- Email: [email protected]
The effect of the dimeric and multimeric forms of fibronectin on the adhesion and growth of primary glomerular cells
Experimental
Cell Research
145 (1983) 265-276
Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/83/060265-12$02.00/0
The Effect of the Dimeric and Multimeric Forms of Fibronectin Adhesion and Growth of Primary Glomerular Cells TERRY RALPH
on the
D. OBERLEY ,l, * JOANNE E. MURPHY-ULLRICH,’ M. ALBRECHT3 and DEANE F. MOSHER*
‘Departments of Pathology and Laboratory Medicine and 2Medicine, University of Wisconsin, Medical School, and ‘School of Pharmacy, University of Wisconsin, Madison, 53706, USA
SUMMARY Primary glomerular cells placed in a chemically defined medium containing Waymouth’s medium MB 752/l supplemented with insulin, transferrin, fibroblast growth factor, nonessential amino acids, sodium pyruvate, and antibiotics showed rapid outgrowth of cells which morphologically resembled well differentiated visceral epithelial cells followed by outgrowth of poorly differentiated cells; morphologic evidence suggests these latter cells are precursor cells of the epithelial cell lineage. Whereas the well differentiated glomerular epithelial cells were never observed to divide by sequential phase microscopic observations, a chemically defined medium was developed for optimal growth of the poorly differentiated cell type. This serum-free medium contained Waymouth’s medium MB 752/l supplemented with insulin, transferrin, selenium, and tibronectin (plus non-essential amino acids, sodium pyruvate, and antibiotics). Using this chemically defined medium, we have compared the effects of dimeric and multimeric fibronectin (high molecular weight disulfide-bonded tibronectin produced by incubation of dimeric fibronectin with 3 M guanidine followed by dialysis against 0.05 M cyclohexylaminopropane sulfonic acid (CAPS) buffer, pH 11) on the adhesion and growth of the poorly differentiated primary glomerular cell type. Dimeric tibronectin (FN) was twice as effective as multimeric FN in promoting glomerular cell adhesion, although both forms of FN promoted cell adhesion better than an uncoated substratum. In contrast, cell growth studies demonstrated that multimeric FN was a more potent growth stimulant than dimeric FN. The differential effects of dimeric and multimeric forms of FN in vitro suggests that these molecules may have different functions in vivo.
Analysis of the growth of glomeruli in culture is made difficult by the fact that glomeruli are composed of four cell types (visceral and parietal epithelial cells, mesangial, and endothelial cells), and each of these cell types may potentially grow out in primary culture. Further, until recently glomeruli could only be grown in high concentrations of calf serum, which made quantitative analysis of glomerular cell growth difficult because of the complex nature of serum. A number of investigators have attempted to characterize glomerular cells grown in calf serum. The most definitive work on identification of glomerular cells grown in calf serum has used [3H]thymidine ([3H]TdR) labelling followed by autoradiography [l]; this study demonstrated that the initial cells seen in culture are the visceral epithelial cells, whereas the mesangial cells grew much later in culture. Foidart et al. [2] also reported studies with 13HlTdR labelling of renal glomeruli in which two major peaks of [3H]TdR incorporation were described, the first involving epithelial cells and the second mesangial cells. Parietal epitheli-
* To whom offprint requests should be sent. Address: Department of Pathology and Laboratory Medicine, 516 SMI, 470 N. Charter St., Madison, 53706, USA.
266
Oberley
et al.
Exp Cell
al cells are seen only rarely growing from glomeruli in calf serum, presumably because most glomeruli do not contain Bowman’s capsule 131. Endothelial cells have never been morphologically identified in glomerular culture and all glomerular cell types are negative for factor VIII antigen [4]. Because only two major cell types are present in glomerular culture and these types grow out of glomeruli at different times, it has been possible for investigators to attain relatively uniform populations of epithelial and mesangial cells [2]. However, there still remains a great deal of controversy concerning the nature of glomerular cells in vitro, since cells identified as ‘epithelial’ in origin lack the morphologic and biochemical features of true epithelial cells; they do not have visceral epithelial cell foot processes [5] or C3b receptors [6]. Nevertheless, the literature presents convincing evidence that these cells must be some form of glomerular epithelial cells since: (1) they produce laminin [7], heparan sulfate proteoglycan [8], type IV collagen [8], and libronectin [81, all properties of visceral epithelial cells and not fibroblasts; and (2) by both scanning (SEM) and transmission electron microscopy (TEM) these cells can be seen to grow unequivocally from glomeruli [9]. There are at least two major possibilities to account for differences in the morphology and biochemistry of glomerular cells grown in vitro in the presence of calf serum. One possibility is that ‘dedifferentiation’ can occur, whereas a second possibility is that serum stimulates the growth of precursors of epithelial cells. To try to resolve this controversy, we have recently begun studying the growth of glomerular cells in chemically defined media. Since Barnes & Sato [lo] have demonstrated that hormones and growth factors allow the growth of many mammalian cell lines, we decided to study whether glomerular cells could be grown in chemically defined media. Glomeruli grown in Waymouth’s medium containing insulin, transferrin, and fibroblast growth factor (FGF) showed two cell types, a typical well differentiated visceral epithelial cell with long cytoplasmic processes and a poorly differentiated cell [ 111. Despite careful observation, in this medium we never observed one of these cell types transforming into the other, and we never observed the well differentiated cell undergoing cell division. Since the well differentiated cell appeared rapidly and did not divide, we interpreted these cells as visceral epithelial cells which simply moved from the glomerulus to the culture surface. The nature of the poorly differentiated cell was uncertain. Since Rojkind et al. [12] has emphasized the importance of the extracellular matrix in cell growth, we decided to add fibronectin as a component of the culture media. We found that glomeruli grown in Waymouth’s media containing insulin, transferrin, and fibronectin demonstrated growth of the poorly differentiated cell into distinct colonies. We have recently demonstrated with immunofluorescence studies that the cells in the center of these colonies are surrounded by a fibronectin and laminin matrix, whereas the cells on the edge of the colonies typically show less extracellular matrix staining [13]. We presently hypothesize that these poorly differentiated cells are precursor cells of glomerular visceral epithelial cells for three reasons: (1) the cells show colony growth, typical of ‘clonogenic’ cells; (2) the edge of the colonies show cells which resemble well differentiated
Res
145 (1983)
Multimeric
Exp Cell Res 145 (1983)
fibronectin
and glomerular
cell growth
visceral epithelial cells; and (3) since it has been shown recently that fetal (but not adult) hepatocytes produce tibronectin [ 141, the fact that the poorly differentiated cells assemble a fibronectin (and laminin) matrix certainly suggests the possibility that they are precursor cells. Despite these arguments, it has not been possible to date to establish conclusively the identity of glomerular cell types. However, the development of a system in which glomerular cells could be grown in serum-free medium has allowed us to examine the role of fibronectin in the adhesion and growth of primary glomerular cells. The following report presents evidence on the role of the dimeric and multimeric forms of fibronectin on the poorly differentiated cell type, which we believe to be a precursor form of the visceral epithelial cell. MATERIALS
AND
METHODS
Animals Adult Hartly guinea pigs of either sex were purchased locally with weights of about 300 g and maintained on laboratory chow and drinking water supplemented with ascorbic acid.
Cell culture Glomeruli were isolated from young adult guinea pigs according to a nylon screening technique previously described [9]. The basic medium contained Waymouth’s media MB 752/I (Gibco) supplemented with sodium pyruvate (1%) (Gibco), non-essential amino acids (1%) (Gibco), and penicillin-streptomycin (100 units/ml) (Gibco). In most experiments the basic media was supplemented with insulin (5 @ml), transferrin (5 &ml), selenium (5 rig/ml) (ah prepared from insulin, transfer&, selenium premix from Collaborative Research), and dimeric or multimeric FN (0.1-25 ug/ml). To demonstrate the morphology of well differentiated epithelial cells in culture, in one experiment the basic medium was supplemented with insulin, transferrin, and FGF without fibronectin [l I]. Glomeruli were placed in culture in 9.6 cm* plastic tissue culture wells (Costar) in an atmosphere of 5% C02-95% air. Each well originally contained 10 ml of media, and 1 ml of new media was added on Monday through Friday. All experiments were repeated on at least two separate occasions. Progress of the cultures was monitored with a Leitz phase microscope. At appropriate intervals, cells were stained in situ with Coomassie brilliant blue R250 (2.5 % in methanol : water : acetic acid; 5.5 : 4.5 : 1; v : v : v) after fixation with 4% glutaraldehyde in phosphate-buffered saline (PBS). For direct visual comparison of cell growth, cells were photographed with a Zeiss bright-field microscope using Panatomic X fdm. All of the results reported in the present study were performed early in glomerular culture before the appearance of mesangial cells (probably actually mesa&al cell precursrs).
