CELL POPULATION KINETICS IN THE RAT JEJUNAL CRYPT

Cell kinetics in the jejunal crypt of the male Wistar rat were studied using autoradiographic techniques with tritiated thymidine and a stathmokinetic technique with vincristine. The migration rate measured by following the movement of the 50 % peak on the labelling index distribution curve with time after injection of tritiated thymidine gave a value of 1.43 ± 0.14 (SE) cell positions per hour, compared with a value from a cumulative birth rate of 1.78 cell positions per hour. The crypt column length was 32.9 ± 0.2 cells and the column count was 22.3 ± 0.2. This measurement gave a total crypt population of 734 cells, compared with an estimate of 650 ± 6 from direct observation of squashed, microdissected crypts. In each crypt 22.5 ± 0.5 mitoses were present, and the crypt cell production rate was 32 cells per crypt per hour; this latter value was confirmed using two independent techniques. The crypt growth fraction calculated from the durations of phases of the cell cycle and the labelling index was 0.62. A value of 0.61 was found from the labelling index distribution curve. As assessed from crypt squashes, there were 403 proliferating cells per crypt.     

GRANULOPOIESIS AND CELL KINETICS IN CHRONIC MYELOID LEUKAEMIA

The present study is an analysis of the kinetic data for chronic myeloid leukaemia derived from personal observations and cases reported in the literature. A comparison is made with the parameters for normal granulopoiesis and the growth pattern of the CML cell population discussed.     

HAEMOPOIETIC STEM CELL (CFU) KINETICS IN THE CULTURE

Haemopoiesis continued for over 2 months in organ culture of embryonal mouse liver, and haemopoietic stem cells (CFU_s) capable of DNA-synthesis were found in it all that time. Between the 10th and 40th day the number of stem cells in the culture was sustained in a steady state. Both in normal and in regenerating adult bone marrow haemopoiesis ceased within a short time in the culture. Induction of proliferation in haemopoietic stem cells combined with undamaged or improved micro-environment resulted in a little better maintenance of CFU_s in the adult bone marrow culture, The results are discussed in the light of current concepts of haemopoietic stem cell regulation.     

CELL POPULATION KINETICS OF THE RECOGNIZABLE ERYTHROID CELLS IN THE RAT

The cell population kinetic parameters defining a simple model of the recognizable part of the erythroid system have been determined. Experimental results using tritiated thymidine and radioactive iron autoradiography have provided estimates of the number of cell divisions, transit times and flow rates for all the recognizable stages of the erythroid system. The accuracy of the estimates and the validity of the model employed are discussed.     

RADIOAUTOGRAPHIC STUDIES OF THE STEM CELL IN THE THYMUS OF THE IRRADIATED RAT

Radioautographic evidence is presented which characterizes the marrow derived stem cell which promotes thymic recovery following irradiation in the rat. These immigrant cells are similar in morphology to blood monocytes and have been called monocytoid, meaning monocyte-like in appearance. The typical cell had abundant pale staining cytoplasm and a nucleus with many invaginations and folds and a fine chromatin structure. There was no prominent nucleolus. The majority of these cells entered the thymus of the irradiated rat via the blood vessels into the septa and made their way through the connective tissue to the outer cortex. Three distinct morphological cell types appeared to be derived from the immigrant cells. These were fibrocyte-like cells which were located within the septa, macrophages located mainly within the medulla and septa, and large blast cells within the cortex, which proliferated giving rise to large thymocytes. The blast cells were characterized as having abundant moderately basophilic (and pyroninophilic) cytoplasm with a distinct cytoplasmic boundary, a large nucleus which still had invaginations and folds, a loose chromatin structure and one or more very prominent nucleoli. They were located in groups primarily within the outer cortex and often adjacent to blood vessels. They were found to be highly susceptible to damage in smear preparations. In contrast, their progeny, the large thymocytes were not highly susceptible to damage in smear preparations but teased out as large round cells with a highly basophilic rim of cytoplasm. The large thymocytes were precursors to medium and small cells. A radioautographic technique for 1 μ tissue sections is also described.     

ON THE DERIVATION OF CELL KINETICS FROM HISTOMORPHOLOGY, OBSERVED IN THE RAT INCISOR ODONTOBLAST

A method to extract cell kinetic information from histomorphology is presented. Each replicating tissue is essentially an ordered structure with an origin where cells are formed and a periphery toward which they are displaced. the displacement path is called the tissue radius. the tissue variables may be studied in two domains, space and time. the first embraces all the states a cell may assume while the second specifies the cell transition rates. During steady state both domains are related linearly. These ideas are illustrated in the rat incisor odontoblast population whose life expectation is determined by the tooth wall shape. the odontoblast cell population paves the interior of the tooth wall delimiting a cone-shaped pulp. Near the root apex the dentine wall is barely visible. As one proceeds distally, the wall thickens while the pulp narrows. Pulp narrowing is associated with odontoblast cell loss whose magnitude may be deduced from the change of the pulp circumference CI(x) (x is the distance from tooth origin). the odontoblast force of mortality μ(x) may be calculated from the instantaneous perimeter change: μ(x) = -CI' (x)/CI(x); where CI'(x) stands for the derivative of CI(x). This equation serves for the construction of the odontoblast life table which may be studied in space and time.     

FUNCTIONAL CELL COMPARTMENTS IN A RAT MODEL FOR HUMAN ACUTE MYELOCYTIC LEUKAEMIA

Functional cell compartments were studied in a rat model for human acute myelocytic leukaemia (AML). This was done by tracing the distribution of injected ⁵¹Chromium-labelled leukaemic cells in the body. It was concluded that two functional compartments can be distinguished in acute leukaemia, i.e., a rapidly exchangeable pool of cells (including the circulating blood pool, the marginal noncirculating blood pool and the rapidly exchangeable tissue pool; RETP) and a slowly exchangeable tissue pool (SETP). The sizes of these various compartments were roughly quantified at various stages of the disease by calculations based on the principle of isotope dilution and organ weight measurements. As the leukaemia progresses, the size of the SETP increases significantly relative to the size of the RETP. Simultaneously, the exchange rates of leukaemic cells between the organs and the blood decrease. The blood transit time of leukaemic cells was also significantly prolonged, as is the case in human AML.     

CELL KINETICS OF GROWTH CARTILAGE IN THE RAT TIBIA II. MEASUREMENTS DURING AGEING

A number of cell kinetic techniques using labelled thymidine and autoradiography have been applied to study growth cartilage in the rat tibia during ageing. No change in the duration of the synthesis phase was found from 4 to 13 weeks of age but there was a reduction in cell proliferation rate during this period. Measurements of labelling index, proliferation zone size and height of hypertrophic cells were used to calculate the growth rate of the bone from 7 days to 1 year. The results agreed well with radiographic measurements of bone growth.     

CELL KINETICS OF GROWTH CARTILAGE IN THE RAT TIBIA I. MEASUREMENTS IN YOUNG MALE RATS

Cell kinetic parameters for the proximal growth plate of the tibia have been measured in young rats. Analysis of a pulse labelled mitosis study gave values of 55 ± 40 hr for the cycle time and 6.5 ± 0.3 hr for the synthesis time in 6-week-old rats. The results of a simulated continuous labelling experiment agreed with this data and provided further information on the size and proliferation rate of the stem cell zone. Diurnal variations in mitotic index and labelling index in the tissue have been investigated.     

THE SPERMATOGONIAL STEM CELL POPULATION IN ADULT RATS

Radioautographed whole mounted seminiferous tubules from adult rat testes were used to analyse undifferentiated type A spermatogonia at various intervals up to 81 hr following a single injection of ³H-TdR. the data obtained led to the identification of the spermatogonial stem cell and to the formulation of a new model for spermatogonial renewal and differentiation. Undifferentiated type A cells were morphologically alike, but were topographically classified as (1) isolated or (2) paired and aligned. Although labeled isolated A cells were scattered over most stages of the seminiferous epithelium, their proliferative activity varied with the stage; their labeling index was 20-30% in stages I and II, but less than 1% in stages VII and VIII. By tracing the labeled divisions of isolated A spermatogonia in time, it was seen that some daughter cells became separated from one another to form two new isolated cells, while others remained together as paired A spermatogonia. Analysis of two successive waves of labeled mitoses revealed that most paired A spermatogonia continued to proliferate forming four aligned A cells, many of which divided again to produce a chain of eight and so on. the greatest incidence of labeling among paired and aligned A spermatogonia occurred in stages XIII-III. In stage I, where the labeling index was 50%, the calculated proliferative fraction was 1 for these spermatogonia. Between stages II and V, they began to leave mitotic cycle, and during stage V this entire cohort morphologically transformed into A₁ spermatogonia. Labeled metaphase curves for undifferentiated A spermatogonia were distinct from any of the curves previously constructed for the six classes of differentiating spermatogonia, especially because of particularly long S and G₂ phases in the former. the cell cycle time of paired and aligned A cells was 55 hr, compared to an average of 42 hr for differentiating types A₂ to B.     

