FULLY FUNCTIONAL FULLY FUNCTIONAL IMMUNE ORGAN IMMUNE ORGAN

GROWN IN MICE FROM GROWN IN MICE FROM LAB-CREATED CELLSLAB-CREATED CELLS

Maleeha Akram08-arid-1772Ph.D. Scholar

Department of Zoology

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ContentsContentsThymus

◦ Structure and Function◦ Types◦ Dysfunctions and Treatment

Thymic involutionGeneration of TECsFunctional attributes of iTECsiTECs can form functional thymusConclusionReference

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ThymusThymusThe thymus is

◦ a soft◦ roughly triangular in

shape◦ specialized organ of

the immune systemIt is located

◦ in the mediastinum of the thoracic cavity between the lungs

◦ anterior and superior to the heart

◦ posterior to sternum3

Structure of thymusStructure of thymus The thymus is of a pinkish-gray

color and lobulated on its surfaces.

It has two distinct but identical lobes– each surrounded by a tough, fibrous

capsule. Within each lobe is a superficial

region of tissue called the cortex and a histologically distinct deep region called the medulla.

Epithelial tissues and lymphatic tissues containing dendritic cells and macrophages make up the majority of both regions of the thymus.

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CortexCortex The cortical portion is

mainly composed of thymocytes (developing T-lymphocytes)

Supported by a network of finely-branched epithelial reticular cells, which is continuous with a similar network in the medullary portion.

This network forms an adventitia to the blood vessels.

Thymocytes

ReticularReticularcellscells

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MedullaMedulla The medulla is

continuous between adjacent lobules

The medulla is paler staining, less densely cellular than the cortex and contains – More mature T-cells– Prominent epithelial

cells– Hassalls corpuscles– Admixed macrophages– Dendritic cells– B lymphocytes6

Thymus functionThymus functionThe thymus serves a vital role in the

training and development of T-lymphocytes or T cells. ◦ T cells defend the body from potentially deadly

pathogens such as bacteria, viruses and fungi.The function of the thymus is to receive

immature T cells– that are produced by hematopoietic stem cells

(HSC) in the red bone marrow – and train them into functional, mature T cells

that attack only foreign cells.It also secretes the hormone thymosin

that stimulates the development of T-cells7

ThymusImmature TLymphocytes Mature T-cells

Thymosin

Stimulates•Development of T-cells•Other immune cells

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Types of T cellsTypes of T cellsHelper T cells (TH cells)

◦ Assist other white blood cells in immunologic processes, maturation of B cells into plasma cells and memory B

cells, activation of cytotoxic T cells and macrophages.

◦ Also known as CD4+ T cells because they express the CD4 glycoprotein on their surfaces.

Cytotoxic T cells (TC cells)◦ Destroy virus-infected cells and tumor cells, and

are also implicated in transplant rejection. ◦ These cells are also known as CD8+ T cells since

they express the CD8 glycoprotein at their surfaces. 9

Process of T-cell Process of T-cell maturationmaturation T cells first reside within the cortex of

the thymus–where they come in contact with epithelial

cells presenting various antigens. The immature T cells that respond to

the antigens corresponding to foreign cells are selected to survive, mature and migrate to the medulla

The rest die via apoptosis and are cleaned up by macrophages. This process is known as positive selection.

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Continued…Continued… Upon reaching the medulla– the surviving T cells continue to mature and are

presented with the body’s own antigens. T cells that bind to the body’s own

antigens test positively for autoimmunity. Autoimmune T cells are eliminated by

apoptosis in a process known as negative selection– resulting in only around 2% of the immature T

cells reaching maturity. T-cells that leave the thymus (via the

cortico-medullarly junction) are singly positive, self-restricted and self-tolerant.11

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Thymus dysfunctionsThymus dysfunctionsPeople without a fully functioning thymus

can't make enough T cells– as a result, they are very vulnerable to

infections.This is a particular problem for bone

marrow transplant patients – Because a functioning thymus is needed to

rebuild the immune system once the transplant has been received.

Some newborns also have malfunctioning or completely absent thymus – Due to conditions such as DiGeorge

syndrome.13

TreatmentTreatment

Thymus disorders can sometimes be treated with – Infusions of extra immune cells– Transplantation of a thymus organ soon

after birth– But both are limited by a lack of donors and

problems matching tissue to the recipient.Being able to create a complete

transplantable thymus from cells in a lab would be a huge step forward in treating such conditions.