Transmission
electron microscopy
(TEM)
Glomeruli were grown in chemically defined media containing insulin, transferrin, selenium and FN. After the cells were confluent, they were detached from the culture substrate with EDTA and trypsin and fixed for 4 h in 4% cacodylate-buffered glutaraldehyde. The cells were secondarily fixed in 1% osmium tetroxide in s-collidine buffer, dehydrated in a graded alcohol series, and embedded in an Epon 812-Araldite epoxy mixture. Ultrathin sections were double-stained with lead citrate and uranyl acetate and examined at 75 kV with a Hitachi H-500 electron microscope.
Scanning
electron microscopy
(SEM)
Cells were prepared for SEM according to a protocol previously described [9]. Cells were grown in 25 cm* Costar flasks and areas of interest cut from the plastic surface. The cells were washed several times with PBS before furation in 0.1 M phosphate-buffered saline (PBS)- 1% ghttaraldehyde (pH 7.4). Cells were fixed for 24-48, dehydrated via an alcohol series, 30 min each in 30%, 50%, 75%, 85 %, 90 %, 95 %, and two 30-min periods in 100 % ethanol. Cells were critical point-dried with 100% ethanol as the intermediate fluid and liquid CO2 as the transitional fluid. The plastic substrate containing cells were cemented onto metal stubs and coated with a 12 nm gold layer. Cells were viewed at 15 kV accelerating voltage on a JEOL 356 scanning electron microscope.
267
268 Oberley et al.
Exp Cell Res 145 (1983)
Fig. 1. SDS polyacrylamide (6% running gel and 3 % stacking gel) slab gel electrophoresis of isolated human plasma fibronectin and its derivatives. a, MW markers (unreduced): ferritin halfunit, 220000 D; BSA, 67000 D. b, Plasma FN (25 ug/well) before reduction. The gel was overloaded to show minor bands. A major band at approx. 440000 is present (arrow). Other bands present are minor components representing aggregates of the monomer and dimer. c, Multimer FN (31 @well) before reduction. Most material is at the top of the stacking and separating gels. A small amount of monomeric (230000 MW) FN is also seen. d, MW markers reduced with mercaptoethanol. e, Plasma FN after reduction (2.5 udwell) (double arrow). The protein is present at MW 230 000. J Multimeric FN after reduction (3 1 ug/well). Again the protein is present at MW 230000. The direction of migration was from top to bottom and the gel was stained with Coomassie brilliant blue R.
Protein
isolation
Human FN was isolated from plasma or the supematant after heat precipitation of a fibronectin- and fibrinogen-rich plasma protein fraction. The solution was passed over a gelatin-Sepharose column with subsequent elution with 1 M sodium bromide, pH 5 [IS]. Purity of FN preparations was assessed by sodium dodecyl sulfate (SDS) slab gel electrophoresis. Dimeric FN was converted to disulfidebonded multimeric FN by incubation with 3 M guanidine followed by dialysis against 0.05 M cyclohexylaminopropane sulfonic acid (CAPS) buffer, pH 11 [3]. The conversion of the dimeric form of FN to its multimeric form was monitored by SDS slab gel electrophoresis (see Results). More details of the preparative methods and characterization of disuhide-bonded FN multimers will be presented elsewhere [3].
Cell adhesion Costar wells (9.6 cm*) were coated with dimeric FN (5 &ml), multimeric FN (5 ug/ml), or media alone, air-dried, washed twice with double distilled water, and sterilized with ultraviolet light. Isolated glomeruli were grown in chemically defined media containing insulin, transfenin, and dimeric FN, until confluency was reached. After brief treatment in 0.01 M EDTA in Tris buffer, the cells were trypsinized until detachment occurred and soybean trypsin inhibitor was then added to stop the reaction. The cells were washed with media alone and plated in the coated wells. 2x 10’ cells were added to each well. No cell clumps were included in the analysis; most were removed by allowing large clumps to settle and pipetting off the single cells in the supematant. After 1 h, the wells were washed with media and the number of attached cells determined by direct counting under phase microscopy. The results were expressed as averages of duplicate determinations.
DNA staining DNA staining tin, selenium, with a solution distilled water
was performed as described by Chen [16]. Glomerular cells grown in insulin, transferand Ebronectin were futed in Camoy’s fixative for 30 min, air-dried and then stained of Hoechst 33258 in PBS (0.5 pg/ml) for 30 min. The cells were rinsed twice in doubleand then mounted in PBS.
Cell growth Method no. I. Glomeruli were added to Costar wells which had been marked on the bottom with grids which were 0.16 cm’ in area. Five of these grids were arbitrarily selected and the number of initial glomeruli plated in each grid was counted. The glomeruli were then fed defined media containing varying amounts of dimeric or multimeric FN daily (Monday-Friday). Each day the number of cells in each of the five marked grids was counted by phase microscopy. Cell growth was then measured by calculating the ratio of the average number of cells observed each day relative to the average in&fat number of glomeruh. This method of determining cell growth was chosen for a number of reasons: (1) Because glomerular cells are of varying sizes and shapes and tend to clump together after dissociation, it was felt that direct counting while the cells were attached and flattened woufd be more accurate than using a Coulter counter.
Exp Cell Res 145 (1983)
Multimeric
fibronectin
and glomerular
cell growth
Fig. 2. Phase microscopy of cells grown in serum-free media without fibronectin. Cells were grown in Waymouth’s medium containing insulin, transferrin, and FGF without tibronectin and examined by phase microscopy. Gtomeruhts (g) is surrounded by (a) well differentiated visceral epithelial cells. The large cell is of the poorly differentiated cell type (arrow); (b) a number of cells of the poorly differentiated cell type. A well differentiated epithelial cell is also seen (arrow). Since both cell types grew from the glomerulus rapidly, it seems likely that both cells are a form of epithelial cell type; previous investigations have shown that mesangial cells require a great deal of time before growing in culture [ 1, 21. (a, b) x 200. (2) Because of the particulate nature of the glomeruli, it was often difficult to pipette exactly equal numbers of glomeruli into each Costar well; therefore the ratio of cells observed relative to the number of initial glomeruli was felt to be more reliable than calculation of the total cell count. (3) Averages of results are presented without calculation of standard deviations because the clonal nature of glomerular growth makes such calculations meaningless; each colony was unique, with a large variation in the total number of cells per colony. Merhod no. 2. To confirm the results of method no.1, direct counts were performed on glomerular colonies. Equal numbers of glomeruli were added to Costar wells (approx. 50 per 0.16 cm’ grid). After either 6 or I5 days following daily feeding, the number of cells in every colony greater than 16 cells was counted, and a histogram was constructed. A cell had to be approximately one cell length or less from the main colony to be included in the cell count.
RESULTS Protein isolation FN isolated from serum or plasma protein fractions [15] on gelatin-Sepharose columns, analysed by slab electrophoresis before and after reduction with mercaptoethanol (fig. 1, lanes b, e), migrated principally as a band of approximate molecular weight (MW) 440000 before reduction, and 230000 after reduction. Multimeric FN (produced by incubation of dimeric FN with 3 M guanidine and dialysis against CAPS buffer, pH 11) migrated with higher MW (greater than 440000) before reduction and 230000 after reduction (fig. 1, lanes c, f). Cell types Cells grown in Waymouth’s medium supplemented with insulin, transferrin, and FGF without FN showed two different cell types, one which resembled a well differentiated visceral epithelial cell and a second cell type which was poorly differentiated (fig. 2a, b). The well differentiated cell type has never been
269
270
Oberlev
et al.