THE EFFECT OF A SINGLE INJECTION OF HYDROXYUREA ON CELL POPULATION KINETICS IN THE SMALL BOWEL MUCOSA OF THE RAT

The effect of a single injection of hydroxyurea (HU) on cell population kinetics in the jejunal crypt of the rat was studied using autoradiography with tritiated thymidine and metaphase arrest with vincristine. HU appeared to act selectively on cells in the S phase producing inhibition of DNA synthesis and cell death. The deficit in proliferating cells was made good by a decrease in cell cycle time and an increase in growth fraction. Particular attention was paid to the basal, slowly cycling (and possibly clonogenic) crypt cells; early in the recovery sequence an increase in cell production rate was found in the base of the crypt. It is proposed that basal crypt cells, having survived cycle-specific insult because of long cell cycle times, proceed to repopulate the depleted proliferative compartment.     

THE CELL CYCLE TIME IN THE RAT JEJUNAL MUCOSA

The regional variation of the duration of cell cycle parameters was studied by constructing fraction of labelled mitoses curves at several levels in the jejunal crypt column of male Wistar rats. Prolonged T_c and T_s values were apparent only in the bottom eight cell positions, and these differences were shown to be significant compared with the remaining cell positions by analysing the data by the method of Gilbert (1972). Above cell position 8 the proliferating crypt cells showed effectively the same phase durations. For the whole crypt column T_c was 11.32 ± 0.14 (SE) and T_s 6.49 ± 0.10. Although variation in phase durations was confined to the basal portion of the crypt, the results essentially confirm the findings of Cairnie, Lamerton & Steel (1965a), and may be interpreted in terms of the slow cut-off model. The demonstration of prolonged T_c values in basal cell positions confirms the presence of a longer cycling subpopulation of cells at the bottom of the crypt.     

FIBROBLAST CELL KINETICS IN THE PERIODONTAL LIGAMENT OF THE MOUSE

Twelve male mice were injected intraperitoneally with tritiated thymidine. Six were sacrificed after 1 hr and six after 7 days. The right manibular incisors were dissected, cut sagittally and dipped in liquid emulsion. In the exposed and stained slides, observation was restricted to the lingual side of the periodontal ligament. Cells were evaluated sagittally from the basal tooth end up to the distance of 5 mm and up to the depth of 100 μm in the direction of socket wall. Cell and grain count was evaluated separately in 100 × 10 μm rectangles, creating a two-dimensional array onto which the periodontal ligament was mapped. The progenitor compartment extends up to the distance of 2400 μm from origin. Fibroblasts leaving this compartment migrate at different velocities, creating a velocity profile across the ligament. Adjacent to the socket wall cell movement is sluggish, whereas the fastest cell movement is exhibited by cells located 20–30 μm from the tooth. The existence of such a profile indicates a continuous renewal of intercellular bonds, consistent with a process of actively pulling the incisor from its socket by the migrating fibrocytes.     

