Similarity between in vitro and in vivo cellular aging

Mechanisms of Ageing and Development, 22 (1983) 71-78

71

Elsevier Scientific Publishers Ireland Ltd.

SIMILARITY BETWEEN I N VITRO AND I N VIVO CELLULAR AGING

T. METSa, E. BEKAERTb and G. VERDONKa aDepartment of Internal Medicine and Geriatrics, Akademisch Ziekenhuis, 9000 Gent ¢Belgium) and bDepartment of Histology, Rijks Universiteit Gent, 9000 Gent (Belgium)

(Received June 8th, 1982) (Revision receivedNovember22nd, 1982)

SUMMARY

In vivo and in vitro cellular aging were compared by determining the division capacity

of individual, cloned cells of (1) the primary stromal population of human bone marrow, obtained from donors of various ages, and (2) the population at various passage levels of an in vitro subcultivated culture of the same origin. We find a strong similarity between the two series of data. This observation provides a further argument that cellular aging in vitro represents a biologically relevant phenomenon.

Key words: In vivo cellular aging; In vitro cellular aging; Human Stromal bone marrow

cells; Proliferation potential; Clonal growths

INTRODUCTION

One of the major contributions in the search for an understanding of the aging process is the observation that cultures of normal, somatic cells have a limited division capacity in vitro [1 ]. During repeated subcultivation the number of cells capable of rapid division decreases, resulting finally in an almost complete arrest of all cell divisions [2-5]. The proposed explanation, associating this phenomenon with aging at the cellular level, is based mainly on the inverse correlation found between the donor age and the number of subcultivations a culture can achieve [6-1 1]. Other arguments are found in the limited replicative capacity of serially transplanted normal tissue [12-14]. These arguments, however, remain indirect and leave possibilities for criticism as to the in vivo implications of the in vitro cellular aging phenomenon [15-18]. Therefore it seemed important to take a more direct approach in comparing cellular aging in vivo and in vitro. We have determined the individual division capacity in populations of primary (i.e. directly taken from the organism) human stromal bone marrow cells (SCs). By using donors of various ages, cell populations were obtained which had been exposed to various 0047-6374/83/$03.00 Printed and Published in Ireland

© 1983 ElsevierScientific Publishers Ireland Ltd.

72 degrees of aging in vivo. Comparable data were obtained for in vitro subcultivated SCs of the same origin, by examining the cell population at each passage. Reports with full details of these two series have been published already [19,20]. Here we compare the in vivo and in vitro aged cell populations and find a strong similarity between the two processes.

MATERIALS AND METHODS SCs were obtained from sternal bone marrow by aspiration puncture at the level of the second intercostal space. The use of an obturated puncture needle prevents contamination by more superficial tissues. The material (0.3-0.5 ml) was handled as described elsewhere [19]. After releasing the nucleated cells from the tissue structure by treatment with collagenase, 0.5% (Gibco), elastase, 0.1% (Gibco) and EDTA, 0.02% (Sigma) during 1 h, they were washed, diluted and seeded at low density in medium MCDB 104 [21,22] (Flow Lab.), supplemented with 10% fetal bovine serum (FBS) (Gibco), penicillin (100 U/ml), streptomycin (100 /ag/ml) and amphotericin B (0.25 /ag/ml). The cultures were incubated for 14 days in an atmosphere consisting of 5% COs, 5% 02 and 90% N2 [23,24] ; they were then fixed and stained with May-GrtinwaldGiemsa stain. Mass cultures were obtained by seeding the remaining cells, not used in the cloning experiment, in Eagle's basal medium, containing 30% FBS [20]. When the culture reached confluency, it was handled as described previously [25]. For cloning, the mass culture was trypsinised (0.25% trypsin); the cells were diluted and then handled as described above. RESULTS We examined the SC population of 28 donors of various ages. The individual division capacity of the isolated primary SCs was evaluated by means of an in vitro cloning assay. Isolated cells were seeded at low density, incubated for 14 days and the formation of clones was microscopically evaluated. For each donor between 60 and 375 clones could be examined. From a 21-year-old donor, whose primary SCs had the most vigorous clonal growth, a mass culture was established, subcultivated and allowed to age in vitro. We were able to passage this culture 17 times. It reached a cumulative population doubling (CPD) of 35.2 (based on the estimation from the cloning of primary cells from this donor that the culture was started from approximately 3.2 X 104 SCs having a large division capacity; i.e. cells able to form clones of at least 65 cells after 14 days). At each passage the individual division capacity of isolated cells was evaluated again by cloning and data analogous to that of the primary SCs were obtained. Cloning of the primary SCs was considered as passage 0 and cloning 3 weeks after passage 17, when the cells failed to reach confluency, as passage 18. Each time at least 150 clones were examined. Seeding at low density, examining large numbers of clones for each population and interrupting

