Three-dimensional co-culture of primary human liver cells in bioreactors for in vitro drug studies: effects of the initial cell quality on the long-term maintenance of hepatocyte-specific functions

In vitro culture models that employ human liver cells could be potent tools for predictive studies on drug toxicity and metabolism in the pharmaceutical industry. A bioreactor culture model was developed that permits the three-dimensional co-culture of liver cells under continuous medium perfusion with decentralised mass exchange and integral oxygenation. We tested the ability of the system to support the long-term maintenance and differentiation of primary human liver cells. The effects of the initial cell quality were investigated by comparing cultures from resected, non-preserved liver with cultures from liver graft tissue damaged by long-term preservation. In cultures originating from non-preserved liver, protein and urea synthesis, glucose metabolism, and cytochrome (CYP450) activities were stable over the 2-week culture period, with maximal activities at the end of the first week in culture. Enzyme induction led to increased 7-ethoxyresorufin O-deethylase activities of up to 20 times the basal value. In cultures from preservation-damaged liver, recovery of metabolic activities was detected during bioreactor culture. After two weeks, most biochemical parameters approached those of cultures from non-preserved human liver. Light microscopy demonstrated the three-dimensional reorganisation of hepatocytes and non-parenchymal cells in co-culture. Long-term maintenance, and even the regeneration of specific functional activities of human liver cells, can be achieved in the bioreactor. This could facilitate the introduction into the pharmaceutical industry of in vitro drug testing with primary human liver cells.     

Cryopreserved rat liver slices: a critical evaluation of cell viability, histological integrity, and drug-metabolizing enzymes

The effects of a cryopreservation procedure on the biochemical, morphological and functional integrity of rat liver slices just after thawing and after 24 h culture were evaluated. Freshly prepared slices were incubated in modified University of Wisconsin solution containing 50% fetal calf serum and 10% dimethyl sulfoxide for 20 min on ice prior to a rapid cooling in liquid nitrogen. After 10-40 days, slices were thawed rapidly at 42 degrees C. Total protein content and (3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) (MTT) reduction were well preserved at thawing, whereas ATP content was markedly decreased relative to freshly prepared slices (-83%). The major microscopic findings in sections of just-thawed liver slices consisted of hepatocellular dissociation and minimal apoptosis. The qualitative profile of antipyrine (AP) metabolism was well preserved in cryopreserved slices, but the amounts of phase I and phase II AP metabolites produced over a 3-h incubation period were markedly reduced relative to fresh slices (-58 to -71%). When cryopreserved slices were cultured for 24 h after thawing, the viability was markedly reduced, as reflected by the almost complete absence of MTT reduction and the loss of ATP content. Histological examinations showed extensive cellular necrosis. The amount of AP metabolites produced by cryopreserved slices was similar after a 3- or a 24-h culture period, indicating that AP metabolism capacities were lost at 24 h culture. In conclusion, our results suggest that cryopreserved rat liver slices may be a useful model for short-term in vitro determination of drug metabolism pathways. Further work is required to extend their use for toxicological studies.     

Hormonal regulation of the production of serum albumin by cultured hepatocytes of rats during prenatal and postnatal period of development

The effects of several hormones on the production of immunoreactive serum albumin (SA) were examined in primary cultures of liver cells obtained from rat fetuses on 21-22 days of gestation of from 3-week old rats. Cortisol, bovine insulin and human growth hormone stimulated SA production in both types of liver cell cultures during 20 h-incubation. L-Triiodothyronine (T3; 10(-9)-10(-7) M) weakly stimulated SA production by hepatocytes from the rats, but markedly inhibited it in cultures of fetal rat liver cells in a dose-dependent manner. In contrast, T3 action on total RNA and protein biosynthesis, estimated as the incorporation of labelled precursors into macromolecules, was stimulatory one in both types of cell cultures. It is concluded that hormonal regulation of SA production is similar in cultured liver cells from fetal and early postnatal rats except for the action of T3. The physiological importance of striking developmental change of T3 action on SA production remains to be determined.     

Development of erythroid colony-forming cells in rat fetal spleen: apparent lack of sensitivity to an in vivo corticosteroid excess as compared to fetal liver

The development of splenic erythroid colony-forming cells from rat embryos in the last 4 days of intrauterine life was examined after 2 and 7 days in a methylcellulose culture system. The number of 2- and 7-day erythroid colonies decreased sharply between, respectively, days 20 and 21 of gestation and days 19 and 20. Concomitantly, a maturation of proerythroblasts and basophilic erythroblasts to mature erythroblasts was detected on smears of splenic cellular suspensions. The effect of a corticosteroid excess induced by a maternal laparotomy was tested on spleen and liver cultures from the same control or experimental fetuses. The ratio of the number of 2-day to the number of 7-day erythroid colonies did not differ in experimental and control splenic cultures, but in liver cultures was significantly lower at days 19 and 20 in experimental than in control cultures.     

