Normal and Leukemic Human Marrow Cells

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1973 42: 701-710

Interacting Cell Populations Affecting Granulopoietic Colony Formation by Normal and Leukemic Human Marrow Cells H. A. Messner, J. E. Till and E. A. McCulloch

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Interacting

Cell

Granulopoietic

Populations

Colons Leukemic

Human

By H. A. Messner,

Marrow was

from

28

separated

plastic

ir:to to

be

(CSA)

than

suspensions,

useful

for

reconstitution

C

added

CSA

procedures

colony

used to

unsepa-

and

assays.

therefore

the

presence.

in the

to

in culture

both

CSA-producing

The

value

factors

of the

could

assessment sponding, the present technique improve

culture

be

cells,

and

be abnormal

Iroi;i

t/i’

from

cells,

were

efficiency

with

procedures

were

acute

to

leukemia

In all four

was

from

to

a defect

in

demonstrated.

in human marrow formation is influenced

derived

four

prior

instances.

cells

for

applied

other

hemic

form by cells.5

in respect to molecular Size and may activity” (CSA).59 In human marCSA-responsive granulopoietic colonythe growth of’ individual marrow specion

concentration

amount

would

of CF’U-C

of exogenous be

independently.

enhanced 1-laskill

CSA if the

but

also

in the

various

et al.’#{176} have

on

cultures. interacting

attempted

such

to glass or plastic. Using human CSA, to develop to

provide

in acute

/n.siitule

evidence

it was methods

that

the

the

O,ziario

possible to for CSA-

latter

cell

class

leukemia.

Alec/lea!

o/

preliminary

this method quantitative

rein the

Seie,ue.

Uniiersiti

vi

Toro,zto,

aizil

((i1z((’r

I,zsiituie.

Canada.

Submitted

Januar,

Supported

h,

Ontario

H. A. Ontario

the

Omitario

/973

(‘ancer Cancer

Vol.

Science, h,

I973.

Grime

revised

Jrom

M T-142()

Treatment

Messner,

o/ Medical

22.

Grain

Cancer

the

Blood.

only the

system

assessed

ofadherence the assay for

Toronto.

and

derived

using density centrifugation to separate human marrow into stimulating, or inhibiting cell populations. In the studies reported paper, CFU-C were separated from CSA-producing cells by

producing may

not

cells

cells marrow Assay

cells

in-

unseparated

colony-forming

CSA-producing

of’ molecules

is dependent

restore

ofgranulopoiesis media’ 4; colony

These active materials are heterogeneous be collectively termed “colony-stimulating row, CSA-producing cells coexist with forming cells (CFU-C)’#{176}: accordingly, mens

of

populations.

patients

used

cultures,

to

treatment.

OM MITTED PROG ENITORS colonies in viscid or semisolid

adherent

numbers

NA

Either

of irradiated

CSA-producing

Quantitative were

or

varying

colony-

cells.

numbers

marrow,

were

and

Cells

CSA-producing

creasing

adherent for

on

or

Normal

E. A. McCulloch

assay

glass

and

dependent

activity

J. E. Till. and

populations

culture

marrow

proved

NA

more

in

stimulating rated

(NA)

The

formation

to

by

Marrow

individuals

adherence

nonadherent

populations. found

nonleukemic by

Affecting

Formation

M.D.:

and

Research

Graduate

Institute. Institute. L’niversitv & Stratton.

42. No. 5 INovember),

Medical

Toronto, of

Toronto

/973;

Institute

Canada.

the

of

Medical

E. the

A.

Ontario

26.

April

Council

and

J. E. Till,

Canada. and

accepted

Research

Foundation,

Student,

Toronto.

2/.

Marc/i the

National

Ph.D.:

Cancer

Science, M.D., Institute.

Grant

236

Institute

lJniversitv

Divisio,z

McCulloch, Cancer

/973.

Canada.

o/

of

o/

loronto.

Biological

F.R.C.P.(C): Toronto.

from

the

of Canada.

and

Research. Institute

Canada.

Inc.

1973

701


702

MESSNER.