73 the experiments before large clones could grow confluent, minimized errors due to cross-contamination of the clones. The interpretation of the results was complicated by the fact that, both in primary cultures and during subcultivation /n vitro, two stromal cell types could be recognized. These two cell types were often present in the same clones and subcultivation in vitro has shown that they belong to the same proliferating system [20]. For convenience we have designated them as type I and type II cells. Type I cells are typically fibroblastlike and have a high division capacity. They give rise to the slowly or non-dividing type II cells, which are larger, have an epithelial-like morphology and larger nuclei, contain abundant intracytoplasmic fibrils and in primary cultures are found to be in close contact with cells from the haematopoietic series (Fig. 1). The two cell types probably correspond respectively to an immature and a differentiated type of reticulum cell, found in histological preparations of human bone marrow [26]. The participation of both cell types in clones derived from primary cells of donors of various ages and from different passages of the/n vitro aged mass culture is schematically represented in Fig. 2. It is evident that larger clones are mainly built up by type I cells, whereas type II cells form the majority of slowly or non-dividing cells and of the small clones. In order to allow a comparison between the in vitro and in vivo aged cells we have each time pooled the results in three groups. For primary cultures the maximum human life span (MHLS) was estimated as 100 years and donors were considered as young (less than 30% of MHLS completed; mean age 22; n = 6), middle-aged (from 30 to 60% of MHLS completed; mean age 46; n = 13) or old (more than 60% of MHLS completed; mean age 74; n = 9). Cloning results of the in vitro aged mass culture were pooled in early (less than 30% of CPD completed; n = 6), middle (from 30 to 60% of CPD completed; n = 6) and late passages (more than 60% of CPD completed; n = 7). A statistical analysis was performed, using the Wilcoxon rank sum test. For technical reasons cloning of the mass culture at passage 6 and 8 was lost. In the calculations we use the mean of the preceding and following passages. Significant differences can be noted as follows:

Between groups A and B: A1 < B 1 (p < 0 . 0 1 )

A4> B4 (p <0.05) B I < C l ( p < 0 05) Between groups B and C: B3 > c3 (p < o.o25) B4 > C4 (p < 0.025) B5 > C5 (p < 0,025) Between groups A and C: A1 C4 (p < 0.005) A5 > C5 (p < 0.05) Between groups D and E: D1 < El (p < 0.005) D5 > E5 (p < 0.005)

74

B

r

• |

| t

Fig. 1. (A) Elongated and triangular fibroblast-like type I cells (I), and epithelial-like type II cell (II) with cells of the haematopoietic series (arrows) on its surface. Mass culture at day 9 of the establishing period; phase contrast, ×250; the bar indicates 25 ~m. (B) Large clone (more than 128 cells) built up exclusively by type I cells, neighbouring a small clone of two type II cells. Primary population of a 68-year-old donor; May-Griinwald-Giemsa, × 25 ; the bar indicates 250 ~m.

75

50-

<30

30 -- 60

> 60

A

B

C

30

i

10

70

D

E

12345

12345

B

r

50-

30

10 12345

Fig. 2. Variation of clones of different types during in vivo and in vitro aging. Clones are classified according to the number of constituting cells: 1-2 (1), 3-32 (2), 33-64 (3), 65-128 (4), more than 128 (5) cells. No distinction was made between cells that had not divided or had only divided once, since cells that are considered to be terminally differentiated may show a remaining, limited division capacity [5,27,28]. Since only minor differences were found for small clones (from 3 to 32 cells) the results of this group were pooled. A further classification of the clones is made according to their cell type composition: clones built up exclusively by type I cells (open bars); clones built up by a majority of type I cells (bars hatched from upper left to lower right); clones built up by a minority of type I cells (bars hatched from upper right to lower left); clones consisting only of type II cells (closed bars). (A, B, C) Cloning of primary SCs of donors, respectively less than 30, from 30 to 60 and more than 60 years old. (D, E, F) Cloning of SCs subcultivated in vitro, when respectively less than 30%, from 30 to 60% and more than 60% of the final CPD was completed.

