1 i ,uAemta Re.search %oi ~. "~.o 5. pp 853-861. 1984 0145-2126/845:~ IN) + OOl i

Printed in (ir¢•l Britain (~-~ 1984 Pergamon Prc~, I Id

E N R I C H M E N T O F H I G H P R O L I F E R A T I O N P O T E N T I A L

C O L O N Y F O R M I N G C E L L S F R O M M O U S E M A R R O W

BY S E L E C T I N G L O W - D E N S I T Y C E L L S E X P R E S S I N G

R E C E P T O R S F O R W H E A T G E R M A G G L U T I N I N

PAUL BAINES, ALICIA LAJMANOVICH and DANIEL HOLLARD

Departement de Recherche Fondamentale. Laboratoire d'H6matologie, Inserm U.217, Centre d'Etudes Nucl6aires 85X, F.38041 Grenoble Cedex. France

{Received 2 February 1984. Accepted in revised form 5 April 1984)

Abstract--Suspensions enriched for high proliferation potential colony forming cells (HPP- CFC's) were prepared from mouse marrow by selecting low.density cells stained with fluores- ceinated wheat germ agglutinin on a fluorescence activated cell sorter. HPP-CFC's were 12-fold more concentrated in the final suspensions and co-enriched with a population of primitive CFU s detected in the spleens of irradiated mice reconstituted 13 days earlier with marrow. This is consis- tent with previous observations suggesting that these populations are closely related. The degree of enrichment for other haemopoietic progenitors was in the order HPP-CFC > day 8 C F U > BFU c > GM-CFC > CFU c.

"Ext ra" colonies developed when human placental conditioned medium (HPCM) was added to cultures of enriched suspensions already containing pregnant mouse uterus extract (PMUE). HPP- CFC's probably formed most of these and we discuss why counting these extra colonies may be more reliable than the conventional "size" assay for HPP-CFC's.

Key words: HPP-CFC, day 13 CFUs, enrichment, buoyant density, Icctin staining, FACS.

INTRODUCTION

1T HAS become clear that in order to understand how haemopoietic colony-forming cells (CFC's) interact with the colony stimulating factors (CSF's) that support their in vitro proliferation, it will be necessary to use more defined culture systems than have been used in the past. Many non-specific and specific growth effects may be contributed by the majority of cells in cultures of unfractionated marrow which do not form colonies and it will be essential to eliminate these. Similarly, it is difficult to evaluate the effects of the diverse proteins present in crude sources of CSF's and present in the serum-supplements currently added to cultures of CFC's. CSF's will have to be purified and serum sup- plements replaced by fully-defined media.

In our laboratory we are preparing to culture high proliferation potential colony- forming cells (HPP-CFC's [4]) in precisely-defined conditions. These progenitor cells pro- liferate to form the largest colonies ( > 1.5 mm dia) of granulocytes and macrophages in cultures supplemented with pregnant mouse uterus extract (PMUE) and human spleen or placental conditioned medium (HSCM, HPCM). HPP-CFC's have been shown to differ from the progenitors which form the majority of smaller colonies of granulocytes and macrophages in the presence of PMUE alone (GM-CFC, M-CFC [3, 5]). Although all

Abbreviations: HPP-CFC, high proliferation potential colony-forming cell; GM-CFC, granulocyte- macrophage colony-forming cell; M-CFC, macrophage colony forming cell; CFU~, colony forming unit in the spleen; HPCM, human placental conditioned medium; WGA, wheat germ agglutinin; FACS, fluorescence activated cell sorter.

Correspondence to: Paul Baines, DRF/Laboratoire d'Hematologie, C.E.N.G. 85 X, F 38041 Grenoble Cedex, France.

853

854 PAUL BAINE$ et al.

th ree p o p u l a t i o n s a p p e a r " c o m m i t t e d " , t he H P P - C F C r e s e m b l e s m o r e c lose ly t he

p l u r i p o t e n t i a l c o l o n y f o r m i n g un i t s in t he sp l een ( C F U ) in b u o y a n t dens i t y [1] a n d cell

cyc le s t a t e [2].

As a f i rs t s tep t o w a r d s c l a r i f y i n g the c o n d i t i o n s r e q u i r e d by H P P - C F C ' s to p r o l i f e r a t e

in vitro, we h a v e e x p l o r e d w a y s o f s e p a r a t i n g these p r o g e n i t o r s f r o m the bu lk o f n u c l e a t e d

cells in m a r r o w . W e r e p o r t h e r e e n r i c h m e n t f o r H P P - C F C ' s in s u s p e n s i o n s o f m a r r o w

cells f r a c t i o n a t e d by a t e c h n i q u e w h i c h se lec ts f o r p l u r i p o t e n t i a l C F U , [9]. T h e d a t a c o n -

f i rm the c lose r e l a t i o n s h i p b e t w e e n H P P - C F C ' s a n d a p r i m i t i v e s u b - p o p u l a t i o n o f C F U

[2]. F u r t h e r , m a n y ex t r a c o l o n i e s d e v e l o p e d w h e n H P C M was a d d e d to P M U E -

c o n t a i n i n g c u l t u r e s o f e n r i c h e d s u s p e n s i o n s . W e r e l a t e this o b s e r v a t i o n to p u b l i s h e d d a t a

a n d sugges t t h a t H P P - C F C ' s f o r m e d m o s t o f these a d d i t i o n a l co lon ie s .

M A T E R I A L S A N D M E T H O D S

Mice and preparation of marrow Femurs from BDFI mice (IFFA CREDO, France) were flushed into Hepes buffered Hanks balanced salt solu-

tion (HHBSS) using a 2-ml syringe and 25 gauge needle. Appropriate fractions of femurs were taken for culture.

Fractionation of marrow The technique was an adaptation of a published technique [9] in which low-density marrow cells are stained

with fluorescent labelled Wheat Germ Agglutinin (WGA). For a medium of final density of 1.0725 g/cmL 9 ml of autoclaved Percoll (Pharmacia) was mixed with I ml of

10-fold concentrated HHBSS pH 6.9 and 7.45 ml of 0.15 M NaCI. The final pH of the mixture was 7.45. Fresh mixtures were prepared before each experiment.

5 x 10' cells were suspended in 2 ml 1.0725 g/cm' Percoll and layered over I ml of 1.0725 g/cm; Percoll. 0.5 ml of 1,060 g/cm ~ Percoll (9 ml autoclaved Percoll, 1 ml x 10 HBSS, 11.4 ml 0.15 M NaCI) was layered over the cells. After centrifugation at 4001[ for 20 rain, cells in the top 3 mi of PercoU were removed, washed and in- cubated with 0.Sp.g/mi of WGA-FITC (Sigma) for 20 rain at 4°C. After two washes, cells were sorted into positive and negative windows (Fig. I) on a FACS IV (Becton & Dickinson) pre-sterilised with alcohol.

Sorting rate was approximately 2000 cells/s and abort rate I ceil/4 drops. After passing a 70 j, tm nozzle, cells encountered an argon laser beam tuned to 488 nm (800 roW). Forward light scatter (FLS) was measured on a photocell (attenuater down) and fluorescence on a photomultiplier shielded by an Oriel 70716 filter of barrier transition range 510-520 nm. Cells were collected in 15 ml siliconised glass centrifuge tubes containing 0.5 ml FCS and aliquots taken directly for assay. Cell yields, calculated by vital dye exclusion, verified the counts given on the FACS.

Preparation of colony-stimulating factors PMUE was prepared by homogenising whole embryos into an equal volume of deionised HzO and taking the

fraction precipitated by 50--100~0 ammonium sulphate as previously described [5]. Dialysed material was heated I h at 60°C, centrifuged at 14,000 g, adsorbed to calcium phosphate gel and eluted with 0.09 M sodium phosphate buffer pH 7.4. After dialysis against water and lyophilization, this material was re-dissolved in water, titrated and frozen at -20 or -70°C as a stock solution of 1.7 mg/ml. We routinely used 50 ~.1 of PMUE solu- tion/mi culture giving a final protein concentration of 85 p.g/ml.

Human placental conditioned medium (HPCM) was prepared by culturing finely minced fresh placental tissue in single strength, alpha modified Eagles medium (Flow) supplemented as described below. Tissue pieces were washed twice with saline before culture to remove excess blood and no serum was added. Supernates were col- lected four days later, dialysed against water, lyophilised and re-dissolved in water before use. HPCM was stored either lyophilised or, after dissolving in water, at -20 or -70°C. The volume and final protein concentration of HPCM in culture were, respectively, 100 gl and 52 v.g/ml.

Colony-forming assays GM-CFC, M-CFC and HPP-CFC were cultured in a a-modified Eagles medium (Flow Labs) containing 25070

foetal calf serum (FCS, Flow Labs) and further supplemented with MEM vitamins (Flow Labs), 200 mM glutamine (Gibco) and 20 g/I gentamicin sulphate (Sigma). Double strength medium was diluted, shortly before addition of cells, in an equal volume of 0.7% liquid agar (Bacto Agar, Difco). Replicate cultures were harvested after 10-12 days of incubation in a 10070 CO, atmosphere. Stimulants were added directly to the dishes.

BFU c and CFU e were cultured in 0.9% methyl-cellulose in lscoves Modified Dulbeccos Medium (IMDM, Gibco), 30% (v/v) FCS (Flow), 2.507o mouse serum, 10% (v/v) deionised BSA (Sigma, fraction V) and 10-' Ms- thioglycerol. Wehi conditioned medium, added to give a final protein concentration of 280ttg/ml (5o7o v/v), acted as a source of BPA. Erythropoietin (Pig Epo, Centre National Transfusion Sanguine, Paris) was added to give final concentrations of I and 0.5 IU/ml respectively in BFU and C F U cultures. C F U were scored after 48 h and BFU c after 7 days of culture at 37°C in a 5070 COt atmosphere. "

CFU assay Mice received 8.3 gray gamma irradiation for use as hosts for the C F U assay [7] and were sacrificed 8 or 13

days later. Spleens were fixed in Tellyesniczky's fluid.

Enrichment o f H P P - C F C 855

Scoring HPP-CFC's In cultures supplemented with both P M U E and H P C M , colonies o f ~ 'eater than 1.5 m m dia were recorded as

derived from H P P -C F C ' s . Smaller colonies were segregated into size categories 1-1.5 m m or < 1 ram. A min imum cut-off for all granulocyte /macrophage colonies o f < 50 cells was employed.

RESULTS

Fractionation of marrow progenitor cell populations Exploratory experiments in our laboratory had indicated that separation of marrow on

Percoll of density 1.0725 g/cm 3 at a pH of 7.45 gave good enrichment for most progenitor cells (Table 1). Scrutiny of the distributions of CFC's in the interface and pellet indicates that fewer BFU~ and C F U were retained at the interface (52 and 49o70 respectively) than day 8 CFU,, day 12 CFU,, HPP-CFC's and GM-CFC's (58, 69, 80 and 65%). Since only 23°7o of total nucleated cells were retained at the interface, the enrichment varies from 1.6× to 2.5x for the former progenitors and from 2.5x to 5.6× for the latter pro- genitors.

After staining low-density interface cells with WGA-FITC, followed by analysis on the FACS, we sorted a window of fluorescent cells of high FLS (Fig. 1) which contained a high proportion of HPP-CFC's and CFU s (Table 1). In suspensions of cells from this window, enrichment for all the different progenitors was, in order of decreasing magnitude, HPP-CFC (12 x ) > day 13 CFU (11 x ) > day 8 CFU~ (9 x ) > BFU~ (5 x ) > GM-CFC (4 x ) > C F U (1 x ). The incidence of most progenitors increased after staining interface cells with WGA-FITC (WGA control, Table 1) but decreased after sorting (Sort Control, Table 1). These observations can be related to the yields of total nucleated cells (Table 1) and yields of progenitors given in Table 2. During WGA-FITC staining, the decrease in total nucleated cell yield was greater than the decrease in CFC yield; hence CFC incidence will rise. After sorting however, the fall in CFC yield was greater than the fall in total nucleated cells (day 8 CFUs excepted); hence CFC incidence will fall. Yields of CFC's also decreased during the density cut procedure and this was especially true for CFU e.

The whole procedure gave yields of between 10 and 19o70 for the earlier progenitors in- vestigated which means that we selected only a small proportion of each population.

Total colony numbers in PMUE-supplemented cultures of enriched suspensions In our laboratory, different batches of HPCM have consistently inhibited proliferation

of a proportion of progenitors from unfractionated marrow which form colonies in the presence of PMUE alone. This is reflected in the figures in the first line of Table 3 where the ratio of numbers of colonies formed in the presence of PMUE + HPCM to the numbers formed in the presence of PMUE alone, is less than one. This ratio, however, in- creased during enrichment and HPCM did not reduce but increased colony numbers in PMUE-supplemented cultures of cells from the fluorescent "window".

DISCUSSION

The enrichment procedure permits the separation of HPP-CFC's from the bulk of nucleated cells present in mouse marrow as well as eliminating many developmentally "late" progenitors from the GM-CFC and CFU e populations.

This procedure also selects for pluripotential CFU and to a lesser extent for BFU e and GM-CFC. It would be expected that closely related progenitors would possess similar buoyant density and similar numbers of receptors for WGA and hence would be enriched to the same extent. Indeed, the order of degree of enrichment - - HPP-CFC > day 13 CFU > day 8 CFU, > BFU > GM-CFC > CFUe m fits well with what is known of the position of these cells in the haemopoietic lineage. Accumulating data indicate that the HPP-CFC closely resembles the more primitive CFU s compartment which is assayed in spleens harvested at day 13 [2]. Day 8 CFU were, on the other hand, enriched only 9-fold

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857

Enrichment o f H P P - C F C

TABLE 2. RECOVERY OF HAEMOPOIETIC PROGENITOR CELLS DURING ENRICHMENT

859

Total progenitor cell yield ( x 10-9

Stage of enrichment Day 8 CFUs Day 13 CFU s HPP-CFC GM-CFC BFU e CFU e

Unfractionated (UF)~ 34 4- 5 37 4- 5 69 ± 10 361 4- 63 41 + 4 237 .4- 32 lmerface,~ II 4- 3 23 4- 4 53 -t- 3 159 4- 23 14 4- 1 54 4- 5 Pellet~ 9 4- 2 13 4- 2 13 4- 3 91 4- 25 13 4- 2 56 + 9 WGA control:[: 7 -4-2 13 4- 2 33 --t- 7 87 4- l0 16 4- 5 34 + 5 Sort control:[: 9 4- 4 9 .4- 3 6 --I- 1 26 4- 6 8 4- 2 6 -4- I Window~ 4 4- l 6 4- i 13 4- 4 18 4- 2 4 ± 2 3 4- 0.5 Window x 100 per UF l 1.8 16.2 18.8 5.0 9.8 1.3

?~; As for Table 1. The final yield as a percentage of the original total number of progenitors is given in the lowest line as window

× lO0 divided by UF.