MATERIALS

AND

28 nonleukemic

individuals

TILL

McCULLOCH

AND

METHODS

Patients Marrow

was

using

an IBM

the table that

separator.

marrows

of the from

the

mia.

In addition,

of all

FCS

plated

over

of

I. It may

conditions

formation

in culture. results

did

the

of absence

of

leukemia

patients

in Table

one

donor from

included,

but

Accordingly,

not

was studied

are listed

In cell

be seen

were

the

basis

assessment. as a white

available: acute

of

philic

granules.

indicate

the any

bias

a diagnosis

of

prior

initiation

to the

leuke-

2.

origin

30”,,

mononuclear

of these

total)

are

Colony

are

difficult

are

of each with

usually

excluded from

No of

From

per

10

the

No. Cells

determined

that

Used

by various

large are

eosino-

peroxidase

because

their

the

loose

significance

colonies

from

as Sources

at

2 The

at

with

Further, similar

on cellulose

peroxidase.

counts

background.

28 Patients

and Nonadherent

Mean ol.

Patients

l’rom

methyl

neutrophils cells

cells,

colonies

performed

been or

cells

mononuclear

CFUC/105

Diagnosis

has

with 7 days.5

as target

in

stain

morphologically

by Marrow

were

peroxidase-positive

vaeuolated

unlike

Adherent

type

Wright’s

for

incubation,

seen

peroxidase-positive

to distinguish

unknown:

Formation

counts

types

contained

large

cell colonies them

The

colony

contained

10,,

of

microscope.

either

medium

cultures cells

14 days

were

Routinely,

culture the

nonadherent 12

progenitors suspensions

medium.

layering

incubating or

composition stained

aspirated alpha

by

and Alter

distinct

cellular

contained

colonies 1.

an inverted

of the

produced

to 30’,.

granulopoietic ol

FCS, and

20,, was

coats

suspensions up

colonies

90,

ol’ten makes

Table

using

to measure

buffy

in agar

control

Approximately to

CSA

morphologically . 12 The

(60’,,

Up

Such

arrangement

CSA. immobilized

scored

used

l’rom

cellulose,

concentrations

individual

was

obtained

methyl

without

Three

maturation.

et al.5

cells

unseparated

marrow

from

colonies

stages

and

were

human cells

negative.

on

with

Iscove

leukocytes

plates.

of

and

using

of 20 cells

examining

of

in increasing

duplicate

majority

only

for these

ol0.8”,,

with

studies,

wasadded

cultures

colony

on

clinical

to serve

in Table

effect

patients

marrow

peripheral

In sensitivity

least

method

Brietly,

concentrations

the cells were

in excess

is summarized of nonleukemic

selected tour

of

was

a variety

as it became

findings

of the

(CFU-C). in final

CSA

from

hematologic

A modification

20’,,

patients.

who

(‘FIJ-(’

in culture plated

a specilic

as part

volunteer

material

with

was used

patients

marrow

The

ssat’ for

had

a normal

patient

patients

diagnoses

inclusion

from

The

from

these

from

of therapy.

from

was obtained

cell

that

none

material

A

obtained

the marrow

instance

mouse

of

Cells Nucleated

20%

CSA

gol. No.

Lowest per

Cells

10

Highest gol. No.

Cells

per

10

Cells

Iron def. anemia

7

38

67

23

Polycythemia rubravera

2

33

25

41

Erythrocytosis

1

57

-

-

Thalass.

1

39

-

-

mm.

Bronchogenic

5

19

3

21

11 11

26

Ca.prostrate Lymphomat

5

33

17

60

Cirrh. hop.

2

19

10

28

Rheum.arthr.

1

20

-

-

Normal

1

36

-

-

ca.

-

Morphological

of the bone

marrow

t Morphological

examination

of the bone

marrow

specimens

did not

28

reveal

any

metastatic

involvement

in any of the patients.

examination

of the bone

cytes in one of the patients. Colony

growth

marrow in this

specimens individual

showed yielded

minimal

infiltration

60 colonies per 1

with

lympho-

nucleated

cells.