Between groups E and F:

E1 < F1 (p < 0.0.5)

E2 > F2 6o < 0.05) E4 > F4 (p < 0.005) Between groups D and F:

D1 < F1 (p < 0.005)

D2 > F2 (p < 0.05) D4 > F4 (p < 0.005) D5 > F5 (tp < 0 . 0 0 5 ) Between groups A and D: No significant differences

76 Between groups B and E: B1 < B2 > B3 > B5 > Between groups C and F: C1 < C2 >

E1 (p < E2 (p < E3 (p < E5 (p < F1 (t9 < F2 (p <

0.005) 0.005) 0.025) 0.025) 0.005) 0.005)

DISCUSSION In order to establish the in vivo significance of the cellular aging phenomenon, observed in vitro, it is necessary to demonstrate a similar behaviour between cells aged in vivo and in vitro. The nearest approach that has been made used populations of secondary ceils from early subcultivations of mass cultures obtained from young and old donors [29]. The proliferation characteristics of cells in these populations resemble respectively those of cells in early and late subcultivations of embryonic fibroblasts. For a still more direct approach the availability of isolated, primary cells is necessary. For most tissues, however, it is technically impossible to dissociate a sufficient fraction as isolated cells. We have found that SCs are suited for this purpose. They present the advantage that they are easily obtained during a routine clinical procedure. As demonstrated previously, the SCs can be released from the tissue structure by mild enzyme treatment [19]. This allows the evaluation, by cloning in vitro, of the division capacity of individual cells from the primary population. By examining the SC population of donors of various ages, the influence of aging in vivo on the division capacity was determined. Comparably, in vitro aged cell populations were obtained by establishing a mass culture of SCs from a young donor. This cell line was subcultivated to the limit of its division capacity. The influence of aging in vitro was evaluated by cloning at each passage. We have previously shown that the cloning experiments allow us to recognize two cell types in the SC population of the human bone marrow [19,20] Type I cells are rapidly proliferating and give rise to the slowly or non-dividing type II cells. Both during in vitro and in vivo aging cells able to form large clones, which consist mainly of type I cells, gradually disappear from the population. They are replaced by non-dividing cells or by cells forming only small clones. These are mainly of type II. In this study we compare the cloning results obtained from populations of in vivo and in vitro aged cells. No significant differences are revealed between the cell populations of young donors and of early in vitro passages. When the middle-aged and old donors are compared respectively to the middle and late in vitro passages, the changes occurring are analogous, but more pronounced for the in vitro aged ceils. This different behaviour can be explained by several factors: (1) Since the mass culture was obtained from a 21-year-old donor the cells had already been influenced by their in vivo evolution. (2) The mass culture was stimulated to the limit of its division capacity, whereas none of the donors had reached the MHLS. (3) The MHLS is probably determined by several factors. In that case cells are not necessarily at the limit of their