Exp Cell Res 145 (1983)
Fig. 3. Morphology of glomerular cells in culture. Glomeruli were grown in a chemically defined medium containing insulin (5 @ml), transferrin (5 ug/ml), selenium (5 rig/ml), and dimeric or multimeric fibronectin (5 &ml). Identical results were obtained with either cell type. (a) Bright-field micrograph of glomerular colony. Cells were of a large uniform size and grew closely opposed. (b) Electron microscopy of glomernlar cells grown in serum-free medium. The cells observed were always attached to basement membrane and not banded collagen, suggesting that they were a form of glomerular cell and not a fibroblast contaminant. The nuclei have prominent nucleoli and the cells have a moderate number of mitochondria. Microvilli are noted on the cell surface. (c) SEM of glomerular cells grown in serum-free medium. Glomeruli were present in the center of all colonies. The cells were epithelial in shape, had a smooth surface, and were extremely flattened. (a) x 112; (b) x9900; (c) x440.
observed in cells growing in calf serum [ll]. Sequential observation of single, well differentiated epithelial cells by phase microscopy over a period of days revealed that although they did migrate, they did not appear to divide (this study and [ll]). The well differentiated cell type grew as widely spaced single cells, appeared rapidly after inoculation of glomeruli in culture, and appeared to require FGF for its appearance. In contrast, the poorly differentiated cell type usually grew as colonies, appeared later in culture, and could be observed in cultures containing only insulin and transferrin without FGF. Despite the appearance of these two
Exp Cell Res 145 (1983)
Multimeric fibronectin
and glomerular cell growth
4. SEM of cell at edge of glomerular colony grown in serum-free medium. Cells are small, with a rounded central cell body and numerous long cytoplasmic extensions (arrow). True foot processes were not seen. X 1000. Fig. 5. DNA stain of glomerular cells grown in serum-free medium. To demonstrate that the cells seen in culture were the result of cell growth and not the simple movement of cells from glomerulus to culture surface. glomerular cells grown in serum-free medium were stained with the Hoechst stain 33258. Mitoses were often seen, indicating true cell growth (arrow). x250. Fig.
cell types, glomeruli grown in the absence of tibronectin showed very poor overall growth and did not become confluent. When dimeric fibronectin was added to a chemically defined medium containing insulin, transferrin, and selenium (without FGF), only the poorly differentiated cell type was observed, and this cell type grew in colonies (fig. 3 a). Morphologic study of cells grown in this identical medium but in which dimeric fibronectin was replaced by multimeric fibronectin showed similar cell outgrowths. TEM and SEM also suggested that cells grown in insulin, transferrin, selenium, and either dimeric or multimeric fibronectin were identical. TEM of cells grown in either fibronectin type demonstrated that although the cells were attached to glomerular basement membrane, the cells did not have typical epithelial cell arbors or foot processes and contained prominent nucleoli, a feature of a poorly differentiated cell type (fig. 3 b). SEM clearly demonstrated a glomerulus at the center of each colony, and the cells observed were extremely flattened (fig. 3 c). However, at the edge of colonies there were cells which morphologically resembled well Table 1. The effect of dimeric us multimericfibronectin glomerular cells
on adhesion of primary
Type of substrate
% of cells attached at 1 h
Dimeric FN
Multimeric
100
52
FN
Medium alone 31
Wells were coated with dimeric or multimeric FN (5 @ml) or medium alone as described in the Methods. 2x lo3 cells were plated in coated Costar wells, incubated at 37°C for 1 h, and then washed. The cells were counted and the % of cells attached at 1 h determined. The values expressed are the average of two determinations.
271
272
Oberley
Exp Cell
et al.
30
I
b MULTIYERIC (lo;0 IfnIl
FN
25
I’
J MULTIMERIC Ii;q Iml)
FN r
MULTIMERIC _’
Y”’
FN lrn’)
/,/
F
/’ ,’
FN
r/’
DAYS
MULTIMERIt tl~qlml) __*/ DIMERIC i25uq
Fk Iml!
IF1 CULTURE
Fig. 6. The effect of dimeric vs multimeric fibronectin on the growth of primary glomerular cells: average number of cells in outgrowths per average number of initial glomeruli in 0.16 cm* area (method no. 1). (a) Approx. 7000, (b) approx. 5000 glomeruli were added to Costar wells (9.6 cm*) containing insulin, transferrin, selenium, and/or (a) 10 &ml of FN (10 cc of media per well at start of experiment); (b) 25 pg/ml FN. Glomeruli were fed daily (1 cc of media per well per day) and cells counted directly by phase microscopy. (n) 5~10~; (b) 2~10~ cells (approx.) were present at day 8 when cells were fed, (n) 10; (b) 25 &ml multimeric FN; the other wells had fewer cells (a). Cells fed 1 &ml x , dimeric; 0, multimeric FN daily; cells fed 10 &ml A, dimeric; 0, multimeric FN daily. (b) Cells fed 1 @ml X, dimeric; 0, multimeric FN daily; cells fed 25 ug/ml A, dimeric; 0, multimeric FN daily.
differentiated glomerular epithelial cells (fig. 4); these cells appeared only after the colony had become large, in contrast to the well differentiated cells seen in a serum-free medium containing FGF but without fibronectin, which rapidly grew (or migrated?) from the glomeruli. Cell adhesion
To test the effect of the dimeric and multimeric forms of FN on cell adhesion, tissue culture wells were coated with the two forms of FN and then trypsinized cells from primary cultures added to each well. The cells were washed at 1 h, and the percentage of attached cells was determined by direct counting (table 1). It was found that although both forms of FN promoted cell adhesion better than an uncoated substratum, dimeric FN was twice as effective as multimeric FN. Cell growth
Glomeruli did not show significant growth in chemically defined medium containing insulin, transferrin, and selenium when incubated at FN concentrations of 0.01 or 0.1 ug/ml, while at FN concentrations of 1 or 10 &ml FN, significant growth occurred. We are certain that this represents real growth and not simple movement of cells from the glomerulus to the culture surface, since the Hoechst stain for DNA showed frequent mitoses (fig. 5). To quantitate this effect, the
Res 145 (1983)
Multimeric
Exp Cell Res 145 (1983)
fibronectin
and glomerular
cell growth
a 30
MULTIMERIC
FIBRONECTIN
(I ug Iml)
DIMERIC
FIBRONECTIN
30
(luglml)
25
25
20
I5
N-IO
n
IO
N-4
5
N-3
N-4 -
N=S
25l500
3Ol350
35l400
o50
SI100
IOII50
ISI200
2Ok 250
NUMBER
401450
OF
o50
CELLS
5IIO0
PER
IOII50
151- 2Ol200 250
251300
3Ol350
35l400
401450
COLONY
b 100 90 In g 60
‘1
MULTIMERIC
FIBRONECTIN
(5ug
Iml)
DIMERIC
FIBRONECTIN
100
(buglml)
90 60
I s 0 0
70
70
I& 60
60
a
50
i# I 40
50
11.45 N-37
40
N-34
is 30
30
20
N.17
20
IO
IO
51T!l
76100
IOII26
IOII60
l6lI75
NUMBER
I76200
POI225
OF
CELLS
o26
PER
2650
5I75
N’4
N=3
N.2 n
76I00
IOII25
I26I50
ISIIT5
II6200
2Ol225
COLONY
7. The effect of dimeric vs multimeric fibronectin on the growth of primary glomcrular cells: histogram analysis of the number of cells per colony in 9.6 cm2 well (Method no. 2). (a) 1; (b) 5 @ml: to demonstrate the effect of (a) multimeric FN at low glomerular density and low concentrations of FN; (b) increasing the tibronectin concentration. (a) approx. 3000; (b) approx. 4000 glomeruli per well were incubated in media containing insulin, transfer-k, selenium, and dimeric or multimeric FN for (a) 15; (b) 6 days. The cells were fixed and stained with Coomassie blue and the number of colonies and their size were determined by direct counting after (a) 15; (b) 6 days. In (a) only four total colonies were present under these low glomerular density conditions in dimeric FN (some single cells and small colonies less than 16 cells were present), but under the same conditions 106 colonies were present in the multimeric FN medium, and these contained an average of 127.8 cells/colony (total number cells counted in multimeric FN= 13 545 cells). In (b) growth was so much more rapid at 5 @ml FN that it was necessary to terminate the experiment much earlier than in (a) so that counting would be feasible. 105 colonies were present after 6 days in dimeric FN (41.6 cells/colony; 4374 cells counted), while 210 colonies were present after 6 days in multimeric FN (52.0 cells/colony; 10928 cells counted). Note that abscissas and ordinates differ in (a) and (b). Fig.
273
274
Oberley
et al.