NORMAL

AND

LEUKEMIC

HUMAN

MARROW

CELLS

a)

U)

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U) 0

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MESSNER,

704

their

3

origin

in human

marrow

ture.

counting

This

Separation the

basis

of

marrow total

convention

of the their

technique

by incubating

suspended

in

10 ml

incubation

2 x

CO2 to keep the pH constant adhered,

supernatant of

input

plastic

was

after

cubating

cells

were

from

l0

tissue

was

carried

removed

this

recovered

culture

Irradiation

(TC

at

Granulocyte

colonies

lyse

after

20 days

in cul-

et al.4

used

to separate

37’C

incubated

for

considered

the

NA in

3001)

for

cell I ml

cells

20#{176}c FCS

under

The

hr

in

preparation. of

alpha

30 mm.

more and

petri

Adherent with

supernatant

cells was

medium. The were recovered

dish,

1 x

for

a mixture

of

cells

that

in

the

50#{176},, and

were FCS

10

dishes

Cells

between

200,,

from

than petri

containing

Usually

on

separated

glass

supernatant,

medium

The

times with alpha l0#{176} of input cells

not

conditions

cells.

suspensions

were

100-mm

a second

be NA

cell

cells and

in

humidified

2 to

marrow

(NA)

marrow

with

at levels of 7.2-7.3.

cells

in

dishes

irradiated

Separation

in a 37Cs irradiator

ofHurnan

Nonadherent obtained from the NA populations where the mean

Marrow

prepared

by

in 35-mm

decanted

and

80% in-

Falcon discarded,

layer was as adherent

then used cells.

Bruce,

Webb.15

as ad-

designed

populations

by Cunningham,

LTS

were a single

prepared experiment,

is compared with that of number of colonies observed that a high proportion NA populations; the

greatly

enriched

for

the view that the separation for colony formation.

2 shows

the

colony-forming

from the

unseparated is shown

marrow plating

suspensions efficiency

marrow in as a function

(80#{176}c)of the original adherence procedure did

CFU-C.

Further,

in the presence of 20#{176}c CSA, the tJA between cell number plated and colony

sistent with requirements

and

by Adherence

(NA) populations 28 patients. For

number. It is evident was recovered in the

Figure

they

McCULLOCH

Procedure

were

pension, tionship

observed.

until

Nonadherent

f#{216}7. nucleated

were

marrow

was

RESU

yield

been

by Brown

medium

and

and the adherent layer washed three herent cells. Usually between 2% and

Cells

not feature

plastic.

out

incubation

to I0

or

of alpha

air

not

used

Mosier

glass

The

had

has

characteristic been

of

to

30 mm. and

colonies

their

has also

adherence

suspensions ce!ls

granulocyte

to retain

AND

by Adherence

modification

A

from

appear

TILL

procedure efficiency

like

population formation. did of the

the

yielded This not original

of

Fig. 1, of cell

suspension not usually original

sus-

a linear r#{233}lafinding is con-

change

the

marrow

cultural suspen-

a)

0. C.. ‘C

0. 5)

a) 0

Fig. 1. Colony formation by unseparated (open circles) and nonadherent (closed circles) normal marrow cells function of cell number. Th. lines

asa 3sl0 Cells per plate

were

of best fit through the origin tained using regression analysis.20

ob-


NORMAL

AND

LEUKEMIC

HUMAN

MARROW

CELLS

705

0

200

200

ISO

iso

0

60

60

40

0

t2O

40

0

I20

0

I00

.

o

8o

:

oj #{149}

0

$

A

0

60

o

:

0

I

.

o

0

8

40

;.