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Thymic involutionThymic involution Unlike most organs that grow until the

age of maturity– the thymus enlarges throughout childhood– but slowly shrinks from the onset of puberty and

throughout adulthood. As the thymus shrinks, its tissues are

replaced by adipose tissue. This process is called thymic involution

The shrinking may be due to the reduced role of the thyroid in adulthood or increased secretion of sex steroids

The immune system produces most of its T cells during childhood and requires very few new T cells after puberty.

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Need for researchNeed for researchThymus transplantations soon after birth

can increase adaptive immunity in patients who are congenitally athymic

But these transplantations are limited by donor tissue supply and histocompatibility

These limitations would be overcome if functional thymic epithelial cells (TECs) could be generated or expanded in vitro, i.e. they are able to ◦ produce mature T-cells◦ produce well-formed organ with the same

structure as a healthy thymus18

History of researchHistory of researchSeveral investigators have reported

derivation of TEC-like cells from pluripotent cells

1. Lai and Jin, (2009) and Lai et al., (2011) used mouse embryonic stem cells to produce the cells that had phenotype of thymic epithelial cells using FGF-7, FGF-10 and BMP-4.

2. Inami et al., (2011) produced thymic epithelial progenitor cells (TEPCs) by phenotype – by culturing induced pluripotent stem cells (iPSCs) with collagen IV coated dishes in the presence of activin A and lithium chloride (LiCl) in mice

3. Parent et al., (2013) used human pluripotent stem cells to generate thymic epithelial cells – by regulating TGFβ, BMP4, RA, Wnt, Shh and FGF signaling19

Continued…Continued… Sun et al., (2013) produced TECs after

transplantation of the pluripotent cell-derived TECs using a transcription factor autoimmune regulator (AIRE) But in the experiment, scientists were

unable to produce cortical and medullary compartments of thymus.

In all these experiments◦ Unable to produce TEC-like cells of an

organized thymus containing all TEC subtypes◦ TEC-like cells had no capacity to support T-

cell development in vitro20

Generation of TECsGeneration of TECsBredenkamp et al., 2014 from

University of Edinburgh carried out their study using cells called fibroblasts taken from mouse embryos (MEFs).

The transcription factor forkhead box N1 ( FOXN1) is critically required for development of thymic epithelial cells

(TECs), during embryonic developmentBy increasing levels of the protein

FOXN1, scientists observed that the morphology of MEFs had changed from fibroblasts’ cell shape to epithelial cells shape.21

The experimentThe experimentRosa26CreERt2/+ mice were taken as

controlsTransgenic mouse line was

developed in which◦ Foxn1 cDNA controlled by CAG promoter was

knocked into ROSA26 locus with a LoxP-flanked transcriptional stop cassette was inserted between CAG promoter and Foxn1 cDNA

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Continued…Continued…These mice were then crossed with

Rosa26CreERt2 generating Rosa26CreERt2/CAG-STOP-

Foxn1-IRES-GFP embryos.From these embryos, primary mouse

embryonic fibroblast (MEFs) were generatedThese MEFs were then treated with

tamoxifen to remove STOP cassette generating Rosa26CreERt2/CAG-Foxn1-IRES-GFP iFoxn1 MEFs

After 10 days of iFoxn1 expression initiation, the morphology of cells changed from elongated, bipolar shape characteristic of fibroblast cells to broader, polygonal shape, characteristic of epithelial cells

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Bright-field (left) and immunofluorescence images (right) showing morphology and K8 staining, 10 days after 4OHT treatment.

Checking the identity of Checking the identity of cellscellsThe identity of these cells was further

confirmed by using◦ Epithelial-specific markers keratin 8 (K8) and◦ Epithelial cell adhesion molecule (EpCAM)

These are expressed by all TECs during early thymus development.

Most of iFoxn1 MEFs, but no control MEFs, showed K8 expression

Approximately 15% iFoxn1 MEFs expressed EpCAM

This suggested that FOXN1 induction had converted the fibroblasts to an epithelial-like state.25

Continued…Continued…

Further, EpCAM+ cells were isolated and analyzed for expression of specific FOXN1-regulated genes◦ TEC- (Dll4, Ccl25 and Kitl)◦ Cutaneous epithelium- (Fgf2 and Krt1)◦ Epithelial V-like antigen (Eva or Mpzl2)◦ non-Foxn1 target TEC-associated genes (Pax9 and

Trp63)The iFoxn1 MEFs but not control MEFs

expressed all genes except cutaneous epithelium genes.

All these expressions suggested FOXN1-mediated conversion of MEFs into TEC-like cells

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Functional attributes of Functional attributes of iTECsiTECsTo test whether induced thymic epithelial

cells (iTECs) were functional or not, scientists cultured a monolayer of iTECs with early T lineage progenitors (ETPs) Lin-CD25-

C-Kit+

Analysis after 12 days of co-culture revealed the presence of CD4+CD8+ double positive, CD4+ and CD8+ single positive cells resembling that of adult mouse thymus

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Continued…Continued…

These T cells expressed both CD3 (role in antigen recognition) and T-cell antigen receptor beta

While, ETPs seeded onto control MEFs did not enter thymopoiesis

Interestingly, the capacity of iTECs to support thymocyte development was dependent on their plating density◦ high density (>500 cellsmm-2) producing >3

times more CD4+ CD8+ T cells within 12 days than a lower density (<250 cellsmm-2)

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iTECs can form functional iTECs can form functional thymusthymus iTEC were then grafted under the kidney

capsule of genetically identical adult miceFetal thymic mesenchyme was included to

ensure that growth factors essential for expansion of the thymus, including FGF10 and IGF, were available within the graft

After four weeks, the cells had produced macroscopic, well-formed organs with the same structure as a healthy thymus, with clearly defined regions (known as the cortex and medulla).

All EpCAM+ cells in the iTEC grafts expressed GFP, reporting the transgenic iFoxn1-IRES-GFP messenger RNA, confirming they were derived from the input iTECs30

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Pan-cytokeratin staining reveals reticular network of epithelial cells throughout the

organs32

Hematoxylin and eosin staining

reveals cortical and medullary

portions of thymus

AdvantagesAdvantages iTECs are a new and readily available source of

TECs, that may provide the basis for thymus transplantation therapies aimed at boosting adaptive immune system function in immuno-compromised patients.

The technique may also offer a way of making patient-matched T cells in the laboratory that could be used in cell therapies.

Such treatments could benefit bone marrow transplant patients, by helping speed up the rate at which they rebuild their immune system after transplant.

The discovery offers hope to babies born with genetic conditions that prevent the thymus from developing properly.

Older people could also be helped as the thymus is the first organ to deteriorate with age.33

Future enhancementsFuture enhancementsThis is an exciting study but much

more work will be needed before this process can be reproduced in a safe and tightly controlled way suitable for use in humans

With further refinements, the researchers hope that their lab-grown cells could form the basis of a thymus transplant treatment for people with a weakened immune system.

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ConclusionConclusionEnforced expression of FOXN1 is

sufficient to convert fibroblasts into iTECs◦an in vitro generated cell type that

exhibits phenotypic and functional properties of in vivo TECs.

iTECs are able to promote full T-cell development in vitro

iTECs generate a properly patterned, functional organ on transplantation in vivo, composed of cortex and medulla of thymus35

ReferenceReference

Bredenkamp, N., S. Ulyanchenko, K. E. O'Neill, N. R. Manley, H. J. Vaidya and C. C. Blackburn. 2014. An organized and functional thymus generated from FOXN1-reprogrammed fibroblasts. Nat. Cell Biol., 1-15.

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Abbreviations usedAbbreviations usedHSC = Hematopoietic

stem cellsMHC = Major

histocompatibility complex

FGF = Fibroblast growth factor

BMP = Bone morphogenetic protein

TECs = Thymic epithelial cells

MEFs = Mouse embryonic fibroblasts

TGFβ = Transforming growth factor β

RA = Retinoic acidWnt = Wingless typeShh = Sonic hedgehog IRES = Internal

ribosome entry siteGFP = Green

fluorescent protein K8 = Keratin 8 EpCAM = Epithelial

cell adhesion molecule37

Continued…Continued…4OHT = 4-

hydroxy tamoxifen

Dll4 = Delta like ligand 4

Ccl25 = Chemokine ligand 25

Kitl = Kit ligandKrt1= Keratin 1Mpzl2 = Myelin

protein zero-like 2

Pax9 = Paired box gene 9

Trp63 = Transformation related protein 63

CD3 = Cluster of differentiation 3

IGF = Insulin like growth factor

iTECs = Induced thymic epithelial cells

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