Lineage- and Stage- Specific Gene Expression

in Human Hemopoietic Cells

by

Erqian Na

A thesis submitted in conformity with the requirernents for the degree of Master of Science

Graduate Department of Medical Biophysics University of Toronto

O Copyright by Erqian Na

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Abstract

Stem cells are capable of maintaining a constant supply of mature lymphoid and myeloid

cells. This smdy describes the establishment of a system for mapping lineage- and stage-

specific gene expression in human hemopoietic precursors. and dernonstrates its ability to

genente information relevant to mechanisrns that control prolifention and differentiation

during hemopoietic development.

Two approaches were used to define patterns of gene expression at specific stages in

hernopoietic differentiation. The first of these is global RT-PCR which amplifies al1

polyA' mRNA from a single cell. Hemopoietic precueors in different cornmitment States

are phenotypically similar and cannot be sepanted from one anocher or purified to

homogeneity using existing methods. The ability to study gene expression at the level of

a single ce11 cm provide an alternative approach to achieving homogeneity. The second

strategy is sibiing analysis. which identifies retrospectively the developmental potentid

of a ce11 subjected to PCR by analysis of the fate of its siblings. To apply these

approaches to human hemopoietic cells. it was first necessary to address an array of

technical challenges. These included identification of culture conditions which could

support colony growth from single isolated cells at high efficiency. emichrnent of

precursors with significant proliferative capacity. and optirnization of conditions for

faithfûl and sensitive PCR. Once these requirements were met. 1 was able to collect more

than 200 cDNA sampies from cells of different lineages and different stages of

devebpment. This cDNA set was initially validated by probing for expression of

constitutive (8-actin. L35) and lineage-special (MPO. a-globin) genes to confimi the

specificity and reiiability of amplification and the accuracy of the assignments. The set

was then used to determine expression patterns of a wide panel of genes (RBTX. CD34

fms. c-kit, KPK1, Bcl-2 family. p53. WAFI. RB. and WT) known to be involved in

reguiarion of proliferation and differen tiation of hemopoietic cells. The results provided

novel infornation on linenge- and stage-specificity of expression of these genes.

Moreover. the work rstnblishrs the potrntial of the system for gining new insights inro

disordered States such as leukemia. and for identification of novel senes controlling

differentiation using subtractive methods.

This thesis is dedicaîed

To my husband Yongxin Ma. for al! his love, understanding, and srrong support throughout my srudies.

Tu my rnother and sisters. fur rheir love and concern.

I am grateful to my supervisor. Dr. Norman N. Iscove, for providing me with the opportunity to work on this project. for his encouragement, guidance. and patience. His rigorous scientific approach to research problems and great scientific sense will continue to affect my work in the future.

I would like to express rny gratitude to Dr. Mark Minden and Dr. Jane Aubin. for serving on rny supervisory cornmittee and advising dunng my study.

1 aiso would like to express my gratitude to the people in the lab for their help and kindness. To Karen for her consmctive cnticism of rny wriring. To Deborah and Mary for their laborarory expertise and Engiish lessons. To Herry. Maryanne. Phyllis. Friedemmn. and Quido. for sharing their knowledge with me. To Katsu and Metdiew. for their fnendship.

Many thanks to Nazir for his support and advice.

I would like to diank many research fellows and students in Ontano Cancer Institute. Weifeng, Yi, Wenrnei. Zhenbo. Yuping, luonin. and Katherin for their help and friendship.

Table of Contents

Abstract Dedication Ac know ledgments List of Abbreviations and Symbols

Chapter I Introduction

1.1 Gene expression and ce11 differentiation

1.2 Human hernopoietic precursor ce11 hierarchy

1.3 Bone rnarrow culture

1.3.1 BM culture conditions

1.3.2 Growth factors

1.4 Enrichment of human bone marrow precursors

1.4.1 Purification by buoyant density

1.4.2 CD34 ce11 surface marker

1.4.3 Other surface markers

1.5 Single ce11 approaches to homogeneity

1.5.1 Global polymense c hain reaction in single cells

1 S.2 Sibling analysis

1.6 Genes selected for expression malysis

1.6.1 Constitutive and lineage-specific genrs

1.6.1.3 Myeloperoxidase

1.6.1.4 cr-Globin 16

1.6.2 Selected genes involved in hemopoietic ce11 growth and differentiation 16

1.6.2.1 Rhombotin 2 16

1.6.2.2 CD34 17

1.6.2.3 fins 18

1.6.2.4 c-kit 18

1.6.2.5 HPKI 19

1.6.2.6 Bci-2 family 19

1.6.2.7 p53 21

1.6.2.8 WAFl 22

1.6.2.9 Retinoblastorna 22

1.6.2.10 WiIm's Tumor 23

1.7 Thesis objective and overview 23

Chapter 2 A System for Analysis of Stage-specific Gene Expression in

Human Hemopoietic Cells

2.1 introduction

2.2 Materials and methods

CeiIs and ce11 lines

Repantion of mononuclear cells (MNC)

Adherent ceil depletion

Irnmunologicai staining and ce11 sortïng

2.2.5 E ~ c h r n e n t of CD34* cells using magnetic beads

2.2.6 Preparation of platelet-poor plasma (PPP)

2.2.7 Preparation of phyto-haemaglutinin stimulated leukocyte

conditioned medium (PHA-LCM)

2.2.8 Cryopreservation of mmow cells

2.2.9 Colony growth in methyl cellulose cultures

2.2.10 Precursor selection with thymidine blockade

2.2.1 1 DNA purification

2.2.12 RNA purification

2.2.13 Amplification of poly A mRNA from single cells

2.2.14 Probes

2.2.15 Southem blot hybridizations

2.3 ResuIts

2.3.1 Growth efficiency in methyl celIulose cultures

2.3.2 Enhancement of the frequency of "useful" colony-star&

2.3.3 Success of sibling analysis

2.3.4 Parameters determining the efficiency of ~Iobal PCR

applied to single cells

2.3.4. I Magnesium

2.3.4.2 Annealing temperature

2.3.4.3 Primer concentration

2.3.5 Collection of cDNA samples h m hernopoietic cells

vii

2.3.6 Use of the cDNA sample set to define patterns of gene expression

in the hemopoietic hierarchy

Chapter 3 Discussion and Future directions

References

Abbreviations

AML BM BSA BSS BFU-E cDNA CD CDK CFU-E CM5637 CFC CSFs CSF- 1 DEPC DMSO DNA EDTA E P FACS FBS 5-FU G-CSF GM-CSF G-6-PD HPK HSC k IL- 1 IL-3 IL-6 IL-1 1 IMDM KLCM LTC-ICS Mec MGDF ML. MNC MoAb MPO mRNA M-CSF NBS

acute myelocytic leukemia bone marrow bovine serurn albumin baianced salt solution burst forming unit-erythroid cellular deoxyribonucIeic acid cluster of differentiation cyclin-dependent kinase colony forming unit-erythroid conditioned medium from humm bhdder carcinoma ce11 Iine 5637 colony foming ce11 colony stimulating factors colony stimulating factor- 1 diethyl pyrocarbonate dimethy IsuIfoxide deoxyri bonucleic acid disodium ethyiene diamine tetraacetate erythropoietin fluorescence actived cell sorter fetal bovine serum 5-fluorouracil granulocyte colony stimulating factor granulocyte colony stirnulating factor glucose-6-phosphate dehydrogenase hemopoietic progenitor kinase hemopoietic stem cell irnmunog Io bin interleukin- 1 interleukin-3 interleukin-6 interleu kin- 1 1 Iscove's modified Dulbecco's medium conditioned medium containincg Kit ligand long-tem culture-initiating ceIls rnethy i cellulose megakaqocyte growth and development factor/Tpo/Mpl ligand Mpl ligand mononuclear ce11 monoclonai antibody myeloproxidase messenger nbonucleic acid macrophage colony stimulating factor newborn bovine serum

PBS PE PCR Pm-LCM PPP RB Rb RBTN rtiIL RNA RT-PCR SCF TBE TdT T P ~ WT

phosphate-bu ffered saline R-phycoerythnn polymense chah reaction phyto-haemagglutinin stimulated leukocyte conditioned medium platelet poor plasma retinoblastoma gene retinoblastoma protein rhombotin recombinant human interleukin ribonucleic acid reverse transcription PCR stem cell factor Tris-borate bu ffer terminal deoxynucleotidyl transferase thrombopoietin Wilms' tumor

Chapter 1

Introduction

In the hemopoietic system. the production of mature blood cells throughout life involves

the highly regulated processes of prolifention and differentiation of a hierarch y of

progenitor cells along multiple distinct ce11 lineages (1). The mechanisrns that regulate

such processes are not fully undentood. It is hypothesized that there exist genes which

drive transitions in self-renewal capacity and lineage determination. Mature hemopoietic

cells have unique morphological and protein marken that distinguish them from

immature cells. Various kinds of precursor cells, on the other hand. are phenotypically

similar. They can be separared from more differentiated cells by methods such as ce11

sorting based on ce11 surface rnarkers. but cannot be separated f?om one another or

purified to homogeneiiy (2). Thus, knowledge of gene expression in the hemopoietic

system is usually obtained by analyzing ce11 populations or ce11 lines. An approach

allowing study at the single ceil level would avoid the use of precunor populations of

mixed composition and guarantee homogeneity.

Two strategies have been recently developed that make it possible to study gene

expression in single cells from murine marrow (3). One of these strategies is global RT-

PCR. which amplifies al1 poly A mRNA from single cells. The other strategy, sibling

analysis. is used to identify retrospectively the developmental potential of individual

precursors subjected to PCR by analyzing the fate of their siblings. This thesis describes

the successful extension of these approaches to human marrow, and focuses on defining

stage-specific and lineage-specific gene expression patterns in normal human

hernopoietic precursors.

In order to apply these two approaches to human marrow. it was necessary to address an

array of technical challenges. First. culture of human BM cells has been improved with

the discovery of various growth Factors in recent yean. but growth of single isolated

precurson with expression of their hl1 growth and differentiation potential in methyl

cellulose conditions has not been reponed. Second, precursors are present at very low

frequency in human BM. Sibling analysis would be more practical if the frequency of

colony-forming cells could be increased prior to sibling analysis. Third, the demands of

PCR and single ce11 work require free access to ceil samples. yet human marrow is only

availabie according to clinicnl schedule. Furthemore, the global PCR method must be

sensitive, reliable, ruid faithful. Sensitive means that even low abundance gene

aanscnpts cm be amplified: reliable means the probability of successful amplification of

cDNA from a single cell is high and consistent: faithful rnems onIy mRNA derived

products are amplified. Before describing the experiments that I conducred successfully

to meet these challenges. 1 will review the relevant background to the work.

1.1 Gene Expression and Ce11 Differentiation

Higher organisms possess about 100,000 different genes, of which only a small fraction

are expressed in any individual cell. It is the choice of which genes are expressed that is

responsible for the transitions from one ce11 type to the next during deveiopment (4).

Bone marrow is an easily accessible example of a differentiating developmental system in

which little is yet known about the genes controlling growth and differentiation decisions.

Deveiopment of a system for analyzing stage-specific gene expression would provide

toois for understanding mechanisms of normal differentiation. as well as for

understanding the defects in these processes that underlie leukemic disease.

1.2 Human Hemopoietic Precursor Cell Hierarchy

Mature cells of the hemopoietic system are relatively short-lived cells that need to be

replaced continuously throughout life. In virro, colony forming assays demonsuare that

precursors divide and form mature cells. In vivo. bone mmow transplanta~ion

demonstrates the existence of multipotential stem cells responsible for the long-term

maintenance of mature ce11 production (5). Since the physiologies of murine and hurnan

hemopoiesis appear to be analogous. our present understanding of human hemopoiesis is

built upon information derived from both murine and human systems. The human

hemopoietic hierarchy is comprised of hemopoietic stem cells. precursor cells and mature

ceIls (Figure 1).