2:

0 0

!:s#{176}

#{149}

40

0

0.100

o 60

0

0

610’

B

Cells per plate

Cells per plate

Fig. 2. (A) Colony formation in unseparated marrow cells suspensions from 28 individuals used as sources of adherent and nonadherent cells. The number of colonies obtained in the presence of 20% CSA (open circles) and the absence of CSA (closed circles) is plotted as a function of cell number. (B) Colony formation by nonadherent marrow cells. The number of colonies in the presence (open circles) and absence of CSA (closed circles) is plotted as a function of cell number.

sions (panel or without and

may

ated

A) and the NA cells CSA. The wide spread reflect

marrow,

every instance required for into

the

the

heterogeneous

colony

formation

(panel colony

counts,

(panel B) for all of the specimens of values observed is evident patient was

An Assayfor The

macrophage

colony

recovery

formation

of

ability

without

populations

of

added

CSA,

NA but

an improved unseparated

ence of increasing concentrations number was insensitive to added

was

cells, yielding assay and

value

four

marrow tions

of the

different cells

were

also

low

or

procedure suspensions

in

almost

almost not

always included

absent

in

was

NA

effective in while yield-

technique

of CSA. For CSA, although

at varying

tested

is illustrated

preparations

of CSA

concentrations:

on 3 x l0

NA

cells.

markedly

reduced

in

numerous

colonies

in its pres-

assay plated

is illustrated in the pres-

for NA

NA cells, however, a linear increase in colony creasing concentrations of CSA. Accordingly, whole marrow for the assessment of CSA. The

of CSA was B). Although

separation the marrow

unsepar-

CSA

of CFU-C.

ence, provided the basis for in Fig. 3; in the experiment

4A,

for

added

with plot

CSA

availability

forming

However,

without

A); in contrast, the addition formation by NA cells (panel

cell suspensions. It is concluded that the removing a cellular source of CSA from ing an acceptable

population.

obtained

plated from the

CSA. cells

the unseparated cells, colony size increased. number was NA cells are

in Fig. were

The were

added

in those It is evident

4. In the

from

4B, the

colony For the

observed with inmore useful than experiments

to cultures of Fig.

colony-

of the

figure,

of Fig. unseparated

same that

preparathe

test


MESSNER,

706

TILL,

AND

McCULLOCH

a’ 0

0. a’

0. 0

Fig. 3. IS

Percent

on NA cells was successf’ul A ssat’ for Just be

used

problem. 2 x lO

ranked only

(‘SA

as NA to

-

cells

assay

CSA

the four preparations, in identifying a preparation

Producing provide

of CSA

while

on colony

10 unsepabone marrow

the test on unseparated with very low activity.

cells

(‘el/s a suitable

CSA-producing

basis cells.

In the first, heavily irradiated NA cells and the mixture tested

suIts of two such experiments are presented shown in Fig. 8, below. A linear relationship added irradiated marrow cells and colony tion was sufficient these experiments

Effect

formation by 3 x rated or nonadherent cells.

to abolish colony are interpreted

for

an

assay

for

We

have

used

(900 for

rads) marrow colony-forming

two

CSA,

by the marrow a measure

may

the

cells were capacity.

added The

to re-

experiment i the number of dose of radia.

cells (see Fig. of’ CSA-producing

a) -

0.

‘a 0. a)

Fig. 4. (A) Effect of four different CSA preparations on colony formation by unseparated marrow cells. Suspensions of 3 x 106 unseparated marrow cells were plated with and without increasing amounts of CSA up to concentrations of 20% per dish. (B) Effect of the same four CSA preparations (A) on colony formation by suspensions of3x i0 NA cells.

also to

in Fig. 5, and a third was found between formation: since the

formation as providing

they

approaches

Percent CSA

5).


NORMAL

AND

LEUKEMIC

HUMAN

MARROW

CELLS

707

Fig. 5. Influence of irradiated marrow and peripheral blood cells on colony formation by nonadherent marrow cells. The open circles (patient 1 ) and open squares (patient 2) represent individual experiments where NA cells were mixed with irradiated (900 rads) autologous marrow. The open triangles demonstrate the influence of homologous irradiated peripheral blood cells when added to NA cells of patient 2. The closed symbols show colony formation when 20% CSA was used in addition to irradiated cells. Irradiation controls are indicated. The values for colony formation by NA cells in the absence of either CSA or irradiated cells are shown at the origin.

capacity added

to

of the marrow under test. As a control the cell mixture: no cumulative effect cells

marrow

was

In a second con

tissue

and

varying

dishes

and

methods

derived from nonadherent

numbers

section.

The dishes were incubated results of one experiment

of marrow

nonadherent To

the

varying amounts of’ marrow, cells in methyl cellulose, 20,,

experiments are creased linearly

f’or each of CSA

cells

pr

plate

point, 20,, CSA was and irradiated bone

observed.

approach,

culture

materials

cells: for a plateau

Irradiated

cells dishes,

cells

removed now

were as

plated

in

described

containing

Fal-

in

the

adherent

cells

were added suspensions of 3 x l0 f’etal calf serum, and alpha medium.

for 12 14 days and colony formation assessed. The of’ this design is presented in Fig. 6 and four further

shown in Fig. 7. Colony formation with the number of marrow cells

values in excess at levels in the

of’ 106 original range of’that designs containing

described CFU-C,

taming

cells

devoid

but

NA cells inof adherent

marrow cells, colony formation found f’or NA cells plated with

Both the experimental which NA populations, CSA-producing

from 3 x l0 used as a source

above were

consist mixed

of with

of’ colony-f’orming

20,

reached CSA.

reconstitutions populations capacity.

in con-

In the

ex-

periments shown in Figs. 5 and 6 the reconstitutions were made with populations derived f’rom the same original marrow aspirate. In addition, in Fig. 5 a reconstitution is demonstrated with homologous irradiated peripheral blood cells (open triangles), indicating that the procedure may be applied to adherent and

NA

cells

f’rom

different

sources.

Fig. 6. Effect of autologous adherent marrow cells on colony forma-

80

tion

o

/

/

40

o

,0

by NA

row cells per plate

0’

source dishes

20

marrow

/

2 x 106

-‘

3

2

I

Source

of adherent

cells

4xl0

cells.

The

colony

number per 3 x i06 nonadherent cells is plotted against the number of marof

adherent

contained marrow

that

served as the cells. Control

adherent cells

but

cells

from

no NA

cells

(closed circle). Values for the unseparated marrow and NA cells plated with 20% CSA only were 59 and 47 colonies/3

x 106

cells.

respectively.


708

MESSNER.

TILL.

AND

McCULLOCH

o 60

So ‘C

4#{176}

0

0

0

Fig.

30

row

A

Colony

7.

cells

(2

autologous C,)

20

#{163}

.

I

is

,.

I

for

acute

described

S ‘

2

CSA

leukemia above:

‘

4

of adherent

(‘el/s

CSA -Producing Assays

e

... ‘

Source

with

e

#{149}

‘

6

8

10*10

Patietzts

that

is, varying

-

numbers

control,

adherent

prepared

assays

f’or

CSA-producing

unseparated

leukemia

cells

with

nies, while complete reconstitution was marrow (Fig. 8). These results are similar plated

on adherent

cells

from

leukemic

Fig. 8. Influence of irradiated unseparated leukemic and normal marrow on colony formation by normal NA marrow cells. The experiment was performed on the same marrow specimens from the normal and Ieukemic patients reprosented by the diamonds in Fig. 7. Open symbols are used to indicate colony formation by NA normal marrow cells when plated together with autologous irradiated marrow cells. Closed symbols indicate that the irradiated cells were derived from the patient with acute leukemia.

four

marrow of four was the second

were and from

in Fig. marrows In one were

marrow

the

culture

individual calf serum,

radiated

-

from

nonadherent cells from a normal layer in methyl cellulose, fetal

7 both

(open

cells

cells

also

cells

tissue

x lO adherent

were

marrow

in plastic

2

layers

over

of’ marrow

served

sources of’ NA cells. The results are shown marrow’s were less effective than normal tion by NA cells ofthe normal individual.

mar-

plated

patients

Leukemia

patients

cell

adherent

made on the The procedure

of adherent

by NA

when

cells. Clinical data for are shown in Table 2.

14”itli A cute

producing cells were prior to treatment.

as a source

a

cells

in four

formation 10)

symbols) or over adherent cells derived from a patient with acute leukemia (closed symbols). The number of colonies is plotted against the number of marrow cells that served as source for adherent

0

0

x

plated alpha the

patients of those

the

leukemic dishes

on top medium.

marrows

and of the As a

used

as

7. In all four cases, leukemic in promoting colony formaof the cases described in Fig.

perf’ormed. normal

achieved to those

The

NA with found

cells

mixture yielded

autologous when NA

of no

ir-

cob-

irradiated cells were

patients.

‘

3slO Irradiated

marrow

cells

per

plate


NORMAL

AND

LEUKEMIC

HUMAN

MARROW

CELLS

709

DISCUSSION

The human and

results presented marrow contains

cells

capable

proliferation eral

of

and

blood

in this both producing

differentiation

of monkeys

in human

pa,.er are consistent with the granubopoietic cobony-f’orming

have

peripheral

required CSA-producing

been

blood

Cline.’t All three cells morphobogically

molecules in culture. by

studied

populations

forming titative

capable

of

assay

procedure

may

CSA and

be expected

CSA:

for example,

(approx.

1300)

Price that

to

CSA

and

CFU-C. The quantitative vide the basis for ofcebls

that

revealed It is not specific

et al.9 have

adhered

to glass,

mononuclear

known

whether

CSA

but

way in our laboratory. studies of marrow from

assay

procedure

for

of normal

populations

with

attributed

to

leukemia

cells

adherent

a failure

to adhere studied,

we

of

with

low

fails

without

with

the

8): thus,

cells

from

marrow

that

by

or

are

plastic.

or

ACKNOWLEDGM assistance.

authors

are

grateful

to

Miss

Judy

Grover

cells

or of a makes

of how

clinically.

(Fig.

are

abnormality part

it

the

A clear

investigated. Uncapacity to popu-

marrow

cells

staining,

characteristics.

of NA 7) cannot

of

Accordingly,

reflects

paper. proExamina-

reconstitution

defective differentiation in leukemia is unknown: however, assay procedure makes it feasible to investigate the problem.

The

mouse

examples

leukemia

from

CSA-producing cells

quanthe

separation. Analycells is at present

applied

with

the

colony-

weight

procedures

to obtain

cells

Whether

abnormal

of of

stimulate

of all such of cell adherent

be

failure

patients

glass

no

or after

of assay

may

CSA-producing

capacity.

and

molecular

to

identifying

leukemia

cells

(Fig.

conclude

those

characterization

fixation,

specific

availability

patients

or

described in this of this population.

is a function

the

to

and Golde

a property property

little

and

but cells,

directly

production

normally

or productive of

with

a CSA CFU-C

without

CSA-producing

NA

by

was functional

from normal was observed in the four patients leukemic marrow failed to restore colony-forming

bations

number

and

periph-

is required since mouse marrespond to all species of human

question using techniques of both NA cells and

under The

tients

either

However,

possible to approach this sis by velocity sedimentatio&9

ditTerence separated

reported human

cells

subpopulation.

Williams,’6

purification

assays for CSA-producing determining the characteristics

large

for

in the

provide the basis for cells. For the molecules,

to facilitate

on

progenitors cells

production with the

Such a procedure purpose, do not

is active

that normal cells (CFU-C)

extend the findings to marrow, indicating site are also glass adherent. provides a reliable means of obtaining

responding

sources. for this

these

LoBuglio’7

capacity in its absence. Such populations assays for both CSA and CSA-producing

ofCSA from human row cells, often used

tion

Moore

and

groups concluded that similar to monocytes

glass adherence. The present studies that the CSA-producing cells in this The adherent separation procedure cell

by

Chervenick

by

view

patients

in the abnormal

four

the

pa-

either

is secondary of

be with

mechanism

of

the availability of an

ENT for

her

conscientious

in

to dilu-

and

excellent

technical


710

MESSNER.

TILL.

AND

McCULLOCH

REFERENCES I.

Senn

parison and

JS, of

McCulloch

colony-forniing

leukernic

Lancet

2:597.

of

human

man

JE:

marrow

in

Corn-

of

normal

cell

culture.