Exp Cell
number of initial glomeruli was counted in each of five arbitrary areas (0.16 cm’), and then the number of cells was counted daily in each area. In comparisons of the average number of cells observed each day with the average number of initial glomeruli, it was apparent that glomerular cells grew faster in multimeric than in dimeric FN at concentrations of l-10 ug/ml (fig. 6a). This stimulating effect of multimeric FN was even more apparent at 25 &ml (fig. 6 b). At these concentrations cell growth was two to three times better in multimeric than in dimeric FN after 8 days of culture. A second method chosen to measure cell growth was to count the number of cells per colony. This type of experiment also demonstrated that growth of glomerular cells was greater in multimeric than dimeric FN (fig. 7); the number of colonies and the number of cells per colony was greater in multimeric than dimeric FN. We have recently demonstrated that the glomerular cell requirement for insulin, transferrin, and selenium may be replaced by glycylhistidyl lysine (GHL) [131. In experiments not shown, multimeric FN also stimulated growth better than dimeric FN in media containing GHL at 50 yg/ml rather than insulin, transferrin, and selenium. Thus, preferential growth stimulation by multimeric FN was not dependent on the presence of insulin, transferrin, and selenium. DISCUSSION Recent articles [17, 181 have provided detailed discussions of ‘stem’ cell properties in tissues in which there is rapid turnover and loss of differentiated cells, examples being small intestine, skin, and bone marrow. Probable properties of stem cells include pluripotentiality, self-renewal, and a probable slow cycling time. Stem cells give rise to ‘clonogenic’ cells, a cell which though capable of cloning, is nevertheless committed to a pathway of differentiation. Clonogenic cells then give rise to transit cells (capable of dividing but morphologically and biochemically differentiated) and finally mature cells (morphologically and biochemically differentiated and incapable of cell division). Whereas there is extensive knowledge of differentiation pathways in rapidly renewing tissues, much less is known about an organ such as the kidney in which cell division and cell growth in the adult animal usually occurs only as a result of cell injury. There is, in fact, however, evidence for ‘stem’ cells in vivo in the adult mammalian kidney. Thorning & Vracko [19] have demonstrated that poorly differentiated cells move into areas of freeze-thaw injury of the adult rat kidney and differentiate according to the basement membrane in which they come into contact; thus these cells become epithelial cells if they attach to the epithelial side of the glomerular basement membrane and become endothelial cells if they attach to the endothelial side. Such results suggest that in organs in which a reparative response is possible, the cell types involved may be ‘stem’ cells, since the cells are pluripotential. We believe that the cells observed in the present study are probably ‘clonogenie’ cells and not true ‘stem’ cells, since the progeny cells at the edge of each colony appear to be uniform in morphology and biochemistry [ 131. The cells at
Res
145 (1983)
Exp Cell Res 145 (1983)
Multimeric
fibronectin
and glomerular
cell growth
the edge do not exhibit the properties of mesangial cells (intense cell surface staining for fibronectin [7, 201 and laminin [7]), but instead they biochemically and morphologically resemble epithelial cells [ 131. Since there is no evidence that these cells are pluripotential, it seems more likely they are ‘clonogenic cells’. There is also biochemical evidence which suggests that the cells in the center of each colony are precursor cells. Quaroni et al. [21] have demonstrated a higher concentration of fibronectin in the small intestinal crypt (an area of precursor cell development) as opposed to the villus (fully differentiated cells). Bender et al. [22], Baumann & Eldredge [14], and Ekblom et al. [23] have all suggested that only fetal or rapidly dividing cells are able to synthesize their own basement membrane components. The ability of the poorly differentiated cell to assemble fibronectin and laminin matrix suggests it is a precursor cell [ 131. Therefore the present study documents a role for tibronectin in the clonal growth of a poorly differentiated glomerular cell type which we believe to be a precursor cell to the visceral epithelial cell. Whereas the evidence that this cell is in fact a ‘clonogenic’ cell is still not conclusive, our present results certainly suggest this may be true. For instance, we have calculated in this study that under optimal growth conditions in the presence of multimeric FN, only approx. 5% of glomeruli placed in culture produce colonies (unpublished evidence). If instead, the cells seen were the result of ‘dedifferentiation’, one would expect all glomeruli to produce colonies. In fact, dedifferentiation does occur when glomeruli are grown in calf serum [5], and it is interesting that cells grown under these conditions grow as sheets of cells rather than colonies [9]. We hope in the future to resolve the controversy concerning the nature of the cell type involved by obtaining immunologic markers for ‘stem’ vs differentiated glomerular visceral epithelial cells. Work in progress in fact suggests that there are precursors of other cell types present in the kidney. We have been successful in growing cells with the morphologic features of poorly differentiated tubular cells in a serum-free medium containing insulin, transferrin, selenium, tri-iodothyronine, and laminin (unpublished observations), whereas Chung et al. [24] has grown biochemically well differentiated tubular cells in a serum-free medium containing insulin, transferrin, and hydrocortisone without an extracellular matrix molecule. We are presently hypothesizing that each precursor cell type will have an extracellular matrix molecule that will stimulate its cell growth under the proper conditions. The present in vitro study suggests a role for fibronectin in glomerular cell adhesion. In fact, immunohistologic studies of glomeruli in vivo have localized fibronectin between basement membrane proper (lamina densa) and visceral epithelial cells [20, 251; we therefore feel that the present in vitro studies are consistent with the hypothesis that fibronectin acts as an adhesive protein to mediate attachment of glomerular epithelial cells to their substrate (plastic in vitro, basement membrane in vivo). Although the present study documents a role for fibronectin in glomerular cell growth in vitro, there is no evidence for such a role in vivo. The requirement of the poorly differentiated cell for fibronectin is specific, since we have not been able to obtain growth with laminin, heparan sulfate proteoglycan, or type IV
275
276
Oberley
et al.
Exp Cell Res 145 (1983)
collagen in identical serum-free media. Yamada et al. [26] has demonstrated that cell surface fibronectin affects the growth of certain fibroblast cell lines in vitro by allowing them to attain a higher saturation density than in plasma fibronectin. It is therefore probable that the different forms of fibronectin can indeed have different effects on cell growth, dependent on the cell types involved. We believe that multimeric tibronectin may possibly have a role in cell growth in vivo, since there is evidence for disultide-loaded fibronectin multimers on cell surfaces [27]; our future research will be directed at establishing whether these multimers have a physiologic role in cell division in vivo and in vitro. This research was supported by grants from the Hereditary Nephritis Foundation, the Kidney Foundation, and the Research and Development Committee of the Department of Pathology to T. D. 0. Support was also received from Grant HL-21644 to D. F. M. This work was done during the tenure of an Established Investigatorship from the American Heart Association and its Wisconsin Affiliate to D. F. M. J. E. M. was supported by a predoctoral traineeship CA-09106 from NIH. The authors would like to thank MS Carol Gabel, MS Ruth B. Johnson, Mr Patrick J. Murphy and MS Fran Simandl for excellent technical assistance.
REFERENCES 1. Norgaard, J 0 R, Renal physiology. The glomerular basement membrane (ed G M Berlyne & S Thomas) p. 220. S. Karger, Basle (1980). 2. Foidart, J B, Dechenne, C A, Mahieu, P, Creutz, C E 8t DeMey, J, Invest cell path01 2 (1979) 15. 3. Kreisberg, J I, Hoover, R L & Kamovsky, M J, Kidney intern 14 (1978) 21. 4. Killen, P D & Striker, G R, Proc natl acad sci US 77 (1980) 994. 5. Norgaard, J 0 R, Lab invest 38 (1978) 320. 6. Scheinman, J I, Fish, A J, Kim, Y & Michael, A F, Am j path01 92 (1978) 147. 7. Oberley, T D, Chung, A E, Murphy-Ullrich, J E & Mosher, D F, J histochem cytochem 29 (1981) 1237. 8. Striker, G E, Killen, P D & Farin, F M, Transplant proc 12 (1980) 80. 9. Oberley, T D, Burkholder, P M, Barber, T A & Hwang, C C, Invest cell path01 2 (1979) 27. 10. Barnes, D & Sato, G, Anal biochem 102 (1980) 255. 11. Oberley, T D, Murphy-Ullrich, J E & Muth, J V, Diagnost histopatho14 (1981) 117. 12. Rojkind, M, Gatmaitan, Z, Mackensen, S, Gimbrone, M A, Dance, P & Reid, L, J cell biol 87 (1980) 255. 13. Oberley, D, Murphy, P J, Steinert, B W & Albrecht, R M, Virchows arch b (cell pathol) 41 (1982) 145. 14. Baumann, H & Eldredge, D, J cell bio195 (1982) 29. 15. Mosher, D F, Schad, P E & Vann, J M, J biol them 255 (1980) 1181. 16. Chen, T R, Exp cell res 104 (1977) 255. 17. Lajtha, L G, Differentiation 14 (1979) 23. 18. Potten, C S, Schofield, R & Lajtha, L, Biochim biophys acta 560 (1979) 281. 19. Thoming, D & Vracko, R, Lab invest 37 (1977) 105. 20. Courtoy, P J, Kamvar, Y S, Hynes, R 0 & Farquhar, M G, J cell biol 87 (1980) 691. 21. Quaroni, A, Isselbacher, K J & Ruoslahti, E, Proc natl acad sci US 75 (1978) 5548. 22. Bender, B L, Jaffe, R, Carlin, B & Chung, A E, Am j path01 102 (1981) 419. 23. Ekblom, P, Thesleff, I, Miettinen, A & Saxen, L, Cell differ 10 (1981) 281. 24. Chung, S D, Alavi, N, Livingston, D, Hiller, S & Taub, M, J cell bio195 (1982) 118. 25. Oberley, T D, Mosher, D F & Mills, M D, Am j path01 % (1979) 651. 26. Yamada, K M, Kennedy, D W & Hayashi, M, Growth of cells in hormonally defined media (ed G H Sate, A B Pardee 8r D A Sirbaska) p. 131. Cold Spring Harbor Laboratory, N.Y. (1982). 27. Perkins, M E, Ji, T H & Hynes, R 0, Cell 16 (1979) 941. Received October 15, 1982 Revised version received January 11, 1983
Rinted
in
Sweden
Cell Research
145 (1983) 265-276
Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/83/060265-12$02.00/0
The Effect of the Dimeric and Multimeric Forms of Fibronectin Adhesion and Growth of Primary Glomerular Cells TERRY RALPH
on the
D. OBERLEY ,l, * JOANNE E. MURPHY-ULLRICH,’ M. ALBRECHT3 and DEANE F. MOSHER*
‘Departments of Pathology and Laboratory Medicine and 2Medicine, University of Wisconsin, Medical School, and ‘School of Pharmacy, University of Wisconsin, Madison, 53706, USA
SUMMARY Primary glomerular cells placed in a chemically defined medium containing Waymouth’s medium MB 752/l supplemented with insulin, transferrin, fibroblast growth factor, nonessential amino acids, sodium pyruvate, and antibiotics showed rapid outgrowth of cells which morphologically resembled well differentiated visceral epithelial cells followed by outgrowth of poorly differentiated cells; morphologic evidence suggests these latter cells are precursor cells of the epithelial cell lineage. Whereas the well differentiated glomerular epithelial cells were never observed to divide by sequential phase microscopic observations, a chemically defined medium was developed for optimal growth of the poorly differentiated cell type. This serum-free medium contained Waymouth’s medium MB 752/l supplemented with insulin, transferrin, selenium, and tibronectin (plus non-essential amino acids, sodium pyruvate, and antibiotics). Using this chemically defined medium, we have compared the effects of dimeric and multimeric fibronectin (high molecular weight disulfide-bonded tibronectin produced by incubation of dimeric fibronectin with 3 M guanidine followed by dialysis against 0.05 M cyclohexylaminopropane sulfonic acid (CAPS) buffer, pH 11) on the adhesion and growth of the poorly differentiated primary glomerular cell type. Dimeric tibronectin (FN) was twice as effective as multimeric FN in promoting glomerular cell adhesion, although both forms of FN promoted cell adhesion better than an uncoated substratum. In contrast, cell growth studies demonstrated that multimeric FN was a more potent growth stimulant than dimeric FN. The differential effects of dimeric and multimeric forms of FN in vitro suggests that these molecules may have different functions in vivo.
Analysis of the growth of glomeruli in culture is made difficult by the fact that glomeruli are composed of four cell types (visceral and parietal epithelial cells, mesangial, and endothelial cells), and each of these cell types may potentially grow out in primary culture. Further, until recently glomeruli could only be grown in high concentrations of calf serum, which made quantitative analysis of glomerular cell growth difficult because of the complex nature of serum. A number of investigators have attempted to characterize glomerular cells grown in calf serum. The most definitive work on identification of glomerular cells grown in calf serum has used [3H]thymidine ([3H]TdR) labelling followed by autoradiography [l]; this study demonstrated that the initial cells seen in culture are the visceral epithelial cells, whereas the mesangial cells grew much later in culture. Foidart et al. [2] also reported studies with 13HlTdR labelling of renal glomeruli in which two major peaks of [3H]TdR incorporation were described, the first involving epithelial cells and the second mesangial cells. Parietal epitheli-
* To whom offprint requests should be sent. Address: Department of Pathology and Laboratory Medicine, 516 SMI, 470 N. Charter St., Madison, 53706, USA.
266
Oberley
et al.
Exp Cell
al cells are seen only rarely growing from glomeruli in calf serum, presumably because most glomeruli do not contain Bowman’s capsule 131. Endothelial cells have never been morphologically identified in glomerular culture and all glomerular cell types are negative for factor VIII antigen [4]. Because only two major cell types are present in glomerular culture and these types grow out of glomeruli at different times, it has been possible for investigators to attain relatively uniform populations of epithelial and mesangial cells [2]. However, there still remains a great deal of controversy concerning the nature of glomerular cells in vitro, since cells identified as ‘epithelial’ in origin lack the morphologic and biochemical features of true epithelial cells; they do not have visceral epithelial cell foot processes [5] or C3b receptors [6]. Nevertheless, the literature presents convincing evidence that these cells must be some form of glomerular epithelial cells since: (1) they produce laminin [7], heparan sulfate proteoglycan [8], type IV collagen [8], and libronectin [81, all properties of visceral epithelial cells and not fibroblasts; and (2) by both scanning (SEM) and transmission electron microscopy (TEM) these cells can be seen to grow unequivocally from glomeruli [9]. There are at least two major possibilities to account for differences in the morphology and biochemistry of glomerular cells grown in vitro in the presence of calf serum. One possibility is that ‘dedifferentiation’ can occur, whereas a second possibility is that serum stimulates the growth of precursors of epithelial cells. To try to resolve this controversy, we have recently begun studying the growth of glomerular cells in chemically defined media. Since Barnes & Sato [lo] have demonstrated that hormones and growth factors allow the growth of many mammalian cell lines, we decided to study whether glomerular cells could be grown in chemically defined media. Glomeruli grown in Waymouth’s medium containing insulin, transferrin, and fibroblast growth factor (FGF) showed two cell types, a typical well differentiated visceral epithelial cell with long cytoplasmic processes and a poorly differentiated cell [ 111. Despite careful observation, in this medium we never observed one of these cell types transforming into the other, and we never observed the well differentiated cell undergoing cell division. Since the well differentiated cell appeared rapidly and did not divide, we interpreted these cells as visceral epithelial cells which simply moved from the glomerulus to the culture surface. The nature of the poorly differentiated cell was uncertain. Since Rojkind et al. [12] has emphasized the importance of the extracellular matrix in cell growth, we decided to add fibronectin as a component of the culture media. We found that glomeruli grown in Waymouth’s media containing insulin, transferrin, and fibronectin demonstrated growth of the poorly differentiated cell into distinct colonies. We have recently demonstrated with immunofluorescence studies that the cells in the center of these colonies are surrounded by a fibronectin and laminin matrix, whereas the cells on the edge of the colonies typically show less extracellular matrix staining [13]. We presently hypothesize that these poorly differentiated cells are precursor cells of glomerular visceral epithelial cells for three reasons: (1) the cells show colony growth, typical of ‘clonogenic’ cells; (2) the edge of the colonies show cells which resemble well differentiated
Res
145 (1983)
Multimeric
Exp Cell Res 145 (1983)
fibronectin
and glomerular
cell growth
visceral epithelial cells; and (3) since it has been shown recently that fetal (but not adult) hepatocytes produce tibronectin [ 141, the fact that the poorly differentiated cells assemble a fibronectin (and laminin) matrix certainly suggests the possibility that they are precursor cells. Despite these arguments, it has not been possible to date to establish conclusively the identity of glomerular cell types. However, the development of a system in which glomerular cells could be grown in serum-free medium has allowed us to examine the role of fibronectin in the adhesion and growth of primary glomerular cells. The following report presents evidence on the role of the dimeric and multimeric forms of fibronectin on the poorly differentiated cell type, which we believe to be a precursor form of the visceral epithelial cell. MATERIALS
AND
METHODS
Animals Adult Hartly guinea pigs of either sex were purchased locally with weights of about 300 g and maintained on laboratory chow and drinking water supplemented with ascorbic acid.
Cell culture Glomeruli were isolated from young adult guinea pigs according to a nylon screening technique previously described [9]. The basic medium contained Waymouth’s media MB 752/I (Gibco) supplemented with sodium pyruvate (1%) (Gibco), non-essential amino acids (1%) (Gibco), and penicillin-streptomycin (100 units/ml) (Gibco). In most experiments the basic media was supplemented with insulin (5 @ml), transferrin (5 &ml), selenium (5 rig/ml) (ah prepared from insulin, transfer&, selenium premix from Collaborative Research), and dimeric or multimeric FN (0.1-25 ug/ml). To demonstrate the morphology of well differentiated epithelial cells in culture, in one experiment the basic medium was supplemented with insulin, transferrin, and FGF without fibronectin [l I]. Glomeruli were placed in culture in 9.6 cm* plastic tissue culture wells (Costar) in an atmosphere of 5% C02-95% air. Each well originally contained 10 ml of media, and 1 ml of new media was added on Monday through Friday. All experiments were repeated on at least two separate occasions. Progress of the cultures was monitored with a Leitz phase microscope. At appropriate intervals, cells were stained in situ with Coomassie brilliant blue R250 (2.5 % in methanol : water : acetic acid; 5.5 : 4.5 : 1; v : v : v) after fixation with 4% glutaraldehyde in phosphate-buffered saline (PBS). For direct visual comparison of cell growth, cells were photographed with a Zeiss bright-field microscope using Panatomic X fdm. All of the results reported in the present study were performed early in glomerular culture before the appearance of mesangial cells (probably actually mesa&al cell precursrs).