T Ceti

Stem ce11

Precursors

B Cell

Macrophage

Neutrophil

Eosinaphii

Mast Cell

Erythrocyte

Megakaryocyie

Mature cells

Figure 1 The Hemopoietic Differentiation Hierarchy

The circle on the frir lefi represents a stem cell. The arrow around the stem cell indicrites its self- renewal ability. It differentiates into precursors and then mature cells. The rightmost circles s i p i & restricted unipotential precursors that divide a number of times to become mature cells. The circles in between signify more primitive stages that have multiiineage, trilineage, or bilineage deveiopment potential.

Stem cells are a rare subpopulation of hemopoietic cells. They are able to self-renew as

well as to differentiate and continuously jenerate progenitor cells of the myeloid and

lymphoid system. They are detected specifically by their capacity to repopulate

hemopoiesis on a long-tenn bais in hosu (6) (7) (8).

Progenitor cells comprise up to 1-5 % of marrow cells. The frequency of multi-lineage.

trilincage, or bilineage progenitor cells is less than that of single lineage progeniton.

These cells become progressively resaicted to distinct lineages during their terminal

differentiation to mature blood cells. They cm be functionally studied in vin-O with

quantitative colony forming assays. In such cultures, an individual progenitor ce11

proliferates to fom a clone of maturing progeny cells which cm be identified and scored

as a colony. The variable size and lineage content of these colonies indicate the

heterogeneity of the progenitor cells. Cells forming smailer colonies are considered to be

more developmentd ty mature than cells forming larger colonies (9) ( 10) ( 1 i ) ( 1 2).

Although many studies have focused on puriQing stem cells and precursors. littie

progress has been made in separating various precursor types from one another, because

of their sirnilar morphology and lack of specific markers. Some ceil surface markers c m

be used ro separate mature cells from precursors with particulm developrnental potential,

such as 8220 (positive and nrgative selection) which marks mature lymphoid cells and

precunors. However this subpopulation is still heterogeneous. containing cells at

different development stages (26).

Mature cells comprise the majority of cells present in hemopoietic tissue. They represent

at least 8 distinct lineages. ïhey have limited or no proliferative potential.

Although the purification of stem cells is hampered by their low frequency and by the

absence of specific markers. the existence of hemopoietic stem cells has been

demonstrated by a variety of techniques. Most of the approaches and strategies now

being applied to humm cells are based on previous experience with murine hemopoietic

cells. The cornpetitive repopulating assay quantitates stem cells based on their ability to

compete with a standard number of unmanipulated cells in the reconstitution of an

irradiated recipient ( 13) (1 4). Radiation-induced chromosomal markers ( 15) and

retrovirus insertion markers ( 16) ( 17) have been used in murine marrow transplantation

and confirmed the existence of stem cells that are able to generate progeny of lymphoid

(T and B) and myeloid lineages. Although such experiments are not suitable in humans.

clinical transplantation has supplied evidence for the existence of multipotential stem

cells with self-renewal capacity in hurnans. Ir is also possible to obtain engraftment of

hurnan marrow in severe cornbined immunodeficient (scid) mice ( 18) ( 19) (20). Clinical

marrow transplantation began in the late 1960's. Some patients have survived for more

than 20 years following transplantation. Genetic rnarkers. such as X Iinked glucose-6-

phosphate dehydrogenase (G6PD) (5) and DNA polymorphisms (2 1) have been exploited

to detemine the origin of cells in patients with leukemia and recipients of BM

transplants. It has been reported that human mmow cells reconstituted lymphopoiesis

and myelopoiesis in both experimental animals (22) (23) (24) and hurnans (25). The

hematopoietic system rernains entirely of marrow donor origin in a host recipient bone

marrow can dso be used to establish secondary gafts. and sequential autoL&s have

been successfully performed (26). Al1 of these snidies indicate that the original rnarrow

contains rnultipotent stem celIs with self-renewal capacity.

1.3 Bone Marrow Culture

1.3.1. BM culture conditions

Recursor cells c m be identified by their ability to form colonies in semi-solid medium.

With a variety of growth factors. semisolid conditions allow precursors to proliferate and

differentiate into colonies that remain physically localized. Investigaton have identified

not only end ce11 colonies but also blast ce11 colonies in these conditions. Blast ce11

colonies contain new blast colony-forming cells that c m be detected by seeding cells

from pnrnary colonies to form secondary ones ( 10) (27).

For detection of cells with extended prolifention capacity. Dexter introduced suspension

culture. As a feeder iayer he used stroma cells which provide a supponive

microenvironment to enable munne progenitor cells to survive and produce mature

myeloid cells (28). This technique currently appears to be the most suitable rnethod for

identifying in vitro relarively ancestral stem cells, particularly when analyzing human

populations (29). Long-term-culture-initinting cells (LTC-ICs) are defined as cells able to

genente myeloid clonogenic ce11 propeny for a minimum period of 5 weeks in long-temi

cultures. The number of human precursors and mature cells continually decrease in these

conditions and totally disappear by 8-10 weeks. Production of lymphoid precurson by

human long-term culture-initiating cells has not yet been shown (30) (3 1) (32).

Suspension cultures have also been explored in which defined cytokines are used in place

of stroma1 feeder layers. Although primitive progenitors c m be induced to differentiate

in such cultures, maintenance and proliferation of long term cells have iiot been achieved,

suggesting that not al1 required stimuli are yet known (33) (34).

1.3.2. Growth Factors

Growth factors or cytokines, called interleukins or colony stimulating facrors (CSFs), are

multifunctional regulators of hemopoiesis (35) (36).

Erythropoietin is the principal hormone regulating erythrocyte production. It was

discovered in the 1950's using in vivo assays. A group of acidic glycoprorein growth

factors was subsequently recognized through their ability to promote colony formation in

vitro. They were named granulocyte-macrophage CSF (GM-CSF), granulocyte CSF (G-

CSF), macrophage CSF (M-CSF) and interleukin-3 (IL-3). GM-CSF supports mainly the

development of colonies composed of granulocytes, macrophages, or a combination of

both ce11 types: G-CSF supports mainly granulocyte colonies; M-CSF supports mainly

macrophage colonies; and IL-3 stimulares formation of multilineaee as well as more

restricted colony types. Individuai human CSFs were biochemically isalated and

mkdady doonedin the49Wk(u).-Additional -th factars.indudUlgIL 1 &6. -

IL4 1. and stem cell factor (SCF). have also been identified (38) (39). Synergistic effects

of the growth factors, such as IL-3 with M-CSF. L i . iL6. IL1 1. GM-CSF, and G-CSF

have been widely reponed (40) (41) (42) (43) (44). Some growth factors have more than

one name. SCF is also known as c-kit ligand. Steel factor. and mast cell growth factor. It

has a broad range of activities. including egects on multipotential stem cells as well as on

cells that are committed ro myeloid, erythroid, and lymphoid lineages. Recently, a

growth factor that is necessary for megakaryopoiesis and the regulation of platelet

production was cloned. It is called Mpl ligand (ML), thrombopoietin (Tpo), and

megakaryocyre growth and development factor (MGDF) (45) (46)(47). Dunng the past

decade, there has been major advance in the identification of growth factors. The

hemopoietic ce11 types identified as responding to different growth factors are

summarized in Table 1.

Table 1 Growth Factors and the Hemopoietic Crlls Responding to Them

Target hemopoietic

precursor cells

Stem ce11

Monocyte-macrophage

Neutrophil

Eosinophil

Mast ce11

Erythroc yte

Megakaryocyte

T ce11

B ce11

Hemopoietic growth factors

SCF. IL 1, IL-6, IL- 1 1, MGDF, Flt-3 ligand

M-CSF, GM-CSF. IL-3, IL-7

G-CSF. GM-CSF, IL-3, IL-6, IL- 1 1, SCF, IL-7

IL-3. IL-5, GM-CSF

IL-3, SCF

Early: SCF. CL-3. IL- 1 1. GM-CSF late: Epo

MGDF, SCF. IL-3, IL-6. IL- 1 1

IL-1 a-2. L-4, rL-10. IL-7

Sm. IL-7. IL-4, IL-6. IL-2

1.4 Enrichment of Human Bone Marrow Precursors

Although it has not been possible to purify hemopoietic progenitors of different kinds

from one anorher with present technologies, it is possible to enncb precunors with

significant growth potential fiom the large number of neariy mature or mature cells in

BM based on methods exploiting differential densities and ce11 sizes. membrane antigen

expression. and cell cycling propenies (9).

1.4.1 Enrichment by buoyant density

Stem cells and precunors are mononuclear cells having a lower buoyant density than

erythrocytes and polymorphonuclear neutrophils. Density separation is 3 usefui

preennchment step in precursor ce11 purification because of its ability to process large

numbers of ceils (2).

1.4.2 CD34 Ce11 Surface Marker

The CD34 antigen is a 1 I O kD surface glycophosphoprotein. It is found on

approximately 1 4 % of normal human rniurow rnononuclear cells (48) (49). Positive

cells include precursors of monocytic. ~ u l o c y t i c . erythroid. lymphoid and

rnegakaryocytic cells and stem cells capable of reconniniting the hemopoietic system in

hurnan transplantation (50) (5 1 ). Thus bis antigen is recogized as a hemopoietic

stemiprogenitor ce11 surface marker. It is also present on smdl vesse1 endothelium and in

basement membranes (52) (53).

Seven anti-CD34 MoABs - My 10, BI.3C5, 12.8, 1 15.2, ICH3, TUK3, and QBENDlO -

have been ciassified according to the epitopes they recognize (5 1). The antibodies have

been used to purify CD34 positive (CD343 cells in conjunction with the fluorescence-

actived ce11 soner (FACS), immunornagnecic beads or affinity columns. The resulting

populations are 10- to 100-fold enriched for in vitro coIony forrning cells (CFC) (54).

1.4.3 Other cell surface markers

CD34' ce11 selection has been the prirnary cool used for the purification of human

precursor cells. Positive cells are highly heterogeneous both functionally and in the other

markers they express. In order to punQ funher the most primitive cells, additional ce11

surface mtigens and staining rengents have been used. Specitically. manferin receptor

(CD7 1 ). CD33. CD38, HLA-DR. CD45RA. c-kit. Thy- 1, combinations of lineaee-

specific mtigens and Rhodamine- 123 staining have been used to separate immature cells

from comrnitted progenitors.

The CD33 antigen is a marker of monocytes and granulocytes. The combined expression

of CD31 and CD33 antigens identifies commined progenitors such as CFU-GM and

BFU-E. while CD34 positiveKD33 negative cells are more primitive, forming blast or

multilineage colonies (55) ( 1 O) (56).

Antigen CD38 is a marker of lymphocytes. monocytes. rnyeloblasts, and very early

erythroblasts. It is expressed on 90% of CD34 positive cells. The CD34TD38-

population lacks differentiation markers and is enriched for more primitive CFC detected

by formation of colonies of undifferentiated cells ("blast colonies") (57).

Rhodamine- 123 (Rh) is a fluorescent dye that stains mitochondrial membranes. Cyciing,

metabolically active cells label more brightly than quiescent cells (26).

Lineage-specific cell surface markers including CD2. CD4. CD8. CD 14. CD 19, CD20,

CD 16, CD56. and glycophonn A are used to identify T. B. NK. myeloid. and mature

erythroid cells (24) (26).

1 .S Single Ce11 Approaches to Homogeneity

15.1 Global Polymerase Chain Reaction in Single Cells

The development of the PCR technique in 1985 has revolutionized molecular biology by

allowing the amplification and analysis of very small amounts of specific DNA (58) (59).

RT-PCR generates a fint sûand of cDNA on mRNA templates using RNAdependent

DNA polymense (reverse transcriptase). and then amplifies the resulting cDNA by PCR.

This technology was used by Brady et al to develop a method that could arnplify cDNA

from al1 polyadenylated (polyA+) RNA vanscripts in samples as small as a single ce11

(60). The technique makes it possible to study gene expression in single hemopoietic

precursors at different stages of development. avoidin_e the problem of heterogeneity

inherent in studies of ce11 populations (3).

In the global RT-PCR process. the first step is to generate a first süand of cDNA on

polyadenylated rnRNA templates by using reverse transcriptase and oligo(dT) primer.