TABLE 3. ENRICHMENT OF CFC's RESPONSIVE TO THE COMBINED STIMULUS OF PMUE AND HPCM

Stage of enrichment

CFC no's /10 ' nucleated cells ratio of CFC no's in PMUE + HPCM cultures

PMUE cultures PMUE + HPCM cultures PMUE Total colonies > 1.5 mm colonies Total colonies

Unfractionated:~ 362 -.t.- 28 66 -'t- 4 188 -1- 8 0.52 lnterfacet 900 4- 108 298 4- 16 728 4- 45 0.81 Pellet1" 106 4- 19 17 "+- 4 82 4- 9 0.77 WGA control +. 1461 -.t- 454 388 -.F 43 1285 -1.- 166 0.88 Sort control~ 384 4- 32 100 4- 11 370 4- 27 0.96 Window:~ 964 4- 167 821 4- 122 2275 ± 317 2.36

Cells from the interface of a 1.0725 g/cm ~ Percoll density cut were stained with WGA-FITC as described in the text. Highly fluorescent cells of high forward light scatter were selected as shown in the window illustrated in Fig. 1 and seeded into cultures containing PMUE alone or PMUE + HPCM.

~Results represent the mean + S.E. of 5 experiments. 1Mean of 3 experiments.

w h i c h is c o n s i s t e n t w i t h e v i d e n c e t h a t t h e s e a r e d e v e l o p m e n t a l l y " l a t e r " C F U s w h i c h lie

in, o r c l o s e r to , a cyc l i ng s t a t e a n d a r e m o r e l ike ly t o d i f f e r e n t i a t e t h a n d a y 13 C F U s [2, 6].

T h a t t h e e n r i c h m e n t o f d a y 8 C F U 5 is f a r h i g h e r t h a n t h e e n r i c h m e n t o f B F U , ( 5 - f o l d )

d o e s n o t n e c e s s a r i l y c o u n t e r t h e s u g g e s t i o n [6] t h a t t h e s e t w o p r o g e n i t o r s a r e c l o se ly

r e l a t ed . It is d i f f i c u l t t o d r a w c o n c l u s i o n s f r o m d i s s i m i l a r e n r i c h m e n t s w h e r e o n e p r o -

g e n i t o r is a s s a y e d in vivo a n d t h e o t h e r in vitro. O u r e n r i c h m e n t p r o c e d u r e m a y e l i m i n a t e

cells a c c e s s o r y to in vitro p r o g e n i t o r cell p r o l i f e r a t i o n a n d w h i c h a r e s u p p l i e d t o a n in vivo e n v i r o n m e n t .

O u r d a t a a r e d e r i v e d f r o m t h e m e a n :k S . E . o f t w o o r m o r e s e p a r a t e e x p e r i m e n t s .

A l t h o u g h v a r i a t i o n b e t w e e n r e p l i c a t e c u l t u r e s o r s p l e e n s w i t h i n a n y o n e e x p e r i m e n t was

sma l l , a l a r g e r v a r i a t i o n in t h e i n c i d e n c e o f p r o g e n i t o r s was o b s e r v e d b e t w e e n ex-

p e r i m e n t s , a t s o m e s t ages o f e n r i c h m e n t . In t h e " S o r t C o n t r o l " t h i s m a y r e f l ec t w e e k t o

week v a r i a t i o n in F A C S o p e r a t i o n . T h e v a r i a t i o n a p p a r e n t b e t w e e n n u m b e r s o f d a y 8

860 PAUL BAINES el al.

CFU s retained at the Percoll interface, could result from the difficulty in enumerating CFU s accurately at this stage of development should their proliferation be more delayed in some weeks by a longer exposure to Percoll or slightly less favourable in vitro conditions. Importantly, these larger variations are only seen in some of the intermediate stages of enrichment. A much smaller weekly variation in the incidence of progenitor ceils was recorded in suspensions of cells selected in our final "window".

Our final concentrations of progenitors are lower than those obtained in the original description of the technique where an 80-fold enrichment of CFU s was reported [9]. This is because the earlier workers reported superior yields and incidences after both WGA- staining and sorting. We have recently improved our yields by reducing further the concen- tration of WGA-FITC, although the incidence of progenitors has not altered (un- published results). During our sorting procedure, CFC's may adhere to the tubing of the FACS or may be damaged by exposure to 488 nm light. The low recovery of CFU e is primarily due to major losses during both the Percoll and sorting steps rather than specific exclusion. CFUe may be particularly fragile cells, ill-suited to manipulations such as these.

Visser et al. [8] have more recently reported additional enrichment for day 13 CFUs by further selecting only those cells which express large quantities of surface H-2 k antigens. It seems likely that this procedure will also enrich for HPP-CFC's and we shall incorporate it in our technique.

In PMUE-supplemented cultures of cells from our final "window", addition of HPCM increased colony numbers. This effect was not observed cultures of unfractionated marrow where HPCM reduced colony numbers. Our interpretation of these data is that few CFC's in normal marrow respond only to the combined stimulus of PMUE + HPCM. Hence only a small increase in colony numbers might be expected when HPCM is added to cultures of unfractionated marrow containing PMUE. In fact any small increase in colony number is obscured by HPCM-mediated inhibition of much larger numbers of GM-CFC's and M-CFC's. However, in PMUE-supplemented cultures of enriched suspen- sions, the greater proportion of HPCM-responsive CFC's is enough to give an increase in total colony number on addition of HPCM.

We propose that HPP-CFC's formed most of the extra colonies observed in the present study. This is suggested by results of experiments using injections of 5-fluorouracil (SFU) which considerably reduces femoral GM-CFC, M-CFC and total nucleated cell numbers. HPP-CFC's proved resistant to 5FU and were greatly enriched in suspensions of marrow from mice treated 2--6 days earlier with the drug [4]. In cultures of these suspensions only a few small colonies developed in the presence of PMUE alone. Addition of human spleen conditioned medium, or in our hands HPCM, increased colony numbers and the majority of the extra colonies exceeded 1.5 mm dia. Here, it was clear that HPP-CFC's formed most of the extra colonies and proliferated only in the combined presence of PMUE + HSCM/HPCM.

From Table 3, 1311 (2275-964) more colonies formed in cultures of enriched suspen- sions when supplemented with PMUE + HPCM than when supplemented only with PMUE. Of these, 821 (63°/0), exceeded 1.5 mm dia and hence would be formed by HPP- CFC's.

This proposal implies that HPCM acts in PMUE-containing cultures of normal marrow by recruiting HPP-CFC's into division to form "new" colonies rather than simply in- creasing the size of pre-existing colonies. Further we can now consider replacing the con- ventional "size" assay for HPP-CFC's with one which counts the "extra" colonies which develop when HPCM is added to cultures of enriched suspensions containing PMUE. This may facilitate isolation of the CSF's involved where many non-specific and specific factors responsible for increasing colony size may be separated at advanced stages of purification.

Enrichment of HPP-CFC 861

Finally, this work represents a step forward in a programme designed to culture HPP- CFC's in "defined" conditions in the presence of purified CSF's. This should simplify the analysis of what cellular and biochemical events are initiated and/or maintained when HPP-CFC's interact with the factors supporting their in vitro proliferation, differentia- tion and maturation.

Acknowledgements--Paul Baines was employed as a Collaborateur Temporaire Etranger by the Com- missariat d l'Energie Atomique, France. Alicia Lajmanovich was employed as an Atlachee Scientifique to the Centre Hospilalier R~gional, Grenoble. We are indebted to Jacques Albert for assistance with the FACS and to Ginette Francois for typing the manuscript.

REFERENCES

I. BAINES P., BOL S. J. L. & ROSENDAAL M. 0982) Physical and kinetic properties of haemopoietic progenitor cell populations from mouse bone marrow detected in five different assay systems. Leukemia Res. 6, 81.

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