New

density

Pike

BL:

cells

marrow

Grune

&

row.

growth

in vitro,

Hemopoietic

York,

Colony

Prolifera-

Stratton,

1970,

J,

Freireich

of rnyeloid

colonies

tients

acute

with

EJ:

from

In

bone

leukemia

vitro

hybrids.

of

remission.

pa-

4.

Brown

CH,

of normal J NatI

Cancer

5.

Iscove

human

Inst

cells

37:1,

colonies:

in

Bone by

blood

cells.

Science

Sachs

induction

L, cells

patients.

Sheridan weight

stimulating

in vitro.

Price

GB,

low

stimulating

Resnitzky

A

leukemic NatI

Acad

Sci

Metcalf

D:

A

low

in lung-conditioned J Cell

and

molecular activity.

monocyte

Physiol

McCulloch

molemedium

LA, weight Blood

colony

81:11, Till

1973

JE:

granulocyte 42:34

1-48,

1971

Bruce laboratory

colonies two

vitro.

cell

Science

Physiol

blood.

HP: cell

animals.

stimulating cells in

80:195, PA,

Phys

N:

Physical

cells from hemopoietic

in tis-

1972 LoBuglio

Stimulators colony

AF: of

Human

granulocyte

formation

in

vitro.

1972

J Clin velocity

Webb

irradiating

Williams

Cline

DW,

Miller

WR,

for

1965

colony-stimulating

by

granulo-

for in

small

178:164,

73:191,

A new

77:277, requirement

from

22, 1972 of

marrow

MAS,

IS. Golde

cells

V:(2)

unit

monocytes:

19.

EA:

cells

in bone

JR.

mononuclear

eral

marrow

37Cs 10:381,

Moore

Science

1971

McCulloch

formation

and

sue. J Cell

and and

H:

1967

Biol

blood

differentia-

from

Proc

granulocyte

formation

DE:

antibody

suspensions

the

JW, factor

Y,

of granulocyte

1970

67:1542,

Barak

230:52,

Haematol

separation of colony vitro colony forming

supernatant

mar-

Green

Transformation

Physiol

convenient

A

Med

marrow

5cr.

17. Chervenick

USA

ony

leukocytes.

DR:

vitro

human

non-leukemic

human

of con-

Effect

human

Boggs

PA,

in hematopoietic

9.

culture:

leukemic

bone

GL,

JE, of

Cunningham

16.

ML,

P: In vitro

8.

and

IS.

1970

Paran

cular

normal

Till

D:

Mosier for

by

stimulat-

in mouse-hamster

Biol]

H,

man.

158:1573,

McCul1och

JE,

(New

J Cell

14.

1971

incubated

169:691,

tion

in

Stimulation

from

growth marrow.

types

Till

from

Chervenick

6.

vitro bone

RNA

to macrophages

in vitro.

1971 JS,

by

medium

Blood

7.

46:989, Senn

marrow

In

human

formation

ditioned

PP:

leukernic

NN,

Colony

LA:

Carbone

and

human

from Eliceiri

Metcalf

cytes

35:61, 1970

cells CP,

and

Studied

forming,

distributions

13.

Galbraith

vitro:

of colony

of ribosomal Nature

mouse

Blood

RD. in

1972

Messner

Density

growth

marrow

in

inhibiting Stanners

12.

Harris

McKnight

Blood4O:394,

Synthesis pp

JS,

interaction

separation

I I.

in Stohl-

Cellular

249-259 3.

Haskill Cell-cell

ing and

WA,

bone

10. PR:

1967

F Jr (ed):

tion.

Till

ability

human

Robinson

2.

EA,

Mi: cell

Invest

RG,

Identification in

51:2981,

Phillips

RA:

sedimentation.

human

of

periph-

1972 Separation J Cell

of Physiol

1969

20. Snedecor

col-

Methods

1973

chap

GW,

(ed 6). Ames,

6, p 169

Cochran Iowa

WG: State

Statistical UP,

1967,