The conditions are designed to limit the lengrh (300-700 bp) of the initiai cDNA

aanscripts to minimize selection against longer cDNA during amplification. This results

in the preservation of the abundance relationships from the original sample to the final

amplified product and maximal efficiency of amplification. The second step is to tail the

cDNA strand with 3' oligo(dA) using terminal aansfense. The third step is to arnplify

the cDNA. Amplification proceeds by generating the second cDNA strand on the first

strand template using a single oligo(dT)-containing primer and Taq polymerase, then by

cyclic denaturation and repctition of the same reaction following standard PCR

procedures. The advanmges of this method are (i) lysis of single cells and al1 subsequent

reactions are carried out in the s m e suspension without any extraction procedures,

eliminating any opportunity for sample loss, (ii) the amplification is performed widiout

the use of sequence-specific primers. (iii) it is sensitive rnough to detect moderate-to-low

abundance (0.02%) messages in single cells, (iv) it is insensitive to initial tmnscript size.

(v) relative abundance relationships in the original sample remain intact in the amplified

product. (vi) the arnplified cDNA material is indefinitely renewable and readily cloned for

genention of libraries and subaction. and (vii) the amplified product c m be probed any

number of times for the presence of different sequences. Since this method amplifies

only a few hundred bases of 3' terminal sequence. codine rezions are not s i~if icuidy

represented in the arnplified product.

1.5.2. Sibling Analysis

The lineage of mature cells c m be recognized by their distinct colony and ce11

morphologies. The identity of cells cm be confirmed by fixing and staining these cells on

glass slides. It is impossible to distinguish precursor types in bone marrow on

morphological grounds. Sibling analysis is designed to identiw whether the examined

cells have growth potential and. if so. what is their differentiative potential.

When human bone mmow precursors are cultured in semisolid or liquid conditions.

precunor cells divide wirhin 2 or 3 days into 4 or 8 cells which are called "colony stuts".

From a colony start, 1-2 cells are subjected to PCR amplification and the remaining

sibling cells are replated individually in secondary cultures and allowed to form colonies.

The fate of sibling cells in these secondary cultures, if uniform, identifies reuospectively

the developmental potential of the ceil subjected to PCR.

1.6 Genes selected for gene expression pattern shidy in this thesis

In the hemopoietic system. the most fundamental question is how multipotential stem

cells and precursors decide to undergo self-renewal or differentiation to produce more

resmcted progeny. A complex pattern of activation and silencing of sets of genes is

expected to be responsible for lineage cornmitment to a defined developmental pathway.

Selecrion of genes for snidy of expression patterns in this snidy was based on three

considerations. First. constitutively and universally expressed genes (B-min. L35) and

lineage specific genes (MPO. globin) were chosen to test the stage- and lineage-fidelity of

the cDNA products generated fiom global RT-PCR of single cells. Second. previous data

denved fiom ce11 populations or ce11 lines have indicated that certain genes are involved

in the proliferation and differentiation of hemopoietic cells, or expressed as a

consequence of cornmitment events (RBNT2 and CD34). Others are cytokine receptor

genes (c-fins and c-kit) or signalling elements (HPKI) whose lineage and stage-

specificity of expression have already been tested in populations of hemopoietic ceils.

investigation of their expression at the single ce11 level will hrther validate Our sample

set, and also extend our knowledge of stage- and lineage-specificity of gene expression in

the normally human differentiating hierarchy. A third group of genes was selected about

which there is less information available concerning their expression in the hemopoietic

hienrchy, but for which there is evidence for roies in either normal or leukemic

hemopoiesis (p53. Bc12, WAFI. Rb and WT).

1.6.1 Constitutive and Lineage-specific Genes

1.6.1.1 8-actin

The functional $-actin gene is located on human chromosome 7. B-actin protein

polymerizes with its isoform y -actin into microfilaments that are a major sauctural

element of the cytoskeleton and present in al1 ceils including hemopoieuc cells in varying

amounts (6 1 ) (62).

The human L35 gene encodes a ribosomal protein (protein number 35 associated with the

large ribosomal subunit). It binds to both initiator and elongator tRNAs and is present in

al1 cells including hemopoietic cells (63).

1.6.13 Myeloperoxidase (MPO)

The human myeloperoxidase gene was cloned in 1986 (64) (65) (66). Its product is a

lysosomal enzyme, present within granules of neutrophils and in lesser amounts in

granules of monocytes. It is only synthesized at the prornyelocytic stage of differentiation

in the rnyeloid series of hemopoietic cells (67). The MPO enzyme has potent bactencidal

activity in the presence of HzOz and halide ions. Clinically, it is used as a marker enzyme

for distinguishing acute myeloid leukemia ( A m ) from acute lymphoblastic leukemia

(ALL), although its expression may not be absolutely Iineage-specific (67) (68).

1.6.1.4 a-globin

The two human adult a-globin genes. ai uz, encode a-globin chains that are erythroid-

specific. They form adult human hemoglobin A (HbA) together wirh two p-globin

chains. Hemoglobin is the oxygen-carrying moIecuIe of red blood cells and comprises up

to 90% of total cellular protein (69).

1.6.2 Selected Genes involved in hemopoietic ceil growth and differentiation

1.6.2.1 Rhom botin 2 (RBTNZ)

The RBTN/Ttg gene family is associated with chromosome translocations in T-ce11

leukemia (T-ALL). RBTN 1 or Ttg- 1 was the first to be identified and was then used to

isolate RBTN2 and RBTN3. They encode proteins çontaining two cysteine-rich regions

known as LIM domains believed to hnction as regulators of transcription (70) (71).

RBTN2 is located at a commonly occumng T- ALL translocation breakpoint t ( 1 1 ; 14)

(p 13; q l I ) also involving die T ceIl receptor (TCR) genes (72). Ir encodes a nuclear

protein that lacks identifiable DNA binding motifs. and thus was proposed to modulate

nmscription through protein-protein interactions. It is widely expressed in fetal tissues.

In the adult hemopoietic system. RNA was detected in T-cell. pre-B-ce11 and myeloid

leukemias and human and rnurine normal myeloid progenitor cells (73). Overexpression

of RBTN2 in transgenic mice resulted in T-cell tumors only (74). RBTNZ is critical for

erythroid differentiation. since homozygous RBTN2 nul1 mutation leads to failure of yolk

sac erythropoiesis and embryonic lethality. In vitro differenriauon of yolk sac tissue from

homozygous mutant mice and sequentiaily targeted double-mumt ES cells exhibits a

block in erythroid developrnent (75).

1.6.2.2 CD34

The CD34 gene encodes a glycoprotein of 110 kd that is expressed selectively by human

hemopoietic progenitor cells and vascular endotheliurn (76). Its tùnction is unclear.

althou* there is evidence for involvement in stromal-hemopoieric ceil interaction (77).

1.6.2.3 c-fms

The c-frns proto-oncogene encodes a 972 amino acid transrnembrane glycoprotein,

identified as the M-CSF receptor and exhibiting ligand-induced tyrosine-specific protein

kinase activity (78) (79). Certain mutations in the c-fms gene upregulate its kinase

activity in a ligand-independent rnanner and lead to cell transformation and

tumongenesis. Murine c-fms has 75% arnino acid identity to its human homolog (80). In

early mouse embryos. c-mis is expressed first in placental trophoblasts, then in the yolk

sac. Later, it is resnicted to macrophages (8 1 ) (82). Arnong human hemopoietic cells, c-

mis expression has been observed on the cells of the monocyte/macrophage lineage and

increases with rnatunty of the cells (83) (84). c-fms mRNA has also been detected in a

Hodgkin's lymphoma-derived ce11 line and nonlymphoid leukemias (85).

1.6.2.4 C-Kit

The proto-oncogene c-kit is idenrified as the cellular homolog of the oncogene v-kit

present in the genome of the acutely transforming feline retrovirus HZ4-feline sarcoma

virus (FeSV) which induces multi-cenmc fibrosarcomas in the domestic cat (86). The c-

kit gene is expressed as a single 5kb transcript and encodes a transrnembrane

glycoprotein. which has been classified as CD1 17 (87). It is a member of a family of

tyrosine kinase recepton which includes c - h s (reco_gnizing M-CSF') and the PDGF

recepton (88). c-kit has been shown to be allelic with the murine white spotting (W)

locus. The lieand for the c-kit product has been cloned and mapped at the munne Steel

(5'0 locus and variously desipated as stem ce11 factor. kit ligand. Steel factor. and mast

ce11 growth factor. Both in vivo and in vitro studies indicate that interaction between c-kit

and kit ligand play important roles in hemopoiesis. Mutations at either the W locus or the

SI locus affect the hemopoietic precursor cornparmient and give rise to hypopiastic

anernia and absence of mast cells. The c-kit product is expressed on marrow progenitor

cells. whereas its ligand is expressed and secreted by fibroblastic cells. Besides

hemopoietic tissue. the c-kit product is detected in normal bnin. testis. mammary

epithelium. gut. small vessels. melanocytes and kidney. c-kit protein and mRNA have

also been reponed in various types of hurnan leukemia cells and cell lines. Activation of

c-kit may affect not oniy proliferation but also differentiation of human normal and

leukemia cells (89) (90).

1.6.2.5 Hemopoietic Progenitor Kinase 1 (HPKI)

The HPKl gene encodes a Ser/Thr protein kinase which activates the SAPWJUNK

signaling cascade (91). The human HPKl gene was recently cloned on the basis of its

homology to the murine HPKI (92). It is expressed predominantly in hemopoietic tissues

including bone rnanow. fetal liver. lymph node. spleen. and thymus (91) (92). Its mRNA

is also detected in CD34", CD34-. CD34"CD38*. CD34"CD38'. and CD34'HLA-DR"

populations purified from human BM or fetal liver (92).

1.6.2.6 BcL2 family

Bcl-2 was originally cloned From the breakpoint of a t ( 14: 18) translocation found in

many hurnan B cell lymphomas. It encodes a protein associated with mitochondrial

membranes. the nuclear envelope and endoplasmic reticulum (93). It is expressed at high

levels in lymphomas. chronic 1 ymphoc ytic leukemia. multiple myeloma. and acute and

chronic myeloid leukemias. It is also expressed in hurnan hemopoietic precursors in fetal

thymus, liver, and bone rnarrow (94). but not in terminally differentiated rnyloid cells

(95). Bcl-2 RNA levels increase in B and T cells in response to proliferation signals (96).

Bcl-2 transgenic mice have efevated numbers of pre-B cells, and these cells survive for

prolonged periods in vitro (97). Overexpression of Bcl-2 suppresses programmed ce11

death induced in hemopoietic precursors by growth factor withdrawal and c m prolong the

viability of terminally differentiated neutrophils (98).

An expanding farnily of Bcl-2 homologs has been identified inciuding Bcl-x and Bax.

They are most highly conserved in two regions. the Bcl-2 homology 1 and 2 (BK1 and

BH2) domains. Two forms of Bcl-x mRNA have been observed. The product of a long

RNA species Bcl-XI, which possesses BHl and BH2. represses programmed ce11 death.

A short form Bcl-x,, which Iacks the B W 1 and BH2 domains as a result of alternative

splicing, acts as a promoter of apoptosis through inhibition of the sumival Function of

Bcl-2 and Bcl-xL (98). Both Bcl-xL and Bcl-xs rnay regulate one or more Bcl-2-

dependent or Bcl-2-independent pathways of apoptotic cell death (99) (100). Bax is a

dominant inhibitor of Bcf-2 and Bcl-xt function. It interacts with boch BcI-2 and Bcl-xL

by heterodimerization that is required for die repression of apoptosis. Bcl-2 mutants that

failed to heterodirnerîze with Bax could no longer repress ce[[ death ( 10 1 ).

Heterodimeric painng of ceII death-suppressing and death-promoting mernbers of the

Bcl-2 family appears to be essential for regulation of ce11 survival and ce11 death.

1.6.2.7 pS3

The human p53 gene encodes a 393 amino acid protein in man ( 102). It acts as a

transcriptional regulator, enhancing the expression of genes that contain specific p53-

binding sites and interactinz with a variety of transcription factors to modulate the

expression of other genes ( 103).

The p53 gene was initially classified os an oncoeene since ( i ) p53 protein was elevoted 5-

to 100-fold in many transformed and tumor cell lines and undetectable in normal cells

(104). (ii) mutant p53 clones were capable of cooperating with the ras oncogene to

transform n t embryo fibroblasts ( 105). and (iii) overexpression of mumnt p53 in

established rat cells caused tumorigenicity in nude mice ( 106). In the late 1980's. it was

discovered that the normal. wild-type p53 gene product could suppress the growth of

tumor cells ( 107). Mutation of p53 is a cornmon generic alteration in many human

cancers ( 108). although mutation is unusual in human AiML. Thus. the p53 gene was

classified as a tumor suppressor gene. p53 plays a role in mmor suppression possibly by

arresting ceil proliferation and inducing ce11 death through apoptosis. Previous studies

show that only wild-type p53 is capable of mediating G I arrest in response to a DNA

damage signal induced for example by irradiation ( 109). It is implicated in the

diffcrentiation of pre-B lymphocytes and c m induce apoptosis in hemopoietic cells ( 1 10)

( 1 1 1).

1.6.2.8 WAEl

The human WAFl gene was cloned in 1993 ( 1 12). It encodes a 2 1 kDa protein, p2 I

(WAFIICIPI). This protein is upregulated by p53 and rnediates growth arrest by

inhibiting the action of G 1 cyclin-dependent kinases (CDK). Mice lacking pZ 1

WAFl/CIP 1 developed nomnlly and did not exhibit spontaneous maiignancies during 7

months of observation. but pz 17'- ernbryonic fibroblasts were deficient in their ability to

mest in G1 in response to DNA damage. Introduction of WAFi cDNA suppressed the

growrh of human bnin. h g . and colon tumor cells in culture ( 1 13) ( 1 14).

1.6.2.9 Retinoblastoma (RB)

RB is a Nmor suppressor gene and is expressed ubiquitously in venebrates. Mutations of

both RB alleles occur in retinoblastoma. an ocular childhood tumor. and other human

Nmon (osteosarcorna. small-ce11 lune carcinoma and breast carcinoma) ( 1 15). The gene

encodes a nuclear phosphoprotein (Rb). The hnctional properties of Rb relate to its

phosphoryiation state. Rb is weakly phosphorylated in the G 1 stage and becomes

phosphorylated as cells enter S phase. The phosphorylated form persists dunng G2 and

M phases. Introduction of unphosphorylated Rb into cells in rmly G 1 blocks their

ennance into S phase. These observations sugesr that Rb is invoived in the control of

cell-cycle progression ( 1 16) ( 1 17) ( 1 18). Rb is also involved in munne development.

Mutant mice lacking inmct Rb show defects in neurogenesis and hernopoiesis and do not

survive ( 1 1 9) ( 1 20). RB is expressed predominantly in pluriporential and commined

precursors of the erythroid and megakaryocytic lineages in munne hernopoiesis (3). It

may play an eryrhroid- and stage- specific functional role in normal human hemopoiesis.

Rb mRNA and protein are detected at a low level in enriched lineage-committed

progenitor cells. Expression is susrained in the erythroid Iineage. but sharply down-

modulated in the grmy locytic senes during erythroid and granulopoietic differentiation in

CU lture ( 1 2 1 ).

1.6.2.10 WILMs' Tumor (WT1)

The W M s ' Tumor gene WT1 was cloned frorn die 1 1 p 13 chromosomal locus involved

in this childhood kidney cancer ( 122). it encodes 3 zinc-finger mscription factor and

has a key role in developmental regulation in the kjdney and gonads ( 123).

W ï 1 is expressed in immature epithelial cells, also widely in acure leukemias.

hemopoietic ce11 lines. normal bone marrow. and CD34' progenitor cells ( 124) ( 125)

(126) (127)- WT1 mutations have been found in about 10% of Wilms' tumors and acure

leukemia patients. suggesting that this gene may be involved in tumorigenesis (1 28). The

fact that leukemia ais0 occurs as a second primaq turnor in Wilms' tumor pauents. and

thrit hemopoietic maiipancies are more common in relatives of chikiren with Wilms'

tumor. indicates the close relationship between these two sysremic diseases.

1.7 Thesis objectives and overview

Stem cells are capable of maintaining a constant supply of mature lymphoid and rnyeloid

hemopoietic cells. IdentiQing genes which are responsible for the ansitions from one

differentiation stage to the next will help to understand the rnechanisms dint control

prolifention and differentiation during hemopoietic development.

The main objective of this smdy wûs to establish a sysrem for mapping lineage- and

stage-specific gene expression in human hemopoietic precurson. The approaches of

global PCR and sibling malysis previously developed in the murinr system. formed the

basis of this thesis. Chapter 2 describes rxperiments addressing specific difficulties in

dealing with human BM. the collection of a set of cDNA samples fiom human

myeloidlerythroid precursors. tests of the accuracy of lineage and stage assignments, and

mapping of RNA level expression of selected genes within the human hemopoietic cell

hierarchy. In chapter 3.1 discuss the results and propose future work.

Chapter 2

A System for Analysis of Stage-specific Gene Expression Patterns

in Human Hemopoietic Cells

2.1 Introduction

The aim of studying gene expression at various points in the hemopoietic hierarchy is to

understand the mechanisms chat control proliferarion and differentiation during

hemopoietic developmenr. 1 utilized a sysrem which combines global PCR and sibling

analysis to extend analysis of pne expression ro specific stases in the humnn

hemopoietic hierarchy . The expenmental paradigm is schematically illusmted as

follows:

Sibling cells

bu\& Sibting colonies

A Precursor cONA

L[000000I .Mature Cell cDNA

Human normal bone marrow precunor cells are emiched and plated in pnmary methyl

cellulose cultures. Dunng 2 or 3 days, precursors divide and f o m colony starts

consisting of 4-8 cells. As shown in the nght side of the figure, 1-2 cells from one colony

start are subjected to global RT-PCR amplification and the other sibling cells are replated

individually inro secondary cultures to form colonies. The likely developmentai potential

of each ce11 consumed in the PCR process cm be identified retrospectively by the fates

(including size and lineage content of sibling colonies) of sibling cells if they are uniform.

cDNA samples are nccordingly collected from individual precurson mapped by this

sibling analysis to vanous points in the hemopoietic hierarchy. The left part of the figure

depicts cDNA sarnples each obtnined from 50- 1 00 mature cells presenr in homopneous

colonies of various lineaees after 1 S days of culture. Finally. the set of cDNA samples

from precursors and mature cells is probed for expression of specific transcripts.

Specific challenees in dealing with human BM cells inciude identification of culture

conditions that allow single isolated cells to express their hl1 growth and differentiative

potential: ennchment of precursors having significant growth potenrial: assuring constant

availability of human bone rnarrow cells: and ensuring maximal efficiency of the PCR

reactions applied to single cells. In this chapter are presented the experimental

approaches taken to address these challenges. a description of the cDNA samples

obtained from human bone marrow cells at different stages of development. and results

tiom expenments using the sample set to define expression patterns of a number of genes

in the hierarchy of normal hemopoietic cells.

2.2 Materials and Methods

2.2.1 Cells and Ce11 Lines

Normal human bone marrow were obtained from transplantation donors and peripheral

blood From healthy volunteers at the Princess Margaret Hospital (Toronto, Ontario,

Canada). AML-5 aiid AML- 1 ce11 lines were supplied by the laboratory of Dr. E.

McCulloch at the Ontario Cancer Institute (Toronto, Ontruic. Canada).

2.2.2 Preparation of Mononuclear Cells (MNC)

Human bone marrow samples were diluted with 1 or 2 volumes of Iscove's modified

Dulbecco's medium (IMDM. Gibco.) prior to density cen~rifûgation. 3-7 rnL of the

suspension were layered on 3 mL of 1.077 @cm3 Ficoll density solution (Gibco) at roorn

temperature and centrifuged at 2000 rpm for 20 minutes. The low density cells were

collected from the interface, washed twice with IMDM or phosphate buffered saline

(PBS). centrifuged at 1200 rpm for 10 minutes. and finally resuspended in IMDM to the

requ ired concentration.

2.2.3 Adherent Ce11 Depletion

MNC at a density of 106/rn~ were cultured in 75 mL plastic flasks (Nunc) with

IMDMISOlc FBS. Following a minimum 12 hour incubation at 3 7 ' ~ in 5% COz, the non-

adherent cells were harvested.

2.3.4 Immunological Staining and Ce11 Sorting

MNC or non-dherent MNC at a density of I O ' / ~ L were incubated with 250 p$mL non-

specific human IgG for 20 minutes to sanirate Fc receptors before imrnunological

staining. The cells were incubated for 30 minutes with mouse anri-human CD34 (ICH-3.

Cedarlane Laborntories Limited. Ontario. Canada). R-phycoerythenn (PEI conjugate:

mouse anti-human CD33 (CD33-433. Cedarlane Laboratories Limited. Ontario. Canada),

FITC conjugate: and mouse anti-human CD38 . biotin conjugate (HIT2. Crdarlane

Laboratories Limited. Ontario. Canada): followed by incubation with Streptavidin-

Quanrurn Red labeled Soar anti-mouse Ig (Sigma Chernical Company. St Louis. MO) for

20 minutes. Al1 incubations were perfomed in pre-chilled balanced salt solution

(BSS)/59 newbom bovine serurn (NBS) at O°C (BSS: 153 mM NaCl, 4.5 miM KCI. 1

miM CaCI2, 0.9 m M MgCl. 6 mM glucose. 1 rnM NaH2POJ). After e x h incubation. the

cells were washed twice with BSSIS%NBS.

Cdls were sorted on a FACstar Plus ce11 sorter ( Bccton Dickenson. San Jose. CA) using

an argon laser with an excitation wave Iength of 488 nm. FITC. PE and streptavidin-

Quantum Red sipals were detected through 525.570 and 670 nm bandpass filters

respectively. Sorted populations were collected into IMDbU5% fetal bovine serum (FBS)

in polystyrene tubes pre-coated with FBS. >YO% purie was confmed by resorting.

23.5 Enrichment of CD34' Cells Csing Magnetic Beads

A Dynal CD34 progenitor ce11 selection system (Dynal INC. Lake Success, NY) was used

to isolate CD34' cells (Figure 1 ). Magnetic beads coated with CD34 specific antibody

rnAb 56 1 (Dynabeads M-450 CD34) were iocubated at equal numbers (4x 10') with

MNC for 30 minutes at 4" C with gentle tilt rotation at 10-20 rpm. 10 mL of isolation

buffer (PBS without ~ a " or M ~ " . 2% bovine semm albumin (BSA). 0.6% sodium

citrate. 100 IUmL penicillin-streptornycin) was added and rosettes were segregated in a

magnetic field. The supematant containing non-rosetted cells was aspinted. Rosetted

cells were resuspended in 100 uL of the isolation buffer and an equal volume (Dynal

CD34 Progenitor Cell Selection System Protocol) of polyclonal iintibodies against the

Fab portion of .MOAB 56 1 (DETACHaBEAD CD34 was added and incubated at room

tempenture for 45 minutes. 2 rnL of isolation buffer was added and the detnched beads

accurnulated by placement of the magner. Released CD3J'cells in the supematant were

trmsferred to a fresh tube ruid woshed twice by adding 10 mL isolation buffer and

cenrrifuging at 1000 rpm for10 minutes nt room temperature. The pellet was resuspended

in isolation buffer nt the desired concentration.

Sipunion of m n î i i m IncuôaUon rr)th and-Fab SepuiUon oî ire6 cind fma CeIl8 for d . ( ~ f i ~ t cd18 i n d b0id8

Figure 1 Purification of CD34' cells by rnagnetic beads.

3.2.6 Preparation of Platelet-poor Plasma (PPP)

Normal hurnan penpheral blood was drawn and mixed with heparin (20 u/mL) ( Sigma

Chernical Co. St Louis.MO) before being centrifuged at 3800 rpm for 10 minutes at room

temperature. The collected supernatant was stored at 4 ' ~ for at l e s i 12 hours. and Further

depleted of platelets following an additional centrifugation step.

