T cells activation and proliferation

Effector stage
CTL-mediated response
TDTH response
Antibody-mediated response
The initiation of graft rejection normally involves the
migration of donor antigen-presenting cells from the graft to
local lymph nodes of patient.
 The Effector stage
Delay-type hypersensitivity---- CD4+TDTH
CTL-mediated cytotoxicity----Tc
Antibody and complement mediated lysis
ADCC-----NK cells
Cytokines release: IL-2, IFN-, TNF-
IL-2: increase TH and TC action
IFN-: enhances DTH, macrophages, MHC II
expression
TNF and TNF-: enhances MHC I expression
Antibody also involved in
rejection: allogeneic cells can
be destroyed by antibody-
mediated cytotoxic reaction
(type II hypersensitivity)
 Rejection and GvHD
Graft rejection--- host defense donor cells
Hyper-acute rejection: within minutes to hours (in 24 hr)
A Type II hypersensitivity reaction
Caused by preexisting host serum antibody
blood transfusion
Repeated pregnancies
Repeated graft
Acute rejection------ in 10 days (days to weeks)
A type IV hypersensitivity
Trigger by donor DC (passenger leukocytes)
Mediated by alloreactive T cells (effector T cells)
Chronic rejection---------month or year
Caused by IgG antibodies to allogeneic HLA class I of the graft
A type III hypersensitivity
Graft verses Host disease ( ; GvHD)
A type IV hypersensitivity reaction
Occurs in hematopoietic stem cell transplantation or liver
transplantation
Grafts immune cells attack host tissues
 Transplant rejection and graft-versus-host disease

Transplant acute rejection and graft-versus-host disease are type IV

hypersensitivity reactions
 (Graft verses Host disease)
(Grafts immune cells attack host
tissues)
THE PREVENTION OF GRAFT
REJECTION
 Matching tissue type

Is major way to prevent rejection

Search donors---- data base
On both donor cells and recipient cells
Blood group compatibility
HLA typing:
Criteria: 3 HLA-I (HLA-A, B, C) and 3 HLA-II (HLA-
DR, DP, DQ)
Microcytotoxicity (CD8 T cell) MHC I match
Mixed lymphocyte reaction (MLR) (CD4 Tcell)
MHC II match
Minor histocompatibility loci
Microcytotoxicity Assay
Mixed Lymphocyte Reaction
The mixed lymphocyte reaction (MLR)
 Genes mismatched on
MHC class II locus is
more important than
gene mismatched on
MHC class I region
 General immunosuppressive therapy

Immune suppressive drugs: used in early post-transplant

period
Side effects:
Non-specific toxicity of drugs

To stay indefinitely on medication

Increased incidence of cancer and infection

Total lymphoid irradiation (used in before transplanted)

Total dosage: 3400 rad
 Corticosterone
the derivatives of the glucocorticoid family of steroid
hormones
1% of genes may be regulated by glucocorticoids
eg. Prednisone, prednisolone, methlprednisolone
Side effects---immune suppression
Lympholysis
Change the lymphocyte circualtion pattern
Decrease phagocytosis, Macrophage activity, NO
Decrease chemotaxis, IL-1,3,4,5,8, TNF, and MHC-II expression
Increase NF-kB inhibitior---I-kB
Inhibit NO synthase
Induction of endonucleases leading to apoptosis in
lymphocytes and eosinophils.
Immature thymocytes sensitive, but mature T cells are resistant to it
 Immune suppressive drugs
Antiproliferative agents (Mitotic inhibitors)
Azathioprine (imuran): a purine antagonist
to compete with inosine monophasphate, so block the synthesis of
inosinic acid (inhibits S phase of cell cycle)
Inosinic acid is the precursor of purine (A or G)
6-mercaptopurine (6-MP), methotrezate, cyclophosphamide
mycophenolic acid (MPA): inhibits inosine monophosphate
dehydrogenase
Specific T-cell activation inhibitors:
Cyclosporin A: inhibit NF-AT then affect IL-2 cytokine production
FK506 (tacrolimus): bind to FK-binding protein 12(FKBP12) and
inhibit NF-AT , less nephrotoxic than ciclosporin, but diabetogenic in
some patients.
Rapamycin (sirolimus): interferes with IL-2R signaling, inhibit mTOR
(inhibit cell proliferation)
Anti-inflammatory drugs:
Corticosterone---- block NF-B action
Rapamycin (sirolimus)