Fine structure of, and ACTH production by, human fetal pituitaries taken at different periods of gestation. An in vitro study

Pituitaries were taken from human fetuses between the 6th and 30th weeks of gestation. Organ and monolayer cultures were prepared. The fine structure of the cultures was examined by electron microscopy and their basal and stimulated ACTH release were studied by radioimmunoassay as a function of time in vitro. It was shown that pituitaries taken from the first trimester of gestation have a capacity of self-differentiation; i.e. there was an increase in the number of cells filled with secretory granules and there was an increase in the number of granules per cell. In contrast, pituitaries taken from older embryos have been losing their secretory granules during the cultivation. We failed to demonstrate any corticotropin responsiveness in pituitary cultures prepared from the 6 to 7-week-old embryos. ACTH release could be stimulated from the 10th week of gestation but in slight measure. Pituitary taken from 16-week-old fetuses revealed an adult-like responsiveness to corticotropin.     

Isolation and identification of germ cells from fetal bovine ovaries

Gonadal cell suspensions were made from bovine fetuses of 35–55-, 56–80-, and 80–130-day age groups corresponding to the periods predominated by primordial germ cells (PGCs), oogonia, and meiotic cells, respectively. Germ cells identified on morphological criteria prior to their isolation from suspensions were compared histochemically and morphologically with cells in cryosections, impression smears, and semithin sections of similar gonads. Oocytes were distinguished by their chromosomal configurations in cell spreads. In suspensions from 35–55-day fetuses, cells considered to be PGCs stood out by their size, large nucleus, intracytoplasmic vesicles, and occasional blebbing. The somatic cells were smaller and contained little cytoplasm and few vesicles. In bovine gonads, in contrast to murine gonads, alkaline phosphatase (AP) activity was not specific enough to identify germ cells once they had entered the gonad. In ovaries from the 56–80-day age group, cells similar to PGCs, but slightly larger and with more cytoplasmic vesicles, were identified as oogonia. The cytoplasmic vesicles stained positively for lipid. In ovaries of 80–130-day fetuses, oogonia, oocytes, degenerating germ cells, and multinucleate germ cells were recognized. Degenerating germ cells exhibited a variety of morphological characteristics and were consistently positive for acid-phosphatase activity. Binucleate germ cells appeared around day 85 of gestation, while multinucleate germ cells were seen from day 95. It was concluded that bovine mitotic germ cells can be isolated from gonadal cell suspensions and that the best time to recover them is between 50 and 70 days of gestation. © 1994 Wiley-Liss, Inc.     

Peroxidase cytochemistry and ultrastructure of resident macrophages in fetal rat liver: A developmental study

The peroxidase cytochemistry and the ultrastructural characteristics of resident macrophages in fetal rat liver have been investigated. Livers of 10-, 11-, 14-, 17-, and 20-day-old fetuses were fixed by immersion or perfusion, incubated for peroxidase, and processed for transmission electron microscopy. Some 17- and 20-day-old fetuses were injected prior to sacrifice with carbon or 0.8-μm latex particles through the umbilical vein. Some livers were additionally processed for scanning electron microscopy (SEM). The endogenous peroxidase was present in the nuclear envelope (NE) and endoplasmic reticulum (ER) of fetal macrophages with a negative reaction in the Golgi apparatus, a distribution pattern identical to that in Kupffer cells of adult rat liver. Such peroxidase-positive cells avidly took up the injected latex and carbon particles and were the only cell type in fetal liver involved in erythrophagocytosis. Furthermore, they were associated with erythropoietic elements, forming close contacts with such cells, especially normoblasts. The peroxidase pattern in leukopoietic cells differed at all stages of maturation from that in macrophages. By SEM the macrophages exhibited ruffles and lamellopodia on their surfaces and protruded often into the lumen of fetal sinusoids. Macrophages in fetal liver underwent mitotic divisions. The macrophages were first seen on gestation day 11, whereas the first mature monocytes were found on gestation day 17. These observations suggest that resident macrophages in fetal rat liver form a self-replicating cell line independent of the monocytopoietic series, although they may both arise from a common precursor cell.     

Influence of doxycycline administration on rat embryonic development during organogenesis

This experiment was performed to evaluate the possible embryotoxic and teratogenic effects of doxycycline during rat development. Twenty‐one female rats were used and distributed into three groups equally (seven animals/group). The low dose group received doxycycline at a dose of 5 mg/kg bw/day orally from the 6th to 14th day of gestation. The high dose group received 10 mg/kg bw/day orally for the same period, the Control group received 1 mL distilled water orally for the same period. The dams were dissected on the 20th day of gestation and their fetuses were subjected to morphological, skeletal, and histological examination. Moreover, DNA damage analysis of liver cells of pregnant rats and their fetuses or fetal skull was assessed by Comet assay. The obtained results showed a significant decrease in fetal body weight, several morphological anomalies, and severe lack of ossification on the skull bones, phalanges, and sternum bone as well as shortness in the ulna and radius bones. Histological studies of pregnant rats revealed congestion and dