/. Embryol. exp. Morph. Vol. 36, 2, pp. 305-313, 1976
Printed in Great Britain
305
The use of variable lactate/malic
dehydrogenase ratios to distinguish between
progenitor cells of cartilage and bone
in the embryonic chick
By P. V. THOROGOOD 1 AND BRIAN K. HALL2
From the Department of Biology, Dalhousie University,
Life Sciences Centre, Halifax, Canada
SUMMARY
The activities of LDH and MDH have been studied, both in differentiated cartilage and
bone from the embryonic chick, and in the pool of mixed osteogenic and chondrogenic stem
cells found on the quadratojugal, a membrane bone. In confirmation of the model proposed
by Reddi & Huggins (1971) we found that the LDH/MDH ratio was greater than 1 in cartilage
and less than 1 in bone. Furthermore we established, for the first time, that ratios occurred in
the chondrogenic and osteogenic stem cells, similar to the ratios in their differentiated
counterparts. Alterations in LDH/MDH resulted from variations in the level of LDH//*g
protein. MDH//tg protein remained constant, even when LDH/MDH was changing. We
interpret these results in terms of adaptation of chondrogenic progenitor cells for anaerobic
metabolism and anticipate that our model will be applicable to other skeletal systems where
stem cells are being studied.
INTRODUCTION
The need to distinguish between different types of skeletal stem cells poses a
problem that has persisted for a number of years. Unequivocal and reliable
methods are necessary in order to characterize and classify progenitor cells and
for a fuller understanding of the cellular dynamics of the skeleton. Precise
knowledge about cell lineages, such as the relationship between cartilage and
bone progenitor cells (a common stem cell population?) and also between
osteoblastic and osteoclastic progenitor cells, would contribute to this understanding (see reviews by Owen, 1970; Hall, 1970, 1975; Hancox 1972; Rasmussen & Bordier, 1974).
In the past a range of techniques has been employed to study stem cells,
based on morphological and/or metabolic characteristics of the cells under
1
Author's address: Developmental Biology Building, Department of Zoology, University
of Glasgow, Glasgow, G12 9LU, Scotland, U.K.
2
Author's address and address for reprints: Department of Biology, Dalhousie University,
Life Sciences Centre, Halifax, Nova Scotia, Canada B3H 4J1.
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P. V. THOROGOOD AND B. K. HALL
study. Early studies employed light microscopy, later augmented with histochemistry (e.g. Pritchard, 1952; Hall 1968 a). Electron microscopy has been
used to attempt ultrastructural classifications of cell types in a variety of osteogenic and chondrogenic tissues (Scott, 1967; Hall & Shorey, 1968; Luk,
Nopagaroonsi & Simon, 1974). Qualitative and quantitative differences in incorporation of labelled precursors of matrix-components have been used to
identify types of different skeletal progenitor cells from each other and from
surrounding soft tissue (e.g. Searls, 1965). In vitro studies have been employed,
primarily to distinguish environmental factors affecting expression of stem cells,
rather than to differentiate between types of stem cells. Many studies have been
based on the response of skeletal progenitor cells to hormones (e.g. Pawelek,
1969). The reaction of osteogenic progenitor cells to hormones such as parathyroid hormone and calcitonin as a means of defining populations of stem
cells has been discussed extensively by Rasmussen & Bordier (1974). However,
equivocality remains as to the nature of stem cell populations with any one, or
combination of, these techniques.
Recently Reddi & Huggins (1971) propounded a model, based on proportionate levels of enzymes, to distinguish between differentiated cartilage and bone.
They used the ratio of lactate dehydrogenase (LDH, 1.1.1.27) activity to malic
dehydrogenase [1.1.1.37] activity to trace the transformation of connective tissue
fibroblasts into cartilage and bone following subcutaneous implantation of
decalcified, lyophilysed bone matrix. They also determined LDH/MDH ratios
in a variety of other connective and skeletal tissues. Their results indicated that
the ratio exceeded one in cartilage but was less than one in bone.