Action: specific inhibits the response to IL-

2/IL-2R signal
Benefits:
It appears to be a tolerance-permissive drugs,
calcineurin inhibitors may inhibit the
tolerance induction
Reduce the incidence of some malignancies
 Specific immunosuppressive
therapy(Target therapy)
Monoclonal antibodies to T cell components or
cytokines
Anti-TCR
Anti-CD4
Anti-IL-2 receptor
Anti-ICAM-1, LFA
Block the co-stimulatory signal to make
alloreactive cells became anergic
CD80, CD86
IDO
Transferring tolerant T cells to a nave recipients
Regulatory T cells are used to prevent alloreactive
T cells: CD4+CD25+Foxp3+
The fetus is an allograft that is
tolerated repeated

Marcus O et al., Cancer Control (2004)

Clinical transplantation
USA Patients needing a transplant outnumber the available organs
Patients needing a transplant outnumber the available organs
Chapter_16.indd 21 3/17/11 5:29 PM
 Pancreatic cell transplantation
Treated for diabetes mellitus
1-year success rates is 55%
Transplanted complete pancreas is not
necessary to restore function
Donor islet cells are implanted into the site
of capsule of kidney or liver sinusoid
Usually combined with kidney
transplantation
Stem cell transplantation
 The earlist definition of the term
stem cell
-Bizzozero (1894)E.B. Wilson (1896),
the cell biologist-

A stem cell is a cell from the embryo,

fetus, or adult that has, under certain
conditions, the ability to reproduce
itself for long periods or, in the case
of adult stem cells, throughout the
life of the organism.

It also can give rise to specialized

cells that make up the tissues and
organs of the body.
 SELF-RENEWAL

differentiation

A single cells that have the capacities:

(1) to self-renew
(2) to differentiate to more mature cells.
 Symmetric cell division

Stem cell Stem cell

SELF-RENEWAL DIFFERENTIATION
(copying) (specializing)

Identical stem cells Specialized cells

 Asymmetric cell division
1 stem cell

Asymmetrical cell division!

1 stem cell 4 specialized cells

Self renewal - maintains Differentiation - replaces dead or damaged
the stem cell pool cells throughout your life
 Sources of Stem Cells

Types: embryonic stem cells, perinatal stem

cells, and adult stem cells
 Potency of stem cells

Totipotent:()
the fertilized egg (zygote) and
probably its immediate progeny
Pluripotent: ()
the embryonic stem cell (ES) is
capable to differentiate into most
cell type in the body
Multipotent :()
Adult (somatic) stem cells have more
limited differentiation ability
Unipotent:
Adult tissue progenitor is able to
differentiate into one specific lineage
 Types of stem cell:
1) Embryonic stem cells


1964
1981
1995
1998
2000
2003
2003
2004
63
Early study of ES cells-- embryonic carcinoma (EC)
cells
1950-1964, researchers had isolated a single type of
cell from a teratocarcinoma (Andrews P, et al., (2005). Biochem Soc
Trans 33 (Pt 6): 152630.)

a tumor now known to be derived from a germ cell.

These cells similarities in morphology and
differentiating potential (pluripotency)
led to the use of EC cells as the in vitro model for early
mouse development
EC cells harbor genetic mutations and often
abnormal karyotypes that accumulated during the
development of the teratocarcinoma.
Martin GR (1980). "Teratocarcinomas and mammalian
embryogenesis". Science 209 (4458): 76876.
 mouse Embryonal Carcinoma cells

In culture, these clones

differentiated into various cell
types.

Retinoic acid treatment Permitted the subcloning of a

pluripotent teratocarcinoma cell
lines could proliferate indefinitely,
produce teratomas, form cystic
embryoid bodies.
chondrocytes endoderm

skeletal muscle myocardium

neural tissue

In 1981, two groups of mouse
embryonic stem cells (ES cells) study:
Martin Evans and Matthew Kaufman
from the Department of Genetics,
University of Cambridge published first
in July, revealing a new technique for
culturing the mouse embryos in the
uterus to allow for an increase in cell
number, allowing for the derivation of ES
cells from these embryos.
Evans M, Kaufman M (1981). Nature 292 (5819): 1546.

Gail R. Martin, from the Department of

Anatomy, University of California, San
Francisco, published her paper in
December, 1981 and coined the term
Embryonic Stem Cell.[9] She showed
that embryos could be cultured in vitro
and that ES cells could be derived from
these embryos.
Martin G (1981) Proc Natl Acad Sci USA 78 (12): 76348

Martin Evans
Stages of early development: Where
ES cells come from
2 Cell 4 Cell

Blastocyst
16 Cell
 Stem Cell-- Break through
Since 1998:
Thomson et al.(University
of Wisconsin, Madison)
cleavage stage human
blastocysts from IVF
Science 1998, 282: 1145

Gearhart et al. (Johns

Hopkins University)
primordial germ stem
cells from terminal
pregnancy 5-9 weeks post
fertilization human
embryo
PNAS 1998; 95:13726

Science, 2000
(blastocyst)
Trophoectoderm
(placenta and amnion)

trophoblast

inner cell mass
Inner cell mass
(all tissues in the body)


Human ES cells isolation

ESC
 human Embryonic Stem cells
blastocyst H9 colony on MEFs
Cell morphology

alkaline phosphatase SSEA-1 SSEA-4

Cell surface markers

TRA-160 TRA-181
ES cells
()
(Sir John B. Gurdon):2060
20%

Somatic nuclear transfer

77
 The 2012 Nobel Prize in
Physiology or Medicine

"for the discovery that mature cells can be

reprogrammed to become pluripotent"
 # 2: Somatic cell nuclear transfer (SCNT)
 !!!

Ian Wilmut, Roslin Institute

66%Keith
Campbell

Dolly in
(05.07.1996 14.02.2003)
Large Offspring Syndrome

Normal fetus from Overgrowth of fetus and

the same ES cell line placenta from nuclear transfer

K. Eggan et al., Proc. Natl. Acad. Sci. U.S.A. 98, 6209 (2001).
 Somatic cell nuclear transfer
()

 Challenges for embryonic stem cells

At present human embryonic
stem cells can only be
obtained by destroying live
human embryos at the
blastocyst stage
They proliferate rapidly and
are extremely versatile, but
there is scant scientific
evidence that embryonic stem
cells will form normal tissues
in a culture dish
Embryonic stem cells are
difficult to develop into a
stable cell line. It
spontaneously accumulate
genetic abnormalities in
embryonic-stem cell cultures
when Embryonic stem cells are Martin Pera
prone to uncontrollable growth
and tumor formation placed in Mature cystic teratoma of
animals the ovary Testicular teratoma
 ES cells

Embryonic Stem Cells-good source of

pluripotent cells, but unethical
Somatic cell nuclear transfer-still
requires oocytes
 Reprogramming
Oct4, Sox2, Klf4, and c-Myc
(OSKM)
Cell 126, 663676 (2006)
 10 factors whose withdrawal resulted in loss
pluripotency these 10 alone = more colonies
Identified a group of 4 genes which in combination
produced pluripotent ES like cells

Oct3/4
Sox2
c-Myc
Klf4
Takahashi and Yamanaka, Cell, Aug 25, 2006
 Sox2- Self Renewal
Oct4- Differentiation switch
Klf4- p53 pathway, Oncogene
c-Myc- Global Histone Acetylation, Oncogene

Four Magic Genes

 Fibroblasts ES cells
Are fully differentiated cells
Can not become any other cell type
Can only divide to make more fibroblasts
Contact inhibition

Fibroblasts
ES-like cells
Are they really stem cells?
Teratoma formation
Dr. Shinya Yamanaka, PhD

Dr. Kazutoshi Takahashi, PhD

Human iPSCs
NATURE| Vol 458|9 April 2009

To avoid insertion mutagenesis!