Transmission
electron microscopy
(TEM)
Glomeruli were grown in chemically defined media containing insulin, transferrin, selenium and FN. After the cells were confluent, they were detached from the culture substrate with EDTA and trypsin and fixed for 4 h in 4% cacodylate-buffered glutaraldehyde. The cells were secondarily fixed in 1% osmium tetroxide in s-collidine buffer, dehydrated in a graded alcohol series, and embedded in an Epon 812-Araldite epoxy mixture. Ultrathin sections were double-stained with lead citrate and uranyl acetate and examined at 75 kV with a Hitachi H-500 electron microscope.
Scanning
electron microscopy
(SEM)
Cells were prepared for SEM according to a protocol previously described [9]. Cells were grown in 25 cm* Costar flasks and areas of interest cut from the plastic surface. The cells were washed several times with PBS before furation in 0.1 M phosphate-buffered saline (PBS)- 1% ghttaraldehyde (pH 7.4). Cells were fixed for 24-48, dehydrated via an alcohol series, 30 min each in 30%, 50%, 75%, 85 %, 90 %, 95 %, and two 30-min periods in 100 % ethanol. Cells were critical point-dried with 100% ethanol as the intermediate fluid and liquid CO2 as the transitional fluid. The plastic substrate containing cells were cemented onto metal stubs and coated with a 12 nm gold layer. Cells were viewed at 15 kV accelerating voltage on a JEOL 356 scanning electron microscope.
267
268 Oberley et al.
Exp Cell Res 145 (1983)
Fig. 1. SDS polyacrylamide (6% running gel and 3 % stacking gel) slab gel electrophoresis of isolated human plasma fibronectin and its derivatives. a, MW markers (unreduced): ferritin halfunit, 220000 D; BSA, 67000 D. b, Plasma FN (25 ug/well) before reduction. The gel was overloaded to show minor bands. A major band at approx. 440000 is present (arrow). Other bands present are minor components representing aggregates of the monomer and dimer. c, Multimer FN (31 @well) before reduction. Most material is at the top of the stacking and separating gels. A small amount of monomeric (230000 MW) FN is also seen. d, MW markers reduced with mercaptoethanol. e, Plasma FN after reduction (2.5 udwell) (double arrow). The protein is present at MW 230 000. J Multimeric FN after reduction (3 1 ug/well). Again the protein is present at MW 230000. The direction of migration was from top to bottom and the gel was stained with Coomassie brilliant blue R.
Protein
isolation
Human FN was isolated from plasma or the supematant after heat precipitation of a fibronectin- and fibrinogen-rich plasma protein fraction. The solution was passed over a gelatin-Sepharose column with subsequent elution with 1 M sodium bromide, pH 5 [IS]. Purity of FN preparations was assessed by sodium dodecyl sulfate (SDS) slab gel electrophoresis. Dimeric FN was converted to disulfidebonded multimeric FN by incubation with 3 M guanidine followed by dialysis against 0.05 M cyclohexylaminopropane sulfonic acid (CAPS) buffer, pH 11 [3]. The conversion of the dimeric form of FN to its multimeric form was monitored by SDS slab gel electrophoresis (see Results). More details of the preparative methods and characterization of disuhide-bonded FN multimers will be presented elsewhere [3].
Cell adhesion Costar wells (9.6 cm*) were coated with dimeric FN (5 &ml), multimeric FN (5 ug/ml), or media alone, air-dried, washed twice with double distilled water, and sterilized with ultraviolet light. Isolated glomeruli were grown in chemically defined media containing insulin, transfenin, and dimeric FN, until confluency was reached. After brief treatment in 0.01 M EDTA in Tris buffer, the cells were trypsinized until detachment occurred and soybean trypsin inhibitor was then added to stop the reaction. The cells were washed with media alone and plated in the coated wells. 2x 10’ cells were added to each well. No cell clumps were included in the analysis; most were removed by allowing large clumps to settle and pipetting off the single cells in the supematant. After 1 h, the wells were washed with media and the number of attached cells determined by direct counting under phase microscopy. The results were expressed as averages of duplicate determinations.
DNA staining DNA staining tin, selenium, with a solution distilled water
was performed as described by Chen [16]. Glomerular cells grown in insulin, transferand Ebronectin were futed in Camoy’s fixative for 30 min, air-dried and then stained of Hoechst 33258 in PBS (0.5 pg/ml) for 30 min. The cells were rinsed twice in doubleand then mounted in PBS.
Cell growth Method no. I. Glomeruli were added to Costar wells which had been marked on the bottom with grids which were 0.16 cm’ in area. Five of these grids were arbitrarily selected and the number of initial glomeruli plated in each grid was counted. The glomeruli were then fed defined media containing varying amounts of dimeric or multimeric FN daily (Monday-Friday). Each day the number of cells in each of the five marked grids was counted by phase microscopy. Cell growth was then measured by calculating the ratio of the average number of cells observed each day relative to the average in&fat number of glomeruh. This method of determining cell growth was chosen for a number of reasons: (1) Because glomerular cells are of varying sizes and shapes and tend to clump together after dissociation, it was felt that direct counting while the cells were attached and flattened woufd be more accurate than using a Coulter counter.
Exp Cell Res 145 (1983)
Multimeric
fibronectin
and glomerular
cell growth
Fig. 2. Phase microscopy of cells grown in serum-free media without fibronectin. Cells were grown in Waymouth’s medium containing insulin, transferrin, and FGF without tibronectin and examined by phase microscopy. Gtomeruhts (g) is surrounded by (a) well differentiated visceral epithelial cells. The large cell is of the poorly differentiated cell type (arrow); (b) a number of cells of the poorly differentiated cell type. A well differentiated epithelial cell is also seen (arrow). Since both cell types grew from the glomerulus rapidly, it seems likely that both cells are a form of epithelial cell type; previous investigations have shown that mesangial cells require a great deal of time before growing in culture [ 1, 21. (a, b) x 200. (2) Because of the particulate nature of the glomeruli, it was often difficult to pipette exactly equal numbers of glomeruli into each Costar well; therefore the ratio of cells observed relative to the number of initial glomeruli was felt to be more reliable than calculation of the total cell count. (3) Averages of results are presented without calculation of standard deviations because the clonal nature of glomerular growth makes such calculations meaningless; each colony was unique, with a large variation in the total number of cells per colony. Merhod no. 2. To confirm the results of method no.1, direct counts were performed on glomerular colonies. Equal numbers of glomeruli were added to Costar wells (approx. 50 per 0.16 cm’ grid). After either 6 or I5 days following daily feeding, the number of cells in every colony greater than 16 cells was counted, and a histogram was constructed. A cell had to be approximately one cell length or less from the main colony to be included in the cell count.
RESULTS Protein isolation FN isolated from serum or plasma protein fractions [15] on gelatin-Sepharose columns, analysed by slab electrophoresis before and after reduction with mercaptoethanol (fig. 1, lanes b, e), migrated principally as a band of approximate molecular weight (MW) 440000 before reduction, and 230000 after reduction. Multimeric FN (produced by incubation of dimeric FN with 3 M guanidine and dialysis against CAPS buffer, pH 11) migrated with higher MW (greater than 440000) before reduction and 230000 after reduction (fig. 1, lanes c, f). Cell types Cells grown in Waymouth’s medium supplemented with insulin, transferrin, and FGF without FN showed two different cell types, one which resembled a well differentiated visceral epithelial cell and a second cell type which was poorly differentiated (fig. 2a, b). The well differentiated cell type has never been
269
270
Oberlev
et al.
Exp Cell Res 145 (1983)
Fig. 3. Morphology of glomerular cells in culture. Glomeruli were grown in a chemically defined medium containing insulin (5 @ml), transferrin (5 ug/ml), selenium (5 rig/ml), and dimeric or multimeric fibronectin (5 &ml). Identical results were obtained with either cell type. (a) Bright-field micrograph of glomerular colony. Cells were of a large uniform size and grew closely opposed. (b) Electron microscopy of glomernlar cells grown in serum-free medium. The cells observed were always attached to basement membrane and not banded collagen, suggesting that they were a form of glomerular cell and not a fibroblast contaminant. The nuclei have prominent nucleoli and the cells have a moderate number of mitochondria. Microvilli are noted on the cell surface. (c) SEM of glomerular cells grown in serum-free medium. Glomeruli were present in the center of all colonies. The cells were epithelial in shape, had a smooth surface, and were extremely flattened. (a) x 112; (b) x9900; (c) x440.
observed in cells growing in calf serum [ll]. Sequential observation of single, well differentiated epithelial cells by phase microscopy over a period of days revealed that although they did migrate, they did not appear to divide (this study and [ll]). The well differentiated cell type grew as widely spaced single cells, appeared rapidly after inoculation of glomeruli in culture, and appeared to require FGF for its appearance. In contrast, the poorly differentiated cell type usually grew as colonies, appeared later in culture, and could be observed in cultures containing only insulin and transferrin without FGF. Despite the appearance of these two
Exp Cell Res 145 (1983)
Multimeric fibronectin
and glomerular cell growth
4. SEM of cell at edge of glomerular colony grown in serum-free medium. Cells are small, with a rounded central cell body and numerous long cytoplasmic extensions (arrow). True foot processes were not seen. X 1000. Fig. 5. DNA stain of glomerular cells grown in serum-free medium. To demonstrate that the cells seen in culture were the result of cell growth and not the simple movement of cells from glomerulus to culture surface. glomerular cells grown in serum-free medium were stained with the Hoechst stain 33258. Mitoses were often seen, indicating true cell growth (arrow). x250. Fig.
cell types, glomeruli grown in the absence of tibronectin showed very poor overall growth and did not become confluent. When dimeric fibronectin was added to a chemically defined medium containing insulin, transferrin, and selenium (without FGF), only the poorly differentiated cell type was observed, and this cell type grew in colonies (fig. 3 a). Morphologic study of cells grown in this identical medium but in which dimeric fibronectin was replaced by multimeric fibronectin showed similar cell outgrowths. TEM and SEM also suggested that cells grown in insulin, transferrin, selenium, and either dimeric or multimeric fibronectin were identical. TEM of cells grown in either fibronectin type demonstrated that although the cells were attached to glomerular basement membrane, the cells did not have typical epithelial cell arbors or foot processes and contained prominent nucleoli, a feature of a poorly differentiated cell type (fig. 3 b). SEM clearly demonstrated a glomerulus at the center of each colony, and the cells observed were extremely flattened (fig. 3 c). However, at the edge of colonies there were cells which morphologically resembled well Table 1. The effect of dimeric us multimericfibronectin glomerular cells
on adhesion of primary
Type of substrate
% of cells attached at 1 h
Dimeric FN
Multimeric
100
52
FN
Medium alone 31
Wells were coated with dimeric or multimeric FN (5 @ml) or medium alone as described in the Methods. 2x lo3 cells were plated in coated Costar wells, incubated at 37°C for 1 h, and then washed. The cells were counted and the % of cells attached at 1 h determined. The values expressed are the average of two determinations.
271
272
Oberley
Exp Cell
et al.
30
I
b MULTIYERIC (lo;0 IfnIl
FN
25
I’
J MULTIMERIC Ii;q Iml)
FN r
MULTIMERIC _’
Y”’
FN lrn’)
/,/
F
/’ ,’
FN
r/’
DAYS
MULTIMERIt tl~qlml) __*/ DIMERIC i25uq
Fk Iml!
IF1 CULTURE
Fig. 6. The effect of dimeric vs multimeric fibronectin on the growth of primary glomerular cells: average number of cells in outgrowths per average number of initial glomeruli in 0.16 cm* area (method no. 1). (a) Approx. 7000, (b) approx. 5000 glomeruli were added to Costar wells (9.6 cm*) containing insulin, transferrin, selenium, and/or (a) 10 &ml of FN (10 cc of media per well at start of experiment); (b) 25 pg/ml FN. Glomeruli were fed daily (1 cc of media per well per day) and cells counted directly by phase microscopy. (n) 5~10~; (b) 2~10~ cells (approx.) were present at day 8 when cells were fed, (n) 10; (b) 25 &ml multimeric FN; the other wells had fewer cells (a). Cells fed 1 &ml x , dimeric; 0, multimeric FN daily; cells fed 10 &ml A, dimeric; 0, multimeric FN daily. (b) Cells fed 1 @ml X, dimeric; 0, multimeric FN daily; cells fed 25 ug/ml A, dimeric; 0, multimeric FN daily.
differentiated glomerular epithelial cells (fig. 4); these cells appeared only after the colony had become large, in contrast to the well differentiated cells seen in a serum-free medium containing FGF but without fibronectin, which rapidly grew (or migrated?) from the glomeruli. Cell adhesion
To test the effect of the dimeric and multimeric forms of FN on cell adhesion, tissue culture wells were coated with the two forms of FN and then trypsinized cells from primary cultures added to each well. The cells were washed at 1 h, and the percentage of attached cells was determined by direct counting (table 1). It was found that although both forms of FN promoted cell adhesion better than an uncoated substratum, dimeric FN was twice as effective as multimeric FN. Cell growth
Glomeruli did not show significant growth in chemically defined medium containing insulin, transferrin, and selenium when incubated at FN concentrations of 0.01 or 0.1 ug/ml, while at FN concentrations of 1 or 10 &ml FN, significant growth occurred. We are certain that this represents real growth and not simple movement of cells from the glomerulus to the culture surface, since the Hoechst stain for DNA showed frequent mitoses (fig. 5). To quantitate this effect, the
Res 145 (1983)
Multimeric
Exp Cell Res 145 (1983)
fibronectin
and glomerular
cell growth
a 30
MULTIMERIC
FIBRONECTIN
(I ug Iml)
DIMERIC
FIBRONECTIN
30
(luglml)
25
25
20
I5
N-IO
n
IO
N-4
5
N-3
N-4 -
N=S
25l500
3Ol350
35l400
o50
SI100
IOII50
ISI200
2Ok 250
NUMBER
401450
OF
o50
CELLS
5IIO0
PER
IOII50
151- 2Ol200 250
251300
3Ol350
35l400
401450
COLONY
b 100 90 In g 60
‘1
MULTIMERIC
FIBRONECTIN
(5ug
Iml)
DIMERIC
FIBRONECTIN
100
(buglml)
90 60
I s 0 0
70
70
I& 60
60
a
50
i# I 40
50
11.45 N-37
40
N-34
is 30
30
20
N.17
20
IO
IO
51T!l
76100
IOII26
IOII60
l6lI75
NUMBER
I76200
POI225
OF
CELLS
o26
PER
2650
5I75
N’4
N=3
N.2 n
76I00
IOII25
I26I50
ISIIT5
II6200
2Ol225
COLONY
7. The effect of dimeric vs multimeric fibronectin on the growth of primary glomcrular cells: histogram analysis of the number of cells per colony in 9.6 cm2 well (Method no. 2). (a) 1; (b) 5 @ml: to demonstrate the effect of (a) multimeric FN at low glomerular density and low concentrations of FN; (b) increasing the tibronectin concentration. (a) approx. 3000; (b) approx. 4000 glomeruli per well were incubated in media containing insulin, transfer-k, selenium, and dimeric or multimeric FN for (a) 15; (b) 6 days. The cells were fixed and stained with Coomassie blue and the number of colonies and their size were determined by direct counting after (a) 15; (b) 6 days. In (a) only four total colonies were present under these low glomerular density conditions in dimeric FN (some single cells and small colonies less than 16 cells were present), but under the same conditions 106 colonies were present in the multimeric FN medium, and these contained an average of 127.8 cells/colony (total number cells counted in multimeric FN= 13 545 cells). In (b) growth was so much more rapid at 5 @ml FN that it was necessary to terminate the experiment much earlier than in (a) so that counting would be feasible. 105 colonies were present after 6 days in dimeric FN (41.6 cells/colony; 4374 cells counted), while 210 colonies were present after 6 days in multimeric FN (52.0 cells/colony; 10928 cells counted). Note that abscissas and ordinates differ in (a) and (b). Fig.
273
274
Oberley
et al.
