FULLY FUNCTIONAL IMMUNE ORGAN GROWN IN MICE FROM LAB-CREATED CELLS Maleeha Akram 08-arid-1772 Ph.D....
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Transcript of FULLY FUNCTIONAL IMMUNE ORGAN GROWN IN MICE FROM LAB-CREATED CELLS Maleeha Akram 08-arid-1772 Ph.D....
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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