2.2.7 Preparation of Phyto-haernagglutinin Stimulateci Leukocyte Conditioned

Medium (PHA-LCM)

Normal hurnan peripherai blood was rnixed with heparin (20 umL) and centnfuged at

2000 rpm for 10 minutes nt room ternperature. The buffy-coat containing the leiikocytes

was collected. 5x10~ cells were incubated ai 37OC with 0.5 mL PHA (5 mlivial. Murex

Diagnostics Ltd.. England). 5 rnL FBS (20%). 175 & 2% methyl cellulose. 39.5 mL

IMDM for 6 or 7 days. n i e culture mediumw-PHA-LCM" was harvesred. cenmfuged at

1000 rpm for 10 minutes. and stored at 4OC.

2.2.8 Cryopreservation of marrow cells

Ce11 peilets were suspended in tieezing solution (20% FBS. 10% dimethyl sulfoxide

(DMSO. Sikgna). 70% Hmk's BSS) ai a final concentration of 1 06- io7/rn~. 1 rnL of ceils

was loaded into a pre-cooled tube ( 1.25 rnL disposable cups. SARSTEDT Numbrecht.

Germany). srored at -70" C overnight and then rransferred to liquid nitrogen.

When cryopreserved cells were used, one tube containine the cells was taken from the

liquid nitrogen tank and quickly placed in a room remperature water bath. Before the

suspension was completely thawed. it was mixed with 10 mL phosphate-buffered saiine

(PBS) and cenmfuged at 1200 rpm for 10 minutes at roorn temperature. The cells were

then resuspended in IMDM/S%FBS at the desired concentration.

2.2.9 Colony growth in methyl cellulose cultures

Cells were plated in 35 mm diameter petn dishes (Nunc) and incubated at 37°C in a

humidified atmosphere of 5% CO?. Three sets of semi-solid culture conditions were used

in the experiments and are summarized in Table 1.

Table 1 Components of Three Methyl cellulose Culture Groups.

Reagents Supplier Quantity Set I Set II

(1 5% FBS)

Set III

(5Yo FBS) (30% Human plasma)

+ Chemical Co., Japan ICN 5%

Human pfasma BSA Transferrrin

Siqma 6 mg/mL + Hoechst, Montreal 120 ug/mL +

-

lnsulin Sigma 10 ug/mL + Sandoz. Canada 50 ngImL 4

Genetics Institute, 100 ng/rnL + Boston, MA Genetics Institute, 50 ng1mL 4

Andover, Massa Amqen Inc 30 ng/mL + Sandoz Pharmaag 30 ng/mL + BaselISwitzerland Amqen Inc. 50 nq/rnL + Genetics Institute. 3 O/O + Kirin Brewery Co., 2 u/mL +

rhl L-3

Erythropoietin

PH A-LCM 1 0% - IMDM +

rhiL: recombinant hurnan interleukin. KLCM: conditioned medium of Kit ligand C.MS637: conditioned medium from human bladder carcinoma ce11 line 5637.

Colonies and clusters were defined as aggregates consisting of 50 or more cells and 8 to

50 cells respectively and scored 18-25 days after initiation of the culture. Erythroid bursts

koom BRT-E were defined as multicentric aggregates composed of several subcolonies.

Erythroid colonies frorn CN-E consisted of unicennic azgregates. Both types of

eryrhroid colony were identified by their red color. Mixed colonies consisted of both

erythroblasts and granulocyteg macrophages. Erythroid. granulocytic. macrophage. and

megakaryocyte colonies were discriminated on the basis of their appearance in the

inverted microscope. Identifications were confirmed by evaluation of individual colonies

spread on glass slides by micropipette and stained with May-Grunwald-Giemsa stain.

22.10 Precursor Selection With Thymidine Blockade

NC (1 x 10'lrn~ ) were cuitured in 75 mL plastic flasks with IMDM/S% FBS, SOngimL [L-

3,30ngPmL GM-CSF. 15% CM5637.2UImL Epo and 100 pg'mL thymidine for 24.48,

or 72 hours. Then cells were washed twice with IMDM and cultured in rnethyl cellulose

conditions as described above with the addition of I O pgmL cytidine.

22.11 DNA Purification

AML-5 cells were resuspended at a concentration of 10'celisim~ in ice-cold Tris-CI

EDTA (TE). 10 volumes of lysis buffer (0.5 M EDTA pH 8.0. 100 pg/rnL proteinase K,

and 0.5 % sarcosyl) were added to the cells which were then incubated at 50°C for 3

hours for lysis. DNA was exnacted twice with an equal voiume of phenol and treated

with 100 py'rnL of DNase-îiee RNase at 37°C for 3 hours. After 2 additional extractions

with phenoi/chloroform. DNA was precipitated in 0.3 M sodium acetate. The DNA pellet

was washed with 70% ethanoi. dissolved in distiiled water. and the DNA concentration

was measured by spectrophotometer.

2.2.12 RNA Purification

AML -5 cells ( 10') were pelletted and transferred into 100 pL of buffer D ( 4 M

guanidium thiocyanate. 25 mM sodium citrate. pH 7.0.0.5 % sarcosyi. 0.1 M B-

mercaptoethanol). 10 pL 2 M sodium aceÿite pH 4.0. 100 pL phenol. 20 pL chlorofotm.

The mixture was thoroughly vortexed and cooled on ice for 15 minutes and centrifuged

for 20 minutes at 4OC at 12.000 rc g. The aqueous phase was collected and mixed with an

equal volume of isopropanol. placed at -20°C for at least one hour. and cenuifuged for 10

minutes at 4°C at 12.000 x g. The RNA pellet was resuspended in 4 M LiCl and

centnfuçed for 15 minutes at 1,000 x 2. The RNA pellet again was resuspended in

(DEPC)-treated HIO. precipitated with 1 vol isopropanol and 0.1 vol 2 M sodium acetate

pH 4.0. washed in 70% sthanol. dried. and resuspended in DEPC-treated H20.

2.2.13 Amplification of Poly A mRNA from Single Cells

cDNA lvsis buffer: 32 m M Tris-HCl pH 8.3. 78 m M KCI. 3.1 m M MgCl?, 0.52% NP-40.

cDNA onmer mix: 12.5 m M e x h dNTP. 0.5 0D260.mL ~ l i g o ( d T ) ~ primer.

RNase inhibitors: 1: 1 mixture of RNAguard (Pharmacia Biotech) md Inhibit Ace (5'-3'

Inc. Boulder. CO.). - - - - - - - - - - - - - - - - -

100 ul of 1 st strmd buffer consists of 96 pi of cm~ lysis buffer.2 @ of cDNATnmm

mix fieshly diluted 1 2 4 with HIO. and 2 pi of RNase inhibitors.

A single ce11 was picked with a finely drawn glass micropipette and deposited into a well

of a Terasaici plate (Nunc, Denmark) that contained 4 pL of 1 st strand buffer. The

procedure was observed under a microscope. The suspension was then transferred into a

pre-chilled 0.5 mL tube and incubated at 65OC for 2 minutes to linearize RNA. room

temperature for 3 minutes and on ice for 2 minutes. Reverse transcriptase. 0.5 pL (1: 1

Moloney murine leukemia virus reverse transcriptase and avian reverse transcriptase,

Boehringer Mannheim) was added and incubated at 37OC for 15 minutes and 6S°C for 10

minutes to stop the reaction. A homopolymeric (dA) tract was added to the 3' tail of the

first strand cDNA by adding 4 pL 2X tailing buffer (800 pL 5X BRL Terminal

Transferase Buffer, 30 pL 100 m M dATP, and 1.17 mL H20) with 10 U terminai

deoxynucleotidyl transferase (TdT) (Gibco/BRL) and incubating at 37OC for 45 minutes.

PCR amplification was conducted in the same 0.5 mL tube in a total volume of 40 pL

containing 4 pL 1OX Taq polymense buffer ( [ 00 m M Tris-Cl, 500 miM K I . 25 mM

MgC12, 0.2% Triton-X100 ), 0.8 p L 25 m M of each dNTP. and 2 1 pM PGdT oligo (5' -

ATG TCG TCC AGG CCG CTC TGG ACA AAA TAT GAA TTC dT(24)-3'). The

reaction was started by addition of 5 U Taq polyrnense to tubes pre-heated to 94°C using

a GeneAmp f CR System 9600 thermal cycler (Perkin-Elmer Cor. CT), covered by

minerai oil and finished with 50 cycles of amplification including 5 cycles of 1 minute at

94OC. 15 seconds at 42OC. 1 minute at 72OC. and 45 cycles of I minute at 94OC. 1 minute

at 60°C, L minute at 7Z0C.

2.2.14 Probes

Probes were derived from the corresponding human cDNAs and contained terminal

3'untransIated sequence. Seven of the probes including B-actin. human nbosomal protein

L35, c-kit. RhombotinZ. p53. Retinoblastoma (RB). and hemopoieric precursor

kinase(HPK1) were gifts frorn different laboratories and cloned in different vectors

(Table 2).

Table2 Human cDNA 3'probes from other sources

S ize

8-Actin L-35 c-Kit Rbtnî

Enzyme Name

P53 Rb

Another nine of the probes were generated by PCR using A m - 5 DNA as ternplate

(myeloperoxidase (MPO) fragment) or by RT-PCR using A-=-5 RNA as tempiate

(CD34. c-mis. cc-globin. Wilms' tumor (WT). WAF- 1. Bcl-2. Bcl-xL, and Bax

fra_gnients).

OC1 Dr. Miyamoto OC1 Dr. Minden OCI Dr. Minden OC1 Dr. Minden

HPK 1 Amgen Dr. Hu

in the RT-PCR process. tïrst-strand cDNA was synthesized by incubating 1 pg of A-ML-5

RNA at 37°C for 1 hour in a final volume of 10 pL in reverse umscriptase buffer (RT-

buffer) (50 dl Tris-KCI. pH 8.3.75 mM KCl. 3 mM MgCl? ) containin_g 100 L? of

.Moloney murine leukemia virus ( M-MLV) reverse transcriptase (GIBCOI BRL.

Source

OC1 Dr. Benchimol 1 p25 16 OC1 Dr. Boehmelt 1 pUC 13

1 1500bp

Vector

pUC 18c Ml3 pUC 19 pGEM-7

BamH 1 & EcoR 1 EcoR I & Hind III

EcoRI Pst 1 & BamH 1 Xba I & BamH I Acc 1 & EcoR I

592bp 583bp

450bp 650bp 600bp

Gaithersburg, MD). 25 pM rmdom primers (Pharmacia. Montreal, Que), 15 U RNase

inhibitor (5'-3' Inc., Boulder. CO), 1 O m M dithiothreitol (DDT, GIBCOIBRL), and 250

pM of each deoxyribonucleoside triphosphate (dNTP. Boehnnger, ~Mannheim GmbH.,

Germany). 1 pi of this first strand cDNA was used as template in a final volume of 100

pi PCR containing I O pL IOX Taq polymerase buffer (100 m M Tris-CI. 500 mM KCI. 25

mM MgCl?. 0.2% Triton-X 100 ). 200 pM of d m . 25 p M of wrget-specific primer(s)

and 5U of Taq pdymerase. Amplification was performed through 30 cycles of

denaturation at 94'C for 1 minine. annealing at 50UC for 30 seconds. and elongation at

72°C for 1 minute.

Primer Designer (Scientiftc and Educational Software) was used to select specific oligo

nucleotide primers based on the full length human mRNA sequences in the Grnebank

data base. Oligos were supplied by Amgen Inc.. Boulder. CO. The human cDNA probes

generated by PCR are summarized in Table 3. Fresh PCR products were subcloned into

the p ~ ~ T ' ' II vector (Original TA Cloning Kit. Invitrogen Inc.) by T4 DNA ligue. The

ligation reactions were transfected into shotM competent becteria (Invitrogen hc). Afier

culture in X-Gd LB agar dishes. colorless clones were selected and propagated. Plasrnids

were punfied using Quiagen Kits and digested with EcoRI. The digested products were

electrophoresed on agarose gels (Figure 2). Fragments of the expected sizes were cut

from die gels. frozen ar -20°C for 1 hour. thawed at 37°C for 5 minutes. loaded into

LTLTFWFREE-MC colums (MiIlipore Corporation. Bedford. MA). and cenrrifuged at

high speed for 5 minutes. DNA concentration and purity were determined by

spectrophotometer.