77 division capacity w h e n the MHLS is reached. (4) In the aged donor group a selective pressure m a y exist for individuals with vigorously growing cells. Such selection certainly does not exist for the in vitro aging cells. It can be c o n c l u d e d that the observed differences are not f u n d a m e n t a l but can be a t t r i b u t e d to some specific conditions o f each series. More i m p o r t a n t is that the changes in p o p u l a t i o n dynamics occurring during in vitro cellular aging do correspond to the situation in vivo. This observation provides a further argument for the idea that cellular aging in vitro represents a biologically relevant p h e n o m e n o n . Current efforts made to elucidate this process therefore seem fully justified. REFERENCES 1 L. Hayflick and P.S. Moorhead, The serial cultivation of human diploid cell strains. Exp. Cell Res., 25 (1961) 585-621. 2 G.S. Merz and J.D. Ross, Viability of human diploid cells as a functio~qof i'n vitro age. J. Cell. Physiol., 74 (1969) 219-222. ~' 3 V.J. Cristofalo and B.B. Shatf, Cellular senescence and DNA synthesis. Thymidine incorporation as a measure of population age in human diploid cells. Exp. Cell Res., 76 (1973) 419-427. 4 J.R. Smith and L. Hayflick, Variation in the life-span of clones derived from human diploid cell strains. J. Cell Biol., 62 (1974) 48-53. 5 T. Matsumura, Z. Zerrudo and L. Hayflick, Senescent human diploid cells in culture: survival, DNA synthesis and morphology. J. Gerontol., 34 (1979) 328-334. 6 L. Hayflick, The limited in vitro lifetime of human diploid cell strains. Exp. Cell Res., 37 (1965) 614-636. 7 S. Goldstein, J.W. Littlefield and J.S. Soeldner, Diabetes mellitus and aging: diminished plating efficiency of cultured human fibroblasts.Proc. Natl. A cad. Sci. U.S.A., 64 (1969) 155-160. 8 G.M. Martin, C.A. Sprague and C.J. Epstein, Replicative life-span of cultivated human cells; Effects of donor's age, tissue and genotype. Lab. Invest., 23 (1970) 86-92. 9 Y. Le GuiUy, M. Simon, P. Lenoir and M. Bourel, Long-term culture of human adult liver cells: morphological changes related to in vitro senescence and effect of donor's age on growth potential Gerontologia, 19 (1973) 303-313. 10 E.L. Schneider and Y. Mitsui, The relationship between in vitro cellular aging and in vivo human age. Proc. Natl. Acad. Sci. U.S.A., 73 (1976) 3584-3588. 11 E.L. Bierman, The effect of donor age on the in vitro life-span of cultured human arterial smooth muscle cells. In Vitro, 14 (1978) 951-955. 12 P.L. Krohn, Review lectures on senescence. II. Heterochronic transplantation in the study of aging. Proc. R. Soc. London Ser. B, 157 (1962) 128-147. 13 D.E. Harrison, Normal production of erythrocytes by mouse marrow continuous for 73 months. Proc. Natl. Acad. Sci. U.S.A., 70 (1973) 3184-3188. 14 C.W. Daniel, Aging of ceils during serial propagation in vivo. Adv. GerontoL Res., 4 (1972) 167199. 15 P.M. Spiegel, Theories of aging. In P.S. Timiras (ed.), Developmental Physiology and Aging, Macmillan, New York, 1972, pp. 564-580. 16 R.R. Kohn, Aging and cell division. Science, 188 (1975) 203-204. 17 R. Ecknauer and H. Raffler, Cell population and aging. N. Engl. Z Med., 296 (1977) 947. 18 C.H. Evans, On the aging of organisms and their cells.Meal. Hypotheses, 5 (1979) 53-66. 19 T. Mets and G. Verdonk, Variations in the stromal cell population of human bone marrow during aging.Mech. Ageing Dev., 15 (1981) 41-49. 20 T. Mets and G. Verdonk, In vitro aging of human bone marrow derived stromal cells. Mech. Ageing Dev., 16 (1981) 81-89. 21 W.L. McKeehan, K.A. McKeehan, S.L. Hammond and R.G. Ham, Improved medium for clonal growth of human diploid fibroblasts at low concentrations of serum protein. In Vitro, 13 (1977) 399-416.

78 22 JAR. Smith and K.I. Braunschweiger, Growth of human embryonic fibroblasts at clonal density: concordance with results from mass cultures. J. Cell. PhysioL, 98 (1979) 597-601. 23 Wm. G. Taylor, A. Richter, V.J. Evans and K.K. Sanford, Influence of oxygen and pH on platIng efficiency and colony development of WI 38 and Vero cells. Exp. Cell Res., 86 (1974) 152-156. 24 TAR. Bradley, G.S. Hodgson and M. Rosendaal, The effect of oxygen tension on haemopoietic and fibroblast cell proliferation in vitro. J. Cell. Physiol., 97 (1978) 5 1 7 - 5 2 2 . 25 T. Mets, M. Korteweg and G. Verdonk, Increased prostaglandin F 2 alpha and E 2 production in late passage WI 38 diploid fibroblasts. Cell Biol. lnt. Rep., 3 (1979) 6 9 1 - 6 9 4 . 26 Y. Tanaka, An electron microscopic study ~f non-phagocytic reticulum ceils in human bone marrow. I. Cells with intracytoplasmic fibrils. Acta Haematol. Jpn., 32 (1969) 275-286. 27 G.M. Martin, C.A. Sprague, T.H. Norwood and W.R. Pendergrass, Clonal selection, attenuation and differentia/ion in an in vitro model of hyperpiasia.Am. J. PathoL, 74 (1974) 137-150. 28 G.M. Martin, C.A. Sprague, T.H. Norwood, W.R. Prendergrass, P. Bornstein, H. Hoehn and W.P. Arend, Do hyperplastoid cell lines "differentiate themselves to death"? In V.J. Cristofalo and E. Holeckova (eds.), Cell Impairment in Aging and Development, Plenum Press, New York, 1975, pp. 6 7 - 9 0 . 29 J.R. Smith, O.M. Pereira-Smith and E.L. Schneider, Colony size distributions as a measure of in vivo and in vitro aging. Proc. Natl. Aead. Sci. U.S.A., 75 (1978) 1353-1356.