Nature, February 2010
Cell, August 2010
Cell Stem Cell, October 2011
 STAPStimulus-
Triggered Acquisition of
Pluripotency cells
Nature. 2014 Jan 30;505(7485):641-7.
Stimulus-triggered fate conversion of somatic cells into pluripotency.
Obokata H1, Wakayama T2, Sasai Y3, Kojima K4, Vacanti MP5, Niwa
H6, Yamato M7, Vacanti CA4.

20141()
Nature

CD45+
LIF(leukaemia inhibitory factor
STAPStimulus-
Triggered Acquisition of Pluripotency cells


STAP
STAP
STAP



STAPES cell

TS cellSTAP


Types of stem cell:
2) Tissue stem cells
Somatic (adult tissue-derived) stem cells
Definition:
Stem cells can be derived from various tissues in adults.

Types:
in bone marrow, blood, skin, muscle, brain, the cornea and retina
of the eye, the lining of the gastrointestinal tract, liver and
pancreas.

The primary role :

to maintain and to repair the tissue
plasticity: to have the potential to make limited range multi-lineage
differentiation from one tissue may give rise to cell types of another tissue.
This is a phenomenon called stem cell

reside in a special microenvironment (niche)

NIH Stem Cells: Scientific Progress and Future Research Directions

 Adult Stem Cells
:
Cell/tissue specialization
Create tissue banks with most common tissue-type
profiles
Minimize /prevent tissue incompatibility
(Limitation):
Stem cells from all cell or tissue types have not been
found in adult human
Low Quantities
Same capacity to proliferate as younger cells do??
?
The multipotent stem cells in tissues
 (adult stem cells)

Also named as somatic stem cells or Tissue-

derived stem cells: skin, hair, etc.
Hematopoietic stem cells (HSC)
Mesenchymal stem cells (MSC)
Multi-potent adult progenitor/stem cells
(MAPC/MASC)
Mesodermal progenitor cells (MPC)
-Neural stem cells: neurons, glial cells
-Epithelial stem cells: skin, linings
 The discovery of HSC
In 1924, Russian-born morphologist
Alexander A. Maximow used
extensive histological findings to
identify a singular type of precursor
cell within mesenchyme that
develops into different types of
blood cells. Alexander Maximow (18741928),

1960, Candidian Scientists Ernest A.

McCulloch and James E. Till first
revealed the clonal nature of
marrow cells in the 1960s.
Heterogeneity of Human HSCs
 Hematopoietic stem cells (HSC)
HSC are produced from hematopoiesis
HSC can differentiate to various blood cells that
include immune cells
0.05% to 0.5% of cells in the bone marrow
Majority remains quiescent
3 basic criteria
Multipotent differentation
High proliferation potential: 5x 107HSC 1x 1013 blood
cells
Self-renewal
Cell Surface Markers of HSCs
CD34+ None of the
markers are tied
CD133+
to unique stem
Thy1+ cell functions or
CD38- truly define the
stem cell
CD117-

Lin-

HSC is multipotent or pluriopotent cells
Turnover time: 30 days (mouse)
Life time in mature cells: 1-120 days
HSC may be differenced into
nonhematopoietic tissues
transdifferentiation concept
Liver
Pancreas
Heart
Brain
kidney
 History

Hematopoietic stem cell transplantation in the mouse

the radiation protection phenomenon (mid-1950s)

Hematopoietic stem cell tranplantation in the dog
Hematopoietic stem cell tranlsplantation in human
patients
19591963 : first allogeneic HSCT in humans
beginning of the Modern Era of HSCT: the end of 1960
 Dr. E. Donnall Thomas

first succsessful HSCT in treatment of acute

leukemias

Thomas ED, Lochte HL, Lu WC, Ferrebee JW. Intravenous infusion of bone marrow in
patients receiving radiation and chemotherapy. N. Engl. J. Med. 1957; 257: 491.
1990

 ANNUAL NUMBERS OF
BLOOD AND MARROW TRANSPLANTS
WORLDWIDE
1970-2002
45,000
NUMBER OF TRANSPLANTS

40,000
35,000
30,000 Autologous
25,000
20,000
15,000
10,000
5,000 Allogeneic
0
1970 1975 1980 1985 1990 1995 2000
YEAR
Bone marrow transplantation unit
Hematopoietic stem cell infusion
 donor
(Syngeneic BMT)