Table3 Human cDNA 3' probes from PCR products

number

MPO 1 Ml7709 1 2342 ( 31 15 / 774bp

5' S m

3' End

Name Primer sequence S ize genebank accession

a-giobin

Srnse GAC TAT ACC AAT CTG CCG AG Antisense GGC GTG ACT GïT AGT TAG GA

Scnse CAT ATC CCC GGG ACT ï T G TCA ACT GC Antisense GTG AAT AAG CAA G U ATC AGG GTG AG

JO0153

Srnse CTC K T GCC GCA ATA CAG GAA CC Antisense CAG AAG AGG CAG CTG GTG ATA AG

Scnse CCA GCT CTG AGC AGA TCA TGA AG Antisense CCA CGT TGA CCT GCT CCA GAA G

7498

Sense CCA AGC TGA GCA CAG AAG ATG G Antisense GCT M T GGT GGC CAA CTG GAG AC

Sznse ATG CTC CAG GTG GCT CTG AGG TG Antisense C.4G TCC AGG CCA GTA TGT TAC AG

7358

Scnse ATA GGG GAT GGT CCA GGA TCT C Anrisense ATC M T ATC CTT GTA AGA TCA ACA CCC

341 bp

-- -

Srnse GCA GGT A I T GGT GAG TCG GAT CG Antisense GAG GAT GTG GTG GAG CAG AGA AGG

Srnse ACA GCT CCT AAG CCA CTG CCT GC htisensc CCG CCC ACT CAG ACT T A TTC AA

Figure 2. Electrophoresis of 9 h u m a 3' cDNA probes which were generzited by PCR, subcfoned into CR^ ï i plasmids and digested by EcoRI. A 100 bp DNA Iadder is shown in the outerrnost lanes.

The identity of each of the 15 probes was confirmed by sequencine.

2.2.15 Southern Blot Hybridizations

Ten microlirers of ampGfodcDJA pro-ct were - electrophoresed - - - - - on a 1.5% agarose gel - - - - - - - - -

in Tns-borate buffer (TBE), denanired in 2X denaturing buffer (0.5 M NaOH / 1.5 M

NaCI). neutralized in 1 M ammonium acetate. nansferred to Hybond M - ~ + nucleic acid

trmsfer membranes (Amersham. LK). and immobilized by UV-irradiation. The filten

were incu bated with prehybridization buffer ( 1.2 M NaCI. Z U ) mM Tris pH 7.4. 16 m M

EDTA. O. 1% Tetrasodium pyrophosphate [Na PPi], 0.2% SDS, 100 pg/mL heparin) at

65OC at least 1.5 hours and then probed with the specific gene fngment of interest labeled

to a high specific activity using '' P and a random primer kit (GibcoiBRL) in

hybridization solution (1.2 M NaCL, 240 m M Tris pH 7.4. 16 m M EDTA. O. 1 % Na PPi,

0.2% SDS, 500 pg/mL heparin. and 10% Dextran sulfate) at 65OC for at least 12 hours.

The filters were washed with 0.2 X SSC (30 m M NaCl, 3 mM sodium citrate. pH 7.0).

0.1 % SDS. and 0.1 % sodium pyrophosphate at 65OC. Auroradiopphy was perfonned

with reflectionm autondiognphy film (NEN Dupont. Boston) to detect hybridization.

23.1 Growth Efficiency in iMethy l Cellulose Cultures

My first objective was to identie culture conditions and ce11 sources that would

eficiently allow single isoiated progenitor cells to express their hl1 growth and

differentimion potential. Since primitive cells are not maximally stimulated by single

growth factors. combinarions of growth factors were explored in this study. Previous

work had demonstrated that Mec cultures supplemented with either 5% FBS or 30%

hurnan PPP (platelet poor plasma) could suppon the zrowth of single rnurine cells and

human bone marrow cells respectively (3) (1 1). Based on these studies. 1 tested various

conditions with 5% FBS. 15% FBS or 30% human PPP in combination with various

growth factors (see Table 1, Materials and Methods) for the stimulation of progenitor

cells in various myeloid linenges at different stages of maturation. 1 also tested simple

purification procedures for ennchment of cells with significant growth potential. MNC

fractionation of whole BM achieved a five-fold enrichment for,colony-forming cells, and

as shown in Table 4. CD34+ selection of marrow MNC yielded a further eight-fold

enrichment.

Table 4. Human Hemopoieiic Coiony Formation In Different Culture Conditions

Population

Fresh MNC

Fresh CD344 Cells

Cells /plate

Culture Coloniedplate Conditions

M E GM G Mixed Total 1 I I 1

15% FBS

5% FBS 14f f .!Y 315i 4.2 3.25IO.S2 25î0.56 0.2Sî0.21

15% FBS 32.72 1.98 23,5& 1.7 'h0.75 4.75*0.% 0.7520.41

M - macrophage: E = erythroid: GM granulocyte and macrophage: G = gnnulocye; and Mixed = erythroid mixed with granulocyte and/or macrophage Results are the mean + SEM of 4 culture plates.

The highesr number of colonies formed h m MNC and CD34' populations was observed

when 30% human PPP was used with iL3. GM-CSF. Epo and PHA-LCM. Although ail

of the expected colony types were observed in ail three conditions. human PPP promoted

the formation of erythroid colonies more efficiently than FBS. FBS used at 5% with IL3.

iL6. IL1 1. G-CSF, GM-CSF. MGDF. KLCM Epo and CM5637 yielded colonies that

were fewer in nurnber and smaller in size than the other two groups. Ahhough the total

number of colonies formed in the presence of 158 FBS with IU, IL6, IL 1 1, G-CSF, GM-

CSF* MGDF* KLCM Epo and CM5637 was fewer than that in the presence of 30%

human PPP. the frcquency and distribution of granulocytes (G), granulocytes and

macrophage (GM), and erythrocytes mixed with granulocytes a d o r macrophages

(Mixed) colonies were comparable if not higher than in 30% human PPP.

These results indicated thar human BM precursors grew bener in 15% FBS and 30%

hurnan PPP conditions. Thus. rhese conditions were used in sibling analysis to initiate

colony srarts and to support the growth of isolated sibling cells in secondary cultures.

Both conditions proved successful in supporting colony formation by single isolated

progenitor cells as detailed below.

Fresh MNC cells and cryopreserved MNC cells from the same donor. 10000/plate. were

culhired in the same conditions. The totaI number of colonies Forrned from fiesh MNC

was higher than the cryopreserved MNC, but the proportions of different colony types

were comparable (Table 1 ). The results indicated that cryopreserved cells would be a

suitable starting point for my purposes.

13.2 Enrichment of the Frequency of ''Useful" Colony Starts

For successful sibling analysis. rnanipulated sibling cells must form colonies to indicate

the developmental potential of the ce11 subjected to PCR. It was essentiai therefore to

maximize the occurrence of colony starts in which sibling cells still have significant

hnher prolifentive potential. In the following discussion of colony starts. 1 will use the

term "useful" to indicate that (1) al1 manipulated siblings fiom a 4 or 8-ce11 colony-start

f om individuai colonies and (2) all of the sibling colonies are identical to each other in

lineage content. The frequency of" useful" coiony stms was determined in cultures of 4

different human BM populations. The results are summarized in Table 5.

Initially. MNC were used for sibling analysis. In total. 103 colony stms were analyzed.

Sibling cells in each colony-start were replated individually into secondq culture. The

colony-forming rate of these sibling cells was poor- only 13 colony starts met the cntena

of "useful" colony stms. Since thymidine treatment was successfully used to enrich for

colony forming cells fiom mouse BM (3). this method w3s tested for its usefuiness on

human MNC. High concentrations of thymidine are lethal to actively cycling cells via

blockade of the pathway generating ÇICTP. When thymidine is removed. primitive

precursors that were initially quiescent may enter the ce11 cycle and proliferate. After 24-

72 houn incubation with thymidine. MNC were washed and cultured in Mec to initiate

colony starts. Only 14 out of 93 colony starts were useful for sibling analysis. a

proportion that was not significantly bener than without thymidine selection. When the

CD34' ce11 population was tested. the kquency of "useful" colony starts improved to

79%.

Table 5. The Frequency of "Usehl" Colony Starts in Populations of Human BM

Population No. of Manipulateci Colony S tarts

- -

MNC

TdR Treated MNC

CD34' Cells

CD34' CD33' Cells 86 CD38'

No. of '6Useful" Colony Starts

Rate of Success (%)

Since primitive hemopoietic precunors are known to express the CD34 antigen. but not

the CD38 and CD33 antigens characteristic of more advanced cells(129) (56). soned

CD34TD33-CD38- cells were also tested. The frequency of useful colony srans from

this population was 72%. not significantly different from that seen with CD34'cells.

Therefore. CD34 selection was sufficient for enriching colony-forming cells to a usable

frequency for sibling analysis.

2.33 Success of Sibiing Analysis

As summarized in Table 6, in total. 405 colony starts were manipulated from CD34+or

CD34+CD33- CD38' populations. Of these. 271 were 4 ce11 colony starts and 134 were 8

ce11 colony s t m . During the process of replating individual sibling cells. some sibling

cells could not be separated from one another or were lost. For these reasons. 45 colony

stans were excluded. 360 colony starts were successfully followed. The rate of

successfbl sibling analysis seen with 4 ce11 colony starts was 80%. The differentiative

fates of al1 siblings from each Ccell start were identical. Sibling cells in 77 8-cells

colony star& fomed individual colonies out of 109 s m s assayed. Al1 sibling colonies

from each 8-ce11 stans h d identical differentiative outcornes except for one start which

produced sibling colonies of differing lineage content. Excluding this instance. the rate of

successful sibling analysis in the group of 8-ce11 starts was 70% (Table 3).

Table 6 The Rate of "Useful" Colony Starts

-- -- - -

NO. of No. of SuccessfÙlIy ~Manipulated Replated Colony Starts Colony Starts

4 Ce11 Colony 27 1 25 1

No. of Rate of " U s e ~ l " Success

Total 1 405

8 Cell -1 Coiony 134 1 09 76 70

6 different sibling outcornes were observed (Table 7). Some cells were bipotential for

granulocytes and macrophages (GM). others were restncted exclusively to neuuophil.

eosinophil, erythroid, or macrophage differentiarion. In 52 s t m . sibling colonies

(referred to as "unknown") could not be identified b y conventional morpho logical

markers (Figure 1). Possible identities included blast cells. dentritic cells or dying cells.

Table 7. Categories of Colony starts and corresponding cDNA Samples

Lineage 1 cDNA Sampies

Since only one coiony-start out of 360 produced non-identical colonies. it is highly likely

that cells subjected to single ce11 PCR would possess the same gowth potential as their

siblings.

Figure l May-GrÜnwald-Giemsa stained ells from coionies grown in methyi cellulose dturrs.

A: Megakaryocytes, B: Eosinophiis, C: Erythroid ce& D: "unknown" cells, E: Macrophage, F: Neutrophils. Original magnification xlûûû

23.4 Parameters Determining the Efficiency of Global PCR Applied to Single Cells

At the beginning of my smdies. problems often occurred with the polyA+ RT-PCR

protocol as originally descnbed (60). YieIds of PCR product were unreliable and false

amplification products were evident which may have been caused by mispnming and

misextension of primers. Therefore. I undertook a series of experirnenu designed to

identify conditions rhat would maximize sensitivity while aiso minimizing the genention

of artefactual products.

The global RT-PCR procedure involves three steps: generation of first strand cDNA on

polyadenylated mRNA templates using reverse transcriptase and oligo(dT) primer.

addition of 3' oligo(dA) to the cDNA strand using terminal transferase. and amplification

of the miled cDNA by cyclic denaturation. annealing, and extension using Taq

polymerase and an oligoo(dT)-containing primer.

I approached the optimization work by first determining conditions for the cDNA

amplification step. and then optimizing the generation of initial cDNA sumds. The

"besf' conditions would be those giving a hi@ yield of the desired product without

nonspecific background due to mispriming or misextension of the pnmers or

misincorporation of nucleotides. Nonspecific background appeared as amplification

product generated in the absence of mRNA template and visible in ethidium bromide

gels. Such background did nor hybridize ceil-specific probes. I varied the annealing

temperature for cDNA amplifkation. and the concentrations of Taq polymerase.

deoxynucleotide triphosphates (dNTPs), and magnesium. I also investigated the

concentrations of reverse transcriptase and oiigo (dT) for generating the first saand

cDNA. Of the panmeters tested, annealing temperature, and the concenaations of

magnesium and oiieo (dT) primer were found to be the most critical.

3.3.4.1 Magnesium

Taq DNA polyrnerase requires fiee magnesium. As well as enzyme activity and fidelity,

the rnagnesiurn concentration affects primer annealing and suand dissociation

temperatures.

In the optimizarion experîments. template cDNA obtained from mouse BM cells by

global RT-PCR was serially diluted From 2x10' molecules to 20 molecules per 40 pL C

reaction. A reaction that was not seeded with cDNA was used as a negative connol. The

amount of template used was expressed in rems of the numbers of cDNA molecules.

The length of amplified product was 300-700 bases. Assurning dim the average size of

the amplified product was 450 bp. 1 ng of amplified cDNA c m be calculated to contain

7x10' molecu1es s shown below:

x 6.023 x 1 on? molecules i mol = 2 x 10' molecules 450 x 680g . mol

The molar weight of 1 bp of double srrand DNA is 680 gimol Avogadro's number is 6.023xlO?~ moiecules/mol

PCR reactions were conducted with magnesium concenations ranging fiorn 1 - 6 mM

with 45 cycles of reamplification. Annealing was tested at 60°C and 42°C. PCR

products were run in 1.7% agarose gels and stained with ethidium bromide. 10" cDNA

molecules of template were loaded on the gel as a positive control. The results are shown

in Figure 2. The arnount of cDNA template is represented in decreasing nurnbers from

2x 109 molecules to zero in each iane. The concentration of magnesium is represented in

increasing increments from the top row to the bonom row. Ampiified PCR products

appear as srneared distributions with most of the product concentrated at the 600 bp

position. In most of the ~ested conditions. even in the absence of cDNA template. non-

specific background was arnplified. More background signal was detected when more

magnesium was present. white reamplification of the trace amount of template was more

efficient at lower rnagnesium concentrations.

2.3.4.2 Annealing Temperature

The temperature required for primer anneaiing depends upon the base composition.

lenm. and concentration of the amplification primers. Increasiq the annealing

temperature enhances annealing specificity and reduces misextension with incorrect

nucleotides at the 3' primer ends. Rearnplification was rnost sensitive and specific nt an

annealing temperature of 60" C rather than 42' C (Figure 2).

On the basis of these results. subsequent amplifications were perforrned with 2 mM

magnesium and 60' C mnealing temperature.

Annealing at 60" C Annealing at 42" C

cDNA template molecules Mg"

Figure 2. Ethidium bromide stained agarose gel of arnpiitïed cDNA products by PCR (35 cycles).

In the leR paneh. the annealhg temperature of PCR was set at 60'~. in the right at 42'~. The num ber above each lane indicates the number of template &NA molecules used, with the exception of the case where 1Ei2 templates were used which was generated in a previous PCR reaction and loaded as a positive controi. A 100 bp DNA ladder is labelled as M (marker). Rows 1 to 6 show different magnesium concentrations. Arnpiified PCR products appear smeared with signais concentrated near 600 bp. The 60 bp primer is also visible.

2.3.4.3 FYimer Concentration

In order to determine the primer concentration that would optimize cDNA synthesis.

oligo (dT) was tested at rhree concentrations with decreasing amounts of RNA templates.

Subsequent PCR amplification was conducted using the previously optimized magnesium

concentration and annealing temperature conditions. The amount of AML-5 RNA used

as template varied from 10 ng to 0.1 pg. A reaction without RNA was performed as the

negative control. RT-PCR products were detected by Southem Blotting and hybridized

with a human b-actin probe. The results are shown in Figure 3. The best amplification

was seen when 25 pM of olieo (dT) was added. At this concentration. 0.1 pg of total

template RNA (lane 6. the smallest amount RNA used in the experiments) could be

amplified. Single ceils contain about 1 pg total RNA and 5% of it is mRNA. Thus the

procedure proved sensitive enough to ampli- mRNA from single cells.

Concentrations of Oligo (dT) primer

Figure 3. Optirnization of concentration of oligo (dm

RT-PCR pmducîs were run on gels, blotted and probed with U~-labelled human p-actin. Oligo (dm primer was tested at three concentrations (50 phi, 25 PM, 12.5 plM). Total RNA derived from ALWL- 5 was amplified by gIobai PCR with 50 cycles as described in Materials and IMethods. Various arnount of template total RICA (10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 0.1 pg) are shown in lanes 1 though 6. Lane 7 is a control reaction that contained al( cornponents of the PCR reaction except for RNA template which was replaced by water. A 100 bp DSA ladder was run in the lane Iabeled "kI".

23.5 Collection of cDNA Samples from hemopoietic cells

In sibling analysis. one or two celis From 4 ce11 or 8 ce11 colony starts are subjected to

global PCR to produce cDNA. The growth and differendation potential of the consurned

cells is identifiec! retrospectively by the fate of their sibling cells. Of the cDNA products

that were generated. 233 hybridized successfully to a human B-actin probe or a human

ribosomal protein L35 probe. The accumuiated precursor sarnple set and the lineage and

stage assignments are summarized in Figure A They were defined as 16 GM. 13

neutrophil. 7 eosinophil. 1 13 erythroid. 5 1 macrophage. and 33 unbown smples.

Additionally, 15 cDNA sarnples were generated from 50- 100 cells taken from terminally

maniring, homogeneous hemopoietic colonies representing 5 distinct lineages. namely

macrophage, neuuophil. eosinophil. megakaryocyre. and erythrocyte.

In total. 242 cDNA sarnples from human BM cells have thus been accurnulated

representing I 2 distinct positions in the hemopoietic differentiation hierarchy. The set

inctudes 27 cDNA samples collected using the same methods by Dr. Harry Atkins. Most

of the samples are from rnonocyric and erythroid precursors. 68 samples were arbitrarily

selected for mapping the expression of a variety of genes by Soudiern blot hybridizaùon

(Figure 4).

Figure 4, Hieranihicd relationships of the hemopoietic cclls sampled For cD1VA.

Each circle represents a stage in the precursor hierarchy. The top circle signifies tripotential precursors. The bottom circles signify mature cells. The circies between are bipotential or unipotential precursors. MF,macrophages; 3, neutroptiils: E, crythroid cells: Eo, eosinophils: .Me, megakaryocytes, Numbers inside of the circles indicate the proportion of sarnples generated that were selected arbitrarily for hybridizlition studies.

2.3.6 Use of the cDNA Sample Set to Define Patterns of Gene Expression in the

' Hemopoictic Hierarchy

The first experiments were designed to determine if trmscnpts known to be constinitively

expressed were successfully captured with the polyA RT-PCR technique.

Two constitutive genes were selected as probes. One is the L35 gene which encodes a

nbosomal protein. The probe hybridized to most of the cDNA samples. panicularly

suongly to erythroid precursor and mature ce11 cDNA (Figure 5A. 5B). The fi-actin gene

encodes a major component of cytoskeletal microfilarnents. Ir also hybndized strongly to

erythroid precursor samples and most of the mature samples. and more variably to

bipotential GM. macrophage. neunophi1 and eosinophil precursors. The results

confimed the presence of cell-derived sequences in the amplified products.

Next. I examined hybridization of probes for two genes having known lineage specificity

of expression. As expected (3). the myeloproxidase (MPO) probe hybridized to

granulocyte and monocyte precursor samples. nlthough the extent of hybridization was

variable sample-to-sample (Figures SA. SB). Similarly. a-giobin expression was detected

only in maruring erythrobiasr sarnples. These results provided supporting cvidence for

the validity of our Iineaze- and stage-assignments. and thus enhanced our confidence that

the system could be used to genente novel information. Therefore. 13 additional genes

known to be involved in hemopoietic cell growth. differentiation or function. were chosen

for mapping on the cDNA sampli: set.

Rhm2 RVA h3s been detected by RT-PCR in CD34'. CD34 cells. and peripheral blood

cells (73). The transcripts are also detecred in both rnouse and human mpotential.

bipotential. and unipotential progenitors and in mature murine colonies of erythrocytes

and rnegakaryocytes (73). Figures 5A. 5C show hybridization of Rbtn2 most snongly and

Figure 5 Gene expression pattern in normal human bone mrirrow cells.

Hybridization of the indicated probes to amplified cDNA samples. Figure SA, results obtained with two cDNA samples kom single tripotential precursors. Figure SB, SC and SD show results with cDNA samples from single bipotential and unipotentinl precursors displayed in the top three rows. The first row consists of 10 samples from bipotential precursors (CM) and 7 samples from neutrophil precursors. The second row consists of 10 samples kom macrophage precursors and 7 f'rorn eosinophil precursors. The third row shows 17 sarnples from erythroid precursars including 5 of BIFU-E occupying Irines 2-5 and 7, and 12 of CFU-E occupying Irine 6 and the rest of the row. 15 cDNA samples derived fiom mature cells in different limages are displayed in the bottom row. The first lane of the first row is a 100 bp DNA ladder, and the Iast lane of the bottom row is a negative controi generated by global RT-PCR without a celt template. GiM = granulocyte and macrophage; N = neutrophile: M = macrophage; Eosi = eosinophile; E = erythroid: Meg = megakaryocyte

Figure 5A

Tripotential Precursors

B-Actin L35 c-kit Rhnt HPK1 W T ~ 5 3 fms

CD34 Bcl-2 Bcl-x Bax Rb MPO a-globin WAF1

Figure 5B

a-Glo bin t '

Figure SC

c-kit

Figure 5D

Bclx

consistentiy to tripotential and committed erythroid precursors. and more weakly to

mature erythroid cells. Hybridization was also observed in 2 of 1 0 macrophage

precunors and 2 of 3 mature macrophage samples. and in mature megakaryocyte samples.

The results support the cntical role for Mm2 in erythroid differentiation identified by

gene ablation in the mouse (75).

1-5% of human rnarrow cells express detectable CD34 antigen. these do not express

terminal differentiation markers. and CD34 is not detected on mature penpheral blood

cells ( 1 30). Marrow cells isolated on the basis of expression of surface CD34 antigen are

ennched for colon y-fonnin_e cells of various kinds, and contain quantitative1 y the great

rnajority of the CFC present in unseparated marrow ( 1 3 1). The available data establish

that expression of CD34 is chmcteristic of mmow cells with sig~ificant growth

potential. There has been no relevant analysis of expression in normal cells at the M A

level. As shown in Figure SC. hybridization of a CD34 probe was detected to amplified

cDNA from 3 of 5 BFU-E and 2 of 12 CN-E. md more weakly also to cDNA kom

maturing erythroblasts. but not to other committed or piuripotential precursors. The

absence of hybndization to most of the precursor types was unexepected. The result rn-

reflect a limitation in sensitivity of detection. or dom-regulation of RNA expression in

the particuIar cyrokine environment used to grow the tested cells.

c-fms RNA is known to be expressed in normal human monocytes (83). in less

differentiated mmow cells presumed to be rnonoblasts ( 132). in human monoblastic ce11

lines (85) and in leukernic blasrs from most patients with AML ( 133). In the mouse.

receptor protein expression is confined in the bone mmow to cells of the monocytic

lineage (80). RNA transcripts are detected in munne committed monocyte precunon as

well as in mature macrophages and not in precursor cells commined to other lineages (3).

but inniguingly are also detectable in multipotential precursor ceils ( 134). As shown in

Figures SA and 5C. hybridization of a c-hns probe was detected in I of 3 mature

macrophages. but not in rheir committed precursors. Transcripts were also detected in 1

of 7 rosinophil precursors. Strong hybndization was also observed to the 2 samples from

tripotential precursor cells. n i e result recapitulates the findings obtained in the mouse

system. indicating that c-fms RNA is expressed strongly in cultured muitipotentiai ceils.

is downregulared as they progess in commitrnent. and rises again in terminally maturing

macrophaees.

