Lectures 1 & 2utminers.utep.edu/raguilera/14-utep-lect1.pdf · 2018. 7. 27. · populations as well as unstained Single positive pop. (one marker detected) Aguilera Lecture 1-2, 2014

Aguilera Lecture 1-2, 2014

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Lectures 1 & 2

http://www.niaid.nih.gov/topics/immuneSystem/Pages/glossary.aspx


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Hematopoietic Stem CellsHematopoietic Stem Cells

Hematopoietic Stem Cell (HSCHSC))


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HSC

CLP

Nat. Rev. Immunol.

Transcription Factors


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Why are HSCs so important?

Without HSC cannot generate an immune system. Irradiation kills these cells and eventually leads to death

Can be used to cure disease. Can irradiate on purpose and repopulate all white cells with just a few HSCs –all would be donor derived

Embryonic stem cells could theoretically be used to create different types of tissues and cells

Why aren’t HSC and Embryonic SC used in humans more often to cure disease?

How can you isolate “pure” populations of HSCs fromcrude populations of cells and from where?

Cell surface molecules serve as cell “markers”Cell surface molecules serve as cell “markers”which are needed for their isolation and identification which are needed for their isolation and identification

(CD45)

B220 = CD45 (tyrosine phosphatase)

IgM


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FluorescenceFluorescenceActivated CellActivated Cell--SorterSorter

Collect or analyzesorted cells

Laser beam

Input cells

Labeled antibodies against cellLabeled antibodies against cellsurface markers are usedsurface markers are used

to isolate cell populationsto isolate cell populations

FACSFACS

FITC, Rhodamine,Texas Red, etc

B220

spleen cells

55%

Surface IgM

FACS Analysis of B Lymphocytes in SpleenFACS Analysis of B Lymphocytes in Spleen

double positiveB220+/IgM+

(mature B-cells)

There are generally single and double positive There are generally single and double positive populations as well as unstainedpopulations as well as unstained

Single positive pop.(one marker detected)


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Objective:To isolate the putative Hematopoietic Stem Cell (HSCHSC)

Results:Using a variety of cell surface markers (antigens),they were able to isolate cells that could differentiate into lymphocyte and myelomonocytic cells

Just 30 of these cells could save 50% of lethally irradiated mice – and reconstitute all blood cell types

Science 1988

Based on the data from prior experiments, the authors reasoned that the HSCHSC should not contain the same types of markers as differentiated cells (ie, lymphocytes, macrophages, etc.)

So they removed all cells by negative selection thatSo they removed all cells by negative selection thatexpressed antigens characteristic of expressed antigens characteristic of BB--cells (B220)cells (B220)and and TT--cells (CD4 & CD8)cells (CD4 & CD8) as well as as well as granulocytes granulocytes (Gr(Gr--1)1) and and myeolomonocytic cells (Macmyeolomonocytic cells (Mac--11))

It was further determined that the putative stem cell expressed low levels of the Thy-1 marker and referred to these cells as Thy-1lo


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It became clear that the Thy-1lo Lineage-minus (Lin-) was enriched for stem cell activity but was still contaminated with more mature progenitor cells

In this paper, they demonstrate that another molecule called Stem Cell Antigen-1 (SCA-1) could be used to further subdivide the putative stem cells into a minor (SCA-1+ ) and a major population (SCA-1-)

They proved that the Thy-1lo Lin- SCA-1+ population was highly enriched in HSC HSC activity

The Thy molecule was originally only found in thymocytes

LinLin--SCASCA--1+1+ThyThy--11lolo

(1) Eliminated T-cells from bone marrow cells with anti-CD4/CD8

(T-cell lineage markers) using antibodies (with magnetic beads)

How did they enrich the HSC population?How did they enrich the HSC population?

(2) Isolated Thy-1 + cells with same strategy –obtained 2% of original pop

(3) Used labeled antibodies (phycoerythrin) to B220, Mac-1, and Gr-1

as well as SCA-1 (biotin) labelled with Texas-red (avidin)

(4) FACS sorted for ThyThy--1 +, SCA1 +, SCA--1+1+ and absence of lineage markers


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cell size cell-cycle

Enriched cells are intermediate in size between lymphocytes Enriched cells are intermediate in size between lymphocytes

and Myeloid cellsand Myeloid cells

Most cells are in G0Most cells are in G0--G1 phase of the cell cycle (resting)G1 phase of the cell cycle (resting)

Analysis of spleen colony formation in lethally irradiated miceAnalysis of spleen colony formation in lethally irradiated mice

12 days after IV injection

One CFU per

10 HSC injected

One CFU per

7200 unseparated

Bone-marrow cells

Colony Forming Unit=CFU


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12 day CFU in the spleen12 day CFU in the spleen

Colonies are generally derived from a single seeding cell

More

differentiated

progenitors

less

differentiated

progenitors

Histological analysis of early and late CFU in spleenHistological analysis of early and late CFU in spleen

Sca-1 minus look more like un-selected bone marrow cellswhile Sca-1+ look more like HSC


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Colony formation in the Thymus (IT) of irradiated miceColony formation in the Thymus (IT) of irradiated mice