We have used this model-which Reddi & Huggins (1971) used for fully
differentiated, histologically identifiable skeletal tissues - and applied it to
differentiating stem cells of the skeleton of the embryonic chick. The system
chosen was the formation of secondary cartilage on a membrane bone - the
quadratojugal (QJ) a site at which both cartilage and bone are produced from
a common pool of progenitor cells. The differentiative fate of this population of
skeletal stem cells has been well established by past work, and the tissue is
accessible to dissection and isolation of the pool of stem cells. This population
commences production of bone at 7 days of incubation, and produces bone plus
cartilage from 11 days onwards. A histological description of these events has
been published previously (Hall 1968 a, Z?). Histochemical and ultrastructural
analyses revealed that within the stem cell population, chondrogenic and osteogenic stem cells could not be distinguished from one another (Hall 1968 a; Hall
& Shorey, 1968): the degree and nature of their differentiation could only be
identified once a matrix of one sort or another had been established. Fortunately
the stem cell population has a degree of flexibility and can be manipulated by
paralysis, in vitro culture, or grafting to an ectopic site, each of which causes a
switch in the fate of the stem cells from a mixed population of chondrogenic
and osteogenic cells to one of solely osteogenic cells (Murray & Smiles, 1965;
LDHjMDH ratios in skeletal stem cells
307
10-day normal
13-day normal
Stem cells
13-day paralysed
Fig. 1. Diagrammatic representation of quadratojugals from a 10-day normal
embryo and from 13-day-old normal and paralysed embryos to show the level of
removal (broken line) of the stem cells, bone (black) and secondary cartilage
(hatched). The inset shows a quadratojugal from a 13-day-old embryo before and
after dissection of the pool of stem cells (ST): the bar equals 2 mm.
Hall, 1968 b). In fact in these abnormal situations the amount of woven bone
formed exceeds that found normally, suggesting that those cells which would
have formed cartilage formed bone instead (Fig. 1).
In addition to the stem cells we also examined differentiated, but embryonic,
cartilage and bone samples from other sites in the skeleton. These samples were
assayed to determine LDH and MDH activities and ratios for the embryonic
skeleton and as a test of the generality of the model proposed by Reddi &
Huggins (1971) for the post-embryonic skeleton.
308
P. V. THOROGOOD AND B. K. HALL
MATERIALS AND METHODS
Eggs of the domestic fowls Gallus domesticus (white Leghorn) were incubated
in a Leahy forced-draft incubator at 37-5 ±0-5 °C and 5 4 ± 2 % R . H . Tissues
from both normal and paralysed embryos were used in the study.
Paralysis
Paralysis was induced by a single dose of 10 mg of D-tubocurarine chloride
(Nutritional Biochemical Corp., Cleveland, Ohio), dissolved in 0-5 ml sterile
0-9 % saline and injected into the air space of eggs incubated for 10 days, a
treatment known to totally immobilize the embryo and to inhibit the formation
of secondary cartilage (Hall, 1972).
Preparation of stem cells from the quadratojugal
Embryos were removed after 10-13 days of incubation, quadratojugals were
dissected out, placed in ice-cold Tris buffer, pH 7-4, 0-5 M and the stem cell
population excised (Fig. 1). The tissue was homogenized in 0-5 ml Tris buffer,
in an ice bath, and 0-1 ml aliquots were used immediately for enzyme assays.
For each of the stages and treatments examined three homogenates were used.
Each homogenate containing a mean number of 51 (30-81) samples of stem
cells.
Preparation of other tissues
Two types of differentiated cartilage and two of bone were also analysed for
levels of activity of LDH and of MDH. They were 13-day sternal cartilage,
epiphyseal cartilage from 17-day tibiae, membrane bone from the shafts of
13-day QJs and endochondral bone from 17-day tibial diaphyses. Each was
dissected out in ice-cold buffer and any adherent connective tissue and muscle
was removed. All tissue samples were rinsed well with buffer to eliminate any
traces of blood and the marrow was flushed from the cavities of the tibial
diaphyses. In the case of the QJ membrane bone only the mid-shaft region was
used, to eliminate any possibility of secondary cartilage or stem cells being included. The tissues were homogenized in ice-cold buffer (five homogenates for
each type of tissue). 0-1 ml aliquots were used for immediate enzyme assays,
the rest being frozen for subsequent protein estimation.
Changes in enzyme activities in the tissues of the paralysed embryos could
be a consequence of a direct effect of the tubocurarine chloride, rather than an
indirect effect of the immobilization. To ensure that this was not the case, tissue
samples of testes - an organ not known to depend on neuromuscular activity
for its development - were dissected out from normal and paralysed embryos,
homogenized as above and subsequently tested for LDH and MDH activity.