Exp Cell
number of initial glomeruli was counted in each of five arbitrary areas (0.16 cm’), and then the number of cells was counted daily in each area. In comparisons of the average number of cells observed each day with the average number of initial glomeruli, it was apparent that glomerular cells grew faster in multimeric than in dimeric FN at concentrations of l-10 ug/ml (fig. 6a). This stimulating effect of multimeric FN was even more apparent at 25 &ml (fig. 6 b). At these concentrations cell growth was two to three times better in multimeric than in dimeric FN after 8 days of culture. A second method chosen to measure cell growth was to count the number of cells per colony. This type of experiment also demonstrated that growth of glomerular cells was greater in multimeric than dimeric FN (fig. 7); the number of colonies and the number of cells per colony was greater in multimeric than dimeric FN. We have recently demonstrated that the glomerular cell requirement for insulin, transferrin, and selenium may be replaced by glycylhistidyl lysine (GHL) [131. In experiments not shown, multimeric FN also stimulated growth better than dimeric FN in media containing GHL at 50 yg/ml rather than insulin, transferrin, and selenium. Thus, preferential growth stimulation by multimeric FN was not dependent on the presence of insulin, transferrin, and selenium. DISCUSSION Recent articles [17, 181 have provided detailed discussions of ‘stem’ cell properties in tissues in which there is rapid turnover and loss of differentiated cells, examples being small intestine, skin, and bone marrow. Probable properties of stem cells include pluripotentiality, self-renewal, and a probable slow cycling time. Stem cells give rise to ‘clonogenic’ cells, a cell which though capable of cloning, is nevertheless committed to a pathway of differentiation. Clonogenic cells then give rise to transit cells (capable of dividing but morphologically and biochemically differentiated) and finally mature cells (morphologically and biochemically differentiated and incapable of cell division). Whereas there is extensive knowledge of differentiation pathways in rapidly renewing tissues, much less is known about an organ such as the kidney in which cell division and cell growth in the adult animal usually occurs only as a result of cell injury. There is, in fact, however, evidence for ‘stem’ cells in vivo in the adult mammalian kidney. Thorning & Vracko [19] have demonstrated that poorly differentiated cells move into areas of freeze-thaw injury of the adult rat kidney and differentiate according to the basement membrane in which they come into contact; thus these cells become epithelial cells if they attach to the epithelial side of the glomerular basement membrane and become endothelial cells if they attach to the endothelial side. Such results suggest that in organs in which a reparative response is possible, the cell types involved may be ‘stem’ cells, since the cells are pluripotential. We believe that the cells observed in the present study are probably ‘clonogenie’ cells and not true ‘stem’ cells, since the progeny cells at the edge of each colony appear to be uniform in morphology and biochemistry [ 131. The cells at
Res
145 (1983)
Exp Cell Res 145 (1983)
Multimeric
fibronectin
and glomerular
cell growth
the edge do not exhibit the properties of mesangial cells (intense cell surface staining for fibronectin [7, 201 and laminin [7]), but instead they biochemically and morphologically resemble epithelial cells [ 131. Since there is no evidence that these cells are pluripotential, it seems more likely they are ‘clonogenic cells’. There is also biochemical evidence which suggests that the cells in the center of each colony are precursor cells. Quaroni et al. [21] have demonstrated a higher concentration of fibronectin in the small intestinal crypt (an area of precursor cell development) as opposed to the villus (fully differentiated cells). Bender et al. [22], Baumann & Eldredge [14], and Ekblom et al. [23] have all suggested that only fetal or rapidly dividing cells are able to synthesize their own basement membrane components. The ability of the poorly differentiated cell to assemble fibronectin and laminin matrix suggests it is a precursor cell [ 131. Therefore the present study documents a role for tibronectin in the clonal growth of a poorly differentiated glomerular cell type which we believe to be a precursor cell to the visceral epithelial cell. Whereas the evidence that this cell is in fact a ‘clonogenic’ cell is still not conclusive, our present results certainly suggest this may be true. For instance, we have calculated in this study that under optimal growth conditions in the presence of multimeric FN, only approx. 5% of glomeruli placed in culture produce colonies (unpublished evidence). If instead, the cells seen were the result of ‘dedifferentiation’, one would expect all glomeruli to produce colonies. In fact, dedifferentiation does occur when glomeruli are grown in calf serum [5], and it is interesting that cells grown under these conditions grow as sheets of cells rather than colonies [9]. We hope in the future to resolve the controversy concerning the nature of the cell type involved by obtaining immunologic markers for ‘stem’ vs differentiated glomerular visceral epithelial cells. Work in progress in fact suggests that there are precursors of other cell types present in the kidney. We have been successful in growing cells with the morphologic features of poorly differentiated tubular cells in a serum-free medium containing insulin, transferrin, selenium, tri-iodothyronine, and laminin (unpublished observations), whereas Chung et al. [24] has grown biochemically well differentiated tubular cells in a serum-free medium containing insulin, transferrin, and hydrocortisone without an extracellular matrix molecule. We are presently hypothesizing that each precursor cell type will have an extracellular matrix molecule that will stimulate its cell growth under the proper conditions. The present in vitro study suggests a role for fibronectin in glomerular cell adhesion. In fact, immunohistologic studies of glomeruli in vivo have localized fibronectin between basement membrane proper (lamina densa) and visceral epithelial cells [20, 251; we therefore feel that the present in vitro studies are consistent with the hypothesis that fibronectin acts as an adhesive protein to mediate attachment of glomerular epithelial cells to their substrate (plastic in vitro, basement membrane in vivo). Although the present study documents a role for fibronectin in glomerular cell growth in vitro, there is no evidence for such a role in vivo. The requirement of the poorly differentiated cell for fibronectin is specific, since we have not been able to obtain growth with laminin, heparan sulfate proteoglycan, or type IV
275
276
Oberley
et al.
Exp Cell Res 145 (1983)
collagen in identical serum-free media. Yamada et al. [26] has demonstrated that cell surface fibronectin affects the growth of certain fibroblast cell lines in vitro by allowing them to attain a higher saturation density than in plasma fibronectin. It is therefore probable that the different forms of fibronectin can indeed have different effects on cell growth, dependent on the cell types involved. We believe that multimeric tibronectin may possibly have a role in cell growth in vivo, since there is evidence for disultide-loaded fibronectin multimers on cell surfaces [27]; our future research will be directed at establishing whether these multimers have a physiologic role in cell division in vivo and in vitro. This research was supported by grants from the Hereditary Nephritis Foundation, the Kidney Foundation, and the Research and Development Committee of the Department of Pathology to T. D. 0. Support was also received from Grant HL-21644 to D. F. M. This work was done during the tenure of an Established Investigatorship from the American Heart Association and its Wisconsin Affiliate to D. F. M. J. E. M. was supported by a predoctoral traineeship CA-09106 from NIH. The authors would like to thank MS Carol Gabel, MS Ruth B. Johnson, Mr Patrick J. Murphy and MS Fran Simandl for excellent technical assistance.
REFERENCES 1. Norgaard, J 0 R, Renal physiology. The glomerular basement membrane (ed G M Berlyne & S Thomas) p. 220. S. Karger, Basle (1980). 2. Foidart, J B, Dechenne, C A, Mahieu, P, Creutz, C E 8t DeMey, J, Invest cell path01 2 (1979) 15. 3. Kreisberg, J I, Hoover, R L & Kamovsky, M J, Kidney intern 14 (1978) 21. 4. Killen, P D & Striker, G R, Proc natl acad sci US 77 (1980) 994. 5. Norgaard, J 0 R, Lab invest 38 (1978) 320. 6. Scheinman, J I, Fish, A J, Kim, Y & Michael, A F, Am j path01 92 (1978) 147. 7. Oberley, T D, Chung, A E, Murphy-Ullrich, J E & Mosher, D F, J histochem cytochem 29 (1981) 1237. 8. Striker, G E, Killen, P D & Farin, F M, Transplant proc 12 (1980) 80. 9. Oberley, T D, Burkholder, P M, Barber, T A & Hwang, C C, Invest cell path01 2 (1979) 27. 10. Barnes, D & Sato, G, Anal biochem 102 (1980) 255. 11. Oberley, T D, Murphy-Ullrich, J E & Muth, J V, Diagnost histopatho14 (1981) 117. 12. Rojkind, M, Gatmaitan, Z, Mackensen, S, Gimbrone, M A, Dance, P & Reid, L, J cell biol 87 (1980) 255. 13. Oberley, D, Murphy, P J, Steinert, B W & Albrecht, R M, Virchows arch b (cell pathol) 41 (1982) 145. 14. Baumann, H & Eldredge, D, J cell bio195 (1982) 29. 15. Mosher, D F, Schad, P E & Vann, J M, J biol them 255 (1980) 1181. 16. Chen, T R, Exp cell res 104 (1977) 255. 17. Lajtha, L G, Differentiation 14 (1979) 23. 18. Potten, C S, Schofield, R & Lajtha, L, Biochim biophys acta 560 (1979) 281. 19. Thoming, D & Vracko, R, Lab invest 37 (1977) 105. 20. Courtoy, P J, Kamvar, Y S, Hynes, R 0 & Farquhar, M G, J cell biol 87 (1980) 691. 21. Quaroni, A, Isselbacher, K J & Ruoslahti, E, Proc natl acad sci US 75 (1978) 5548. 22. Bender, B L, Jaffe, R, Carlin, B & Chung, A E, Am j path01 102 (1981) 419. 23. Ekblom, P, Thesleff, I, Miettinen, A & Saxen, L, Cell differ 10 (1981) 281. 24. Chung, S D, Alavi, N, Livingston, D, Hiller, S & Taub, M, J cell bio195 (1982) 118. 25. Oberley, T D, Mosher, D F & Mills, M D, Am j path01 % (1979) 651. 26. Yamada, K M, Kennedy, D W & Hayashi, M, Growth of cells in hormonally defined media (ed G H Sate, A B Pardee 8r D A Sirbaska) p. 131. Cold Spring Harbor Laboratory, N.Y. (1982). 27. Perkins, M E, Ji, T H & Hynes, R 0, Cell 16 (1979) 941. Received October 15, 1982 Revised version received January 11, 1983
Rinted
in
Sweden