C-kit protein is detectable on 1 - 1% of low density mononuclear cells in human mmow

(89). and most of these crlls are CD34'. The c-kit' hction comprises as many as half of

the CFC (135). c-kit RNA is detected in primary AiML biast populations and in leukemic

ceil lines (89). but has not been documented in normal mmow cells. Its expression was

difficult to map in the cDNA set obtained from singie murine precursor cells because of

the AT-richness of the 3' LTR (3). On the basis of the protein d m on sorted cells. RNA

expression w u expected in early human hemopoietic precursors but not in maturing cells.

Figures 5A. 5C show hybndization of a 3' human c-kit probe to most of my cDNA

sarnples. including terrninally mature cells. The result was unexpected. raising concem

about the specificity of the probe. Scannine of the probe sequence for possibie repeat

motifs or similarity to other genes, and hybndization tests to Northern and Southern blots

of human RNA and DNA. would be helpful in determining whether specificity is

questionable or not.

The hemopoietic progenitor kinase 1 (HPK 1 ) gene encodes a serineithreonine kinase

(9 1). Xlthough the protein plays a role in signal transduction. its roIe in hemopoietic

differentiation and proliferation is not yet known. Human HPK 1 expression was detected

predominantly in hemopoietic cells including BM. lymph node md thymus (92). RNA

was detected by RT-PCR in CD34'. CD34-. CD34*CD38'.CD34'CD38-. and

CD34'HLA'DR' subpopulations of normal BM (92). Strong HPKl expression was

detected throughour the cDNA sample set in al1 tested rnyloid precurson and mature cells

(Figure SA. SD) .

Bci-2 protein has been detected immunohistochemically in CD3bCD38-humm rnarrow

ceil populations highly enriched in CFC. The small ce11 subset of this fraction was

negative for Bcl-2 but positive for Bcl-x (98). RNA-level expression of Bcl-2 in normal

marrow cells has not been reported. Bcl-x RNA was detected in both CD3-k and 0 3 4 -

marrow populations by RT-PCR (98). B u protein has bcen detected in normal rnarrow

low density cells ( 136). but RNA data have not - bren reported. In my analysis. no

hybridization was detected to probes for Bcl-2. Bcl-x or B a (FigureSA. 5D). dthough

the samr probes dencted the expected trmscnpts on Nonhem blots prepared from the

AML- 1 and AiML-5 leukemia ceil Iines (data not shown). The faihre of detection could

refiect a sensitivity limir of the Southem blotting approach and funher experimentation

by specific RT-PCR may be required.

There are contradictory reports as to the presence or absence of p53 protein in normal

hurnan CD3C marrow cells ( 137) ( 138). Expression of p53 protein has been documented

in mature monocytes. neutrophils. B and T lymphocytes ( 138). and RNA in quiescent

monocytes and stimulated lymphocytes ( l38). As shown in Figure 5A and 5D. p53 RNA

expression WM detected in tripotential ( 2 2 ) and commitred erythroid precursors (1115

BN-E. 6i 12 CN-E) and variably in most terrninally maniring cells in al1 tested lineages.

but was below detection limit in eranulocytic and monocytic precursors.

Expression of WAFlipll RNA has been derecred in normal BiM mononuc1em cells,

myeloid and lymphoid ce11 lines. and primvy leukemia cells ( 139). My probe hybridized

to cDNA samptes From mature macrophages (2'3) and rnegakaryocytes ( l i3 ) (Figure 5D).

but not to cDNA from precursor cells except for 2 of 17 erythroid samples. tts expression

was thus seen mainly in terrninally mature celis with little funher proliferatîve potencial.

and only in sarnples chat were also positive for p53 RNA.

Rb mRNA is reponed not to br rxpressed in a CD3.1' human marrow kriction e ~ c h e d

for plunpotenrial CFC. to be upre_oulated during subsequent generation of cornmirted

erythroid and granuiocytic CFC in culture. and to bc sustruned through erythropoietic but

downregulated during granulopoietic differentiation ( 1 2 1 ). The analysis was performed

using a sensitive RT-PCR approach. In cDNA samples from rnunne hemopoietic

precursor cells. RB mRNA expression was detected by blot hybridization in piuripotential

cells but was similarly sustained only into the erythroid and megakaryocyte linea-s (3).

In rny analysis, hybridization was detected only in a single eosinophilic precunor ceil

(Figure 5D). Funher experimentation would be needed to validate the sensitivity of the

probe. including positive controls (Southern and Nonhem blors). and possibly analysis iit

higher sensitivity by specitic RT-PCR.

Expression of WT mRNA has been detected by RT-PCR in prima- A.ML populations

'and in hemopoietic ce11 lints. but not in low density mononuclear celis from normal

mmow ( 1%) ( 117). For rhat reason. expression was proposed as a valuable indicator of

residual disease. More recently. expression was also detecred in CD34' but not CD34

low density cells fiom normal rnarrow ( 128). As shown in Figure 5D. expression was

readily drtectable in nearly ail our samples regardless of maturation stage or lineage.

This result will require confirmation with Northern and Southem blot controls for

specificity of the probe.

Chapter 3

Discussion and Future Work

As stem cells produce daughter cells that differentiate dong each lineage. it is Iikely that

different portions of the genorne are selectively expressed or repressed in each cell type.

Each ce11 must "know" which genes to express. how actively to express them. and when

to express thern. In order to identifj these patterns. it wouid be necessary to have

homogeneous populations of early hemopoietic cells at sequential stages of

differentiation dong the differenr lineages. Since current methods are nor able to purifj

different kinds of precursors ro homogeneity. knowledee of genr expression in the

hemopoietic system is usually obtained by analyzing ce11 populations or ce11 lines.

Analysis of p e expression patterns in single cells offers a means of guaranteeing

homogeneity.

At the time this study bezan. the feasibiiity of global RT-PCR on single cells with

identification by sibling fates had already been demonstrated with murine hemopoietic

cells. This success stimulared the idea of studying human hemopoietic cells. cDNA

smpies collected Born human BM cells in different stages of development could be used

to extend information on stage-specificity of gene expression. it also has potential for

generating new insights into disordered States such as leukemia. When the target shifted

from mouse rnarrow to human rnarrow. new challenges emerged: culture of isolated

human marrow cells in rnethyl cellulose conditions. efficiency of sibling analysis.

availability of human marrow. and specific conditions of global PCR.

One of achievements in this thesis was to identify semi-solid culture conditions that

suppon the expression of the hl1 growth and differentiation potential of single isolated

hurnan BM precursor cells. Using the optimized conditions. the most frequent colonies

obtained in primiuy marrow cultures were erythroid. followed by monocyte/macrophage.

occasional GM. G. mixed. and mepkaryocyte colonies. M a t ce11 colonies were not

idenrified. The proponion of colonies formed was maintained regardless of any

additional precursor ennchment steps. Therefore. i t is not surprising that the types of

sibling colonies obtained in second. cultures were simiiarly distributed. Unfomnately.

rnixed colonies. mu t ce11 colonies and meeakriryocyte colonies were not identified in rny

sibling analysis. and corresponding cDNA samples were not obrained.

Ennchment of bone marrow precursors was required to obtain a usable frequency of

colony formetion by sibling cells. About eighty percent of colony stms selected tiom

CD3C or CD34'CD33-CD38' populations formed colonies in sibling analysis.

CD3CCD33'CD38- seiection had no advantage over the simple selection of CD%' cells.

It has been shown that cryopreserved human bone marrow cells reconstitute recipient

hemopoiesis safely and satisfactorily ( 1 JO). My study was able to be carried out with a

continuous supply of CD34' or CD34'CD33'CD38' populations by using cryopreserved

ce11 technoiogy.

Initially, an ofien encountered problem with global RT-PCR was the occurrence of

artehctual amplification products. In negative control samples that were set up without

cDNA or cells as template. B-actin or L35 hybridization signals were detected. These

were likely due to exogenous DNA contamination or caq-over of DNA fiom previously

ompiified templates. Various methods were ndopted to eiiminate the risk of

contamination. 4 separate biosafety hood with UV germicidal lamps and sets of supplies

and pipetton were used exclusively for setting up amplification reactions: positive

displacement pipettes were used in the whole process; al1 reagents including DNA and

RNA free water were aliquoted to minimize the number of repeated sarnplings and stored

in a PCR product free area. These methods and precautions effectively eliminated

contamination. Another problem in PCR was the presence of non-specific background

amplification that could be due to mispriming or misextension of the primers. Increasing

the annealing temperature enhances discrimination aeainst incorrectly annealed primes

and reduces misextension of incorrect nucleotides at the 3' end of primers. The results of

experiments for optimization of PCR conditions in this study demonsuûted that high

mneding temperatures were important for specific amplification. Under optimized

conditions. the PCR protocol maintained high stringency without losing efficiency.

By overcoming the difficulties in dealing with human mmow samples, the most

imporiant achievement of this study was the successful collection of PCR products from

single precursors and homogeneous populations of mature cells from human BM. The

cDNA set was tested by hybridization to probes for constitutive genes (p-actin, L35), and

for stage- and lineqe-special genes (MPO. a-globin). The results confirmed the

specificity and reliability of amplification and the accuncy of the assignments. Finally.

the cDNA set was used to gain new information about a set of genes having regulating

roles in hemopoiesis. Some of the results confirmed specificiries reported previously.

such as the expression of RBTNZ mainly in erythroid precursors. Other results were

unexpected. such 3s the absence of hybridization of probes for Bcl-2 family genes over

the whole sarnple set. cven though their expression is detectable in normal hemopoietic

tissue. It is possible that transcnpts were indeed represented in the arnplified cDNA, but

ar levels below the threshold of detection by Southern blotting. It has recently been

rstablished in our lab ( 134) thnt transcnpts for most cytokine receptor also escape

detection on blots. but cm be shown to be present by specific secondary PCR

amplification of the p h a v global cDNA. Further. it is possible diar the specific

conditions of culture used ta genente the original colony stms r n q have favoured down-

regufation of Bcl-2 hrnily transcnpt levels.

A saiking feature of our cDNA set is the cell-to-ce11 variation of cnnscript abundance

even in samples of the same iineage and same stage. Similar results were reponed by

othes usine the same technology (3) ( 141) (142). Since large fluctuations in expression

were not seen when global PCR was applied to lysates of pooled cells in dilutions

equivalent to less than one ce11 (3). it appears that the variation does not xise From the

PCR procedure itself. However. cell damage or differential degradation of RNA

amscripts during processing cannot be excluded. On the other hand. the variations may

represent normal heterogeneity ce11 to cell. Regardless of the cause of the variation. it is

necessary to analyze gene expression nt any particular developmental stage with adequate

numbers of individual cDNA samples to minimize interpretationai bias.

In summary. combining the strategies of global PCR in single cells with sibling analysis.

this thesis established a system for study of specific gene expression in normal human

hemopoietic ceils. Since the approach is carried out at a single ce11 level. it provides a

unique way to gain information on the expression of known genes in the human

hemopoietic lineage at different stages that could not be obtained by analysis of mixed

populations. It also provides a mode1 for malyzing and cornparhg jene expression

patterns in diseases such as leukemia.

Future work will include. first. continued generation of cDNA sampies. Since more

primitive precursors may not be physiologically responsive to conventional hemopoietic

CSFs and require the stimulus of other growth factors. funher optimization of the semi-

solid culture conditions will probably be needed to capture more primitive. multipotential

precursors. as well as basophil and megakaryocyte precursors. Second. subuaction and

cloning of novel genes cm be carried out usine the eaisting collection of cDNA samples.

Third. there is scope for improvernent of the global PCR method. Globai PCR of single

cells currently only yields 300-700 bp of 3' sequence. and may not include any coding

sequence. If longer cDNA c m be generated from single cells. it wouid be easier to

compare the subtracted sequences with known sequences in the available sequence data

base. Recent advances invoiving improved reverse transcriptase enzymes and protocois

(143). together with the use of proofreading 3' exonucleases in the PCR reaction (1441,

could probably be incorporated into our global RT-PCR procedure to yield significantly

longer PCR products.

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