(This rationale is based on the possible effect of curare on the total LDH
activity of the testes. If the enzyme were affected directly by curare, different
LDHjMDH ratios in skeletal stem cells
309
isozymic forms, and therefore the different tissue-specific isozyme patterns,
might vary in their sensitivity. However, the predominance of the isozymes
LDH1 and LDH2 in chick tissues at this stage of development (Schultz & Ruth,
1968) suggests that a such possibility is unlikely to affect the present results
or their interpretation.)
Assays ofLDH and MDH
LDH activity in the 0-1 ml aliquots of homogenate was measured by the
method of Wroblewski & LaDue (1955) based on the reduction of pyruvate to
lactate following the procedure laid down in the Sigma Tech. bulletin no. 340 u.v. The initial velocity of oxidation of reduced nicotinamide - adenine dinucleotide (NADH) was measured at room temperature over 3 min at 340 nm
on a Pye Unicam S.P. 1800 spectrophotometer and plotted on a chart recorder.
Decrease in light absorption per minute was calculated. Rate of decrease of
optical density is proportional to the rate of oxidation of NADH, which in
turn is a function of the amount of LDH present.
MDH activity was measured by the procedure of Siegel & Bing (1956) based
upon the reduction of oxalacetate to malate. Spectrophotometric technique was
identical to that described previously for LDH.
The results are expressed as International Units (IU) of enzyme. An 1U is
defined as that amount of enzyme which will convert one /oriole of substrate per
minute under the conditions specified above (the substrate for LDH was pyruvate, that for MDH was oxalacetate). Units of enzyme were related to pig
of protein, the protein concentration in the homogenates having been determined by the folin-phenol procedure of Lowry, Rosebrough, Farr & Randall,
1951).
The ratio of LDH activity to MDH activity was calculated for each of the
tissue samples, expressed as means ± standard error, and Student's t tests
performed where appropriate.
RESULTS
The data obtained by Reddi & Huggins (1971) indicated that fully differentiated cartilage had an LDH/MDH ratio in excess of one whereas bone and
connective tissue had a ratio of less than one. Our data from the isolated stem
cells of the QJ established that determined but cytologically ' undifferentiated'
progenitor cells showed similar ratios.
Isolated stem cells taken from the QJs of 10-day embryos had a mean LDH/
MDH of 0-58; those from 11-day embryos had a mean ratio of 0-57 (Fig. 2).
However, by 12 days of incubation LDH/MDH had risen significantly to a
mean of 0-88 and by 13 days had risen even further to 1-12. Thus as some of the
cells within the population of progenitor cells became determined for chondrogenesis from 11 days onwards, the LDH/MDH rose to a value in excess of 1.
Stem cells from embryos paralysed from 10 days onwards failed to show this
rise in LDH/MDH. Instead their ratios remained at approximately 0-5, that is
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P. V. THOROGOOD AND B. K. HALL
1-2
11
Normal
10
0-9
0-8
0-7
0-6
0-5
0-4
0-3
0-2
01
12
13
(Injection)
Time (days)
Fig. 2. LDH/MDH ratios in stem cell populations of quadratojugals taken from
.10-, 11-, 12-, and 13-day normal embryos and from 11-, 12-, and 13-day embryos
paralysed at 10 days. (Values are means ±2 S.E.)
at the same level as Reddi & Huggins (1971) found for differentiated bone.
These stem cells are osteogenic, paralysis of the embryo having prevented the
switch to chondrogenesis which normally occurs at 10^-11 days.
LDH/MDH ratios in stem cells from both 12- and 13-day embryos paralysed
at 10 days were significantly different from the values obtained from stem cells
of untreated embryos. We calculated from the data in Fig. 2 that LDH/MDH
in chondrogenic stem cells first significantly differed from that in paralysed
embryos at approximately 11 days 10 h of incubation. Thus during normal
development it is at this stage that chondrogenic cells become recognizable
from osteogenic cells on the basis of this metabolic criterion.