(HLA )

HLA (Matched Unrelated

BMT)

HLA

(peripheral mobile blood cells)

G-CSF

The action of G-CSF

Hematopoietic Stem cell therapy is
one type of cellular therapy

,
50-100 cc

, HLA

:,

The sources of Hematopoietic stem cells

Bone marrow
Mobilized peripheral blood
Umbilical cord blood
Fetal liver and fetal spleen (in mouse)
Embryonic stem cells (Vodyanik et al., Blood, 2004)
Fetal bone marrow (in human)
 The discovery of MSC
An ex vivo assay for examining the
clonogenic potential of multipotent marrow
cells was later reported in the 1970s by
Alexander Friedenstein (19241998),and
colleagues. In this assay system, stromal
cells were referred to as colony-forming
unit-fibroblasts (CFU-f).
 Human Mesenchymal Stromal Cells
(MSC)

A postnatal (adult) stem cell

Phenotype:
adherent, fibroblast-like,
non-hematopoietic markers:
positive for CD105, CD29, CD73, CD44, CD166, SH-2, SH-3, SH-4
Negative for CD3, CD7, CD14
Sources: bone marrow, cord blood, blood, adipose tissue,
lung, fetal liver
Capacity: differentiate in vitro and in vivo into mesenchymal
tissues (bone, cartilage, tendon, muscle, adipose tissue, bone
marrow stroma)
 Mesenchymal Stem Cells
Mesenchymal Stem Cells (MSCs), cells that reside within the
bone marrow, are multi-potent.
MSCs have the ability to differentiate and generate cartilage,
muscle, tendon, ligaments, fat, and bone.
Osteogenesis:
Osteogenic induction: Beta-glycerophosphate, ascorbic
acid and dexamethasone

Phase (2 wks)

Alkaline Phosphatase (2 wks) Von Kossa (2 wks)

 Chondrogenesis
Pellet culture with TGF-beta 1 induction

Safranin-O staining (21 days)

Type 2 Collagen immunohistochemical staining

HRP stain: positive staining appears brown (3 wks)
 Adipogenesis:
Adipogenic induction: IBMX and hydrocortisone

Phase (3 wks) Oil Red O (3 wks)

 Are there other sources for
mesenchymal stem cells besides bone
marrow?
Trabecular bone (Sottile 2002)
Synovial membrane (De Bari 2001)

Adipose tissue (Zuk 2001)

Umbilical cord blood ?

Placenta

amniotic fluid or amniotic membrane

Adipose stem cells
 Stem cells can acquire from
placenta

From 38-40 wks health placenta

Stem Cell, 2005; 23:3-9

Human Stem cells can be acquired from amniotic
fluid or amniotic membrane

Human Reproduction Vol.19, No.6 p 1450-1456, 2004

Isolation of Multi-Potent Mesenchymal
Stem Cells from Umbilical Cord Blood
Blood 103 (5): 1669-1675, 2004

MSCs from UCB can give rise to

progenies originated from all three
germ layers!
Plasticity: Neuroglial Differentiation of UCB-MSCs

10ng/ml NGF + 1mM IBMX +

Day 8 200uM butylated
hydroxyanisole + 50mg/ml
ITS+ premix

Astrocyte Oligodendrocyte Neuron

CD4+ and CD8+ T-cell proliferation is
inhibited by mesenchymal stem cells

Scand J Immun, 2004, v 60, pp307315

Blood. 2007;110:3499-3506
The clinical experience of MSC in
HSC transplantation

Chou et al., Cell Transplantation, 2013

Allogenic MSCs as a stem cell drug!

1.Roitt's Essential Immunology, Peter Delves,
Seamus Martin, Dennis Burton, Ivan Roitt
(12th Edition), 2011
2. Janeways Immunobiology, Murphy Kenneth;
Garland Science Publish (8th Edition), 2012.
3. Cellular and Molecular Immunology, Abbas,
Lichtman, Pillai, Elsevier (7th Edition), 2012
4. Kuby Immunology, Owen, Punt, & Stranford;
Freeman Publish (7th Edition), 2013