SPERMATOGONIAL STEM CELL RENEWAL IN THE MOUSE: II AFTER CELL LOSS

The repair of the mouse seminiferous epithelium after cell loss has been studied in seminiferous tubules mounted in toto . Cell loss was inflicted by injection of Myleran in a dose of 10 mg/kg body weight. In stages 7–8, in which we mainly counted, the numbers of A_isolated (A_is), A_paired (A_pr), A_aligned (A_al) and A₁ spermatogonia and resting primary spermatocytes decreased after injection. After about 24 days normal numbers of A₁ spermatogonia were found again. Thereafter a substantial overshoot in the number of A₁ spermatogonia was found.
While normally most of the A_pr and A_al cells differentiate into A₁ spermatogonia in stages 3 and 4 and do not divide until stage 9, during repair they pass through one more division during stages 6 and 7. Normally, during these stages divisions of these spermatogonia are rare. Owing to this extra division the transformation of A_pr and A_al into A₁ spermatogonia is delayed from stage 3 or 4 to stage 8, i.e. still before stage 9, in which A₁ spermatogonia divide. From 16 days after the injection onwards the extra division takes place less generally and more and more cells transform into A₁ spermatogonia at the normal time.     

THE RELATIONSHIP BETWEEN STEM CELL SEEDING EFFICIENCY AND POSITION IN CELL CYCLE

The seeding efficiency of colony-forming cells from normal, regenerating and velocity-sedimented cycling and non-cycling narrow preparations was compared. Colony-forming cells in cycle were found to exhibit a 50% reduction in splenic seeding when compared to normal marrow or sedimented non-cycling cells. The results of this study indicate that the spleen colony assay underestimates the total number of colony-forming cells by a fraction which is directly related to the number of cells in cycle.     

KINETICS OF THE INNER ENAMEL EPITHELIUM IN THE ADULT RAT INCISOR

Rats were injected with tritiated thymidine and sacrificed at various time intervals up to 24 hr. the extracted incisors were decalcified, cut sagittally, dipped into liquid emulsion exposed for 14 days, developed and stained. the counting consisted of an exact mapping and numbering of the cells.
The inner enamel epithelium consists of two compartments: proliferative and mature cells. the first may be further subdivided into blasts and metablasts. Each dividing blast yields one blast and one metablast. the metablasts continue to divide further, at least once, yielding again two metablasts.
The kinetic parameters of this population are: generation time 22 hr, synthesis time 4.5 hr, mitotic time 30 min, G₂ time 2 hr and G₁ time 15 hr. the daily cell production of the proliferative compartment equals its size.     

STUDY OF CELL DEATH IN FRIEND LEUKAEMIA

Cell death of splenic Friend leukaemic cells has been studied in vivo, using ¹²⁵I-UdR and ³H-TdR pulse labelling. The evolution of the splenic specific activity has been measured by autoradiography and external counting during 40 hr after injection of the labelled precursor. These two techniques show the existence of a large reutilization of ³H-TdR (50%), which is measurable as soon as 7 hr after the injection. The DNA turnover rate is rapid, 83-8 % of the splenic cellular DNA being renewed per day. These results confirm that most of the cells produced in the Friend leukaemic spleen are rapidly lost; they also demonstrate that this cell loss is mainly due to a massive death, which occurs in proerythroblastic and erythroblastic compartments after one or two cell divisions. Friend leukaemic cells, which are characterized by a limited capacity of proliferation and a short lifespan, do not appear to be malignant.     

A DISTINCTIVE CELL CONTACT IN THE RAT ADRENAL CORTEX

Extensive cell contacts which resemble septate junctions occur between cells in the three major zones of the rat adrenal cortex. Characteristically, they extend between small intercellular canaliculi and the periendothelial space, frequently interrupted by gap junctions and rarely by desmosomes. Zonulae occludentes have not been identified in the adrenal cortex. Along this distinctive cell contact, the cell membranes of apposing cells are separated by 210–300 a bisected by irregularly spaced 100–150-A extracellular particles which are often circular in profile. In lanthanum preparations, these particles appear to form a continuous chain throughout the intercellular space and are visualized as an alveolate structure in sections parallel to the plane of the cell membrane. The cell membrane in the area of septate-like contact does not differ from nonjunctional areas of the cell membrane in freeze-fracture replicas. The cell contact retains its integrity after cell dispersion and after the separation of cell membranes from disrupted cells. The intercellular particles also persist after brief extraction in lipid solvents. Besides adherence, possible functions of this adrenal contact include maintenance of the width of the extracellular space, the provision of channels between intercellular canaliculi and the bloodstream, and utilization as cation depots. Similar structures are also present between adrenal cortical cells of several other species and between interstitial cells of the testis. This type of cell contact may, in fact, be a typical feature of steroid-hormone-secreting tissues in vertebrates.