The change in the ratio of the enzyme activities observed in the stem cells
between 10 and 13 days of incubation is a reflection of increasing levels of LDH
with time without a concomitant change in the level of MDH (Table 1). The
level of MDH in the stem cells from 13-day paralysed embryos did not differ
significantly from that in untreated control embryos. In contrast the level of
LDH in stem cells from 13-day-old paralysed embryos was significantly lower
(P < 0-05) than that in stem cells of normal 13-day embryos. The level was
LDHjMDH ratios in skeletal stem cells
311
Table 1. Activities of LDH and of MDHj/ig protein and LDHjMDH for progenitor cells from the quadratojugal and test is for normal and paralysed embryos,
and for differentiated cartilage and bone from normal embryos
LDH//tg
Tissue assayed
10-day normal
13-day normal
13-day paralysed
MDH//*g protein
Progenitor cells from the quadratojugal
0-41 ±008 (3)
0-71 ± 009
0-67±003 (3)
0-60 ±003
0-32±003 (3)
0-59 ±005
LDH/MDH
0-58
1-12
0-54
13-day normal
13-day paralysed
Testis
1-88±005 (4)
1-65 + 0-25(7)
1-96 ±009
1-89 ±0-20
0-96
0-87
13-day sternum
17-day tibial epiphysis
Cartilage
1 0 2 ± 0 0 9 (5)
200±0-19 (5)
0-80 ±005
1-12 + 010
1-28
1-78
Bone
0-38 ±0-15 (5)
0-53 ±0-22
0-72
2-64 ±0-21 (7)
311 ±0-29
0-85
13-day shaft of
quadratojugal
17-day tibial diaphysis
Values are means ± S.E.M. (#i), LDH and MDH are expressed in international units.
approximately half the normal value and had remained at the level typical of
stem cells from 10-day embryos (Table 1).
To confirm that an organ not known to be dependent upon embryonic movement for its development had normal LDH/MDH, testes from both normal and
paralysed embryos were examined. Ratios of LDH/MDH did not differ between
the testes of normal and paralysed embryos (Table 1).
As an internal control for the assay procedure and to check the ratios obtained by Reddi & Huggins (1971), we measured LDH/MDH from differentiated cartilage and bone (Table 1). We found that LDH/MDH in cartilage
(sternal and epiphyseal) was greater than one and that in bone (intramembranous and endochondral) the ratio was less than one, confirming the results
of Reddi & Huggins (1971).
DISCUSSION
Our data demonstrate that the model proposed by Reddi and Huggins (1971)
for LDH/MDH in the skeletal tissues of post-natal mammals is also applicable
to the avian embryonic skeleton. Furthermore we have shown that we can use
this criterion to distinguish not only between differentiated cartilage and bone,
but also between differentiating progenitor cells of cartilage and bone. It is this
finding which is of general usefulness, for although there are many techniques
available for characterizing differentiated cells of cartilage and bone, there are
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P. V. THOROGOOD AND B. K. HALL
very few reliable criteria available for characterizing skeletal progenitor cells of
different types (see Introduction). It is hoped that this model will be applicable
to analysis of other skeletal systems in which a mixture of stem cell types occurs
(e.g. fracture repair, endochondral ossification and ectopic cartilage and bone
formation).
Variations in the ratios obtained (namely increasing to above one as chondrogenic precursors arise, but remaining around 0-5 in osteogenic precursors) must
reflect metabolic changes in the cells concerned. As the level of activity of MDH
remained constant, irrespective of whether the stem cell population was chondrogenic, osteogenic, or a mixture of both, we can eliminate MDH as the metabolic
variable. However, as the activity of LDH//*g protein remained constant in the
solely osteogenic population of stem cells (before 11 days and after paralysis)
but rose as chondrogenic precursors appeared in the population (normal QJ
from 11 days onwards), we can reasonably conclude that chondrogenic precursors possess higher levels of LDH activity than do osteogenic precursors.
Because LDH is known to be a key enzyme in anaerobic metabolism (Delbruck,
1970), and because of the high levels of the enzyme in chondrogenic but not in
osteogenic precursors, we conclude that the former are better adapted for
anaerobic conditions than are the latter. Differentiated bone cells exist in a
vascularized, highly oxygenated environment whereas cartilage cells survive in
an avascular, low oxygen environment (Bassett, 1964). Whilst it has been known
from other studies (Hadhazy, Olah & Krompecher, 1963; Shaw & Bassett,
1967) that differentiated cartilage cells are better adapted for existence in an
anaerobic environment than are differentiated bone cells, our results show, for
the first time, that chondrogenic progenitor cells are similarly adapted. Therefore, in order to further test this model, and as a means of investigating the
initial differentiation of the common progenitor cells, it would be appropriate
to study variations in LDH/MDH after alterations in the vascular and gaseous
environment of skeletal tissues in vivo and/or in vitro.
Research supported by National Research Council of Canada grant no. A 5056 to B. K.
Hall. The expert technical assistance provided by Ms. Sharon Brunt is gratefully acknowledged.
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(Received 4 February 1976, revised 13 May 1976)