Approximately one CFU-T was transferred per 5 HSCs

If inject 10 cells, more than 95% of thymi contained colonies

If inject BM cells, it would take 5000 or 8000 cells to see one colony

40 HSC can repopulate both B, T and myeloid lineages 40 HSC can repopulate both B, T and myeloid lineages in irradiated micein irradiated mice

The LyThe Ly--5.25.2antigenantigen

detects onlydetects onlydonor cellsdonor cells

Six weeks after IVSix weeks after IV50% of blood cells50% of blood cellswere donor derivedwere donor derived60% T cells60% T cells50% B cells50% B cells50% Gr50% Gr--1+1+


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Protection from lethal irradiationProtection from lethal irradiation

50% of mice survived with injection of 30 cells50% of mice survived with injection of 30 cellsYou would need 1You would need 1--3 x 103 x 1044 BM cells to achieve thisBM cells to achieve this

SCASCA--1+ populations in the BM and the purified population1+ populations in the BM and the purified population

very few in very few in unsorted BMunsorted BM


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There is a similar HSC population in HumansThere is a similar HSC population in Humans

So What?So What?

What is the big deal about stem cells?What is the big deal about stem cells?


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��HSCs can not only save lethally irradiated miceHSCs can not only save lethally irradiated micebut also humansbut also humans

��Theoretically a single cell can reconstitute all blood cellsTheoretically a single cell can reconstitute all blood cells

��Can save cancer patientsCan save cancer patients--been done already been done already

��Can be used for gene therapy? Add a missing geneCan be used for gene therapy? Add a missing gene

��Can be used to cure AIDS? Well maybe not but who knowsCan be used to cure AIDS? Well maybe not but who knows

��Can regenerate the nervous system/ brain stem cells? Can regenerate the nervous system/ brain stem cells?

��Embryonic stem cell can generate animals (transgenics)Embryonic stem cell can generate animals (transgenics)

Stem cells can give rise to all cellsStem cells can give rise to all cells


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Unfortunately as we get old, HSC lose ability to make more lymphocyte precursors

WNT SIGNALLING AND HAEMATOPOIESIS:A WNT–WNT SITUATION

Frank J.T. Staal* and Hans C. Clevers ‡

The evolutionarily conserved WNT-signalling pathway has pivotal roles during the

development of many organ systems, and dysregulated WNT signalling is a key factor in the

initiation of various tumours. Recent studies have implicated a role for WNT signal transduction

at several stages of lymphocyte development and in the self-renewal of haematopoietic stem

cells. Here, we outline new insights into the WNT-signalling pathway, review its role in the self-renewal

of haematopoietic stem cells and in the development of T and B cells, and discuss

controversies and future developments with regard to WNT signalling in the thymus.

NATURE REVIEWS | IMMUNOLOGY VOLUME 5 | JANUARY 2005 |


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Wnt

NATURE | VOL 423 | 22 MAY 2003 | www.nature.com/nature

Inhibition of Wnt signalling results in inhibition of

engraftment of donor cells

Axin inhibits

intracellularWnt signalling

Purified Stem cells (Ly5.1)


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If HSC and stem cells are so great, why aren’t they used more often to save people?

Rejection is a serious problem for transplant patients


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Allogeneic rejection of non-self tissue is T-cell mediated

Allograft is a graft from an allogeneic/non-self donor of same species-Alloantigens are defined as polymorphisms at the MHC locus –are the non-self antigens of allograft

T-cell mediated Tissue Rejection


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Figure 1: Models of hematopoietic differentiation.From Nature Immunology 11,569–570(2010)Kenneth Dorshkind

(b) A revised model of human hematopoiesis, showing that the development of lymphoid and myeloid

cells is not as dichotomous as presented in a. The report confirms the existence of a CMP from which

GMPs and megakaryocyte-erythroid progenitors are generated. GMP progeny include macrophages and

DCs. However, consistent with evolving mouse models, lymphoid-specified progenitors, called MLPs

here, retain myeloid potential. Thus, macrophages can be derived from MLPs and GMPs. DCs are also

shown here to be developmentally heterogeneous, with origins in both MLPs and GMPs


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Figure 1 Strategies to enhance immune function in the elderly improve the development, expansion, and function of T cells. Sex steroid modulation via surgical or chemical castration in aging and transplanted mice or humans enhances lymphoid progenitor gene...

Amanda M Holland , Marcel RM van den Brink

Rejuvenation of the aging T cell compartment

Current Opinion in Immunology Volume 21, Issue 4 2009 454 - 459

http://dx.doi.org/10.1016/j.coi.2009.06.002


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Table 1. Summary of age-related changes in T cells.

CD4 T cellsCD4 T cells

Reduced TCR signaling intensity

Reduced expansion in response to TCR stimulation

Reduced Th1 and Th2 effector differentiation

Reduced cognate helper function

Retain the ability to differentiate to Th17

CD8 T cellsCD8 T cells

Reduced TCR repertoire diversity

Development of clonal expansions

Reduced antitumor responses

Regulatory T cellsRegulatory T cells

Increased numbers

Retain/gain function with age

Downregulate antitumor responses

May contribute to Th17 skewing

Current Opinion in Immunology 2009, 21:414–417

Lectures 1 & 2utminers.utep.edu/raguilera/14-utep-lect1.pdf · 2018. 7. 27. · populations as well as unstained Single positive pop. (one marker detected) Aguilera Lecture 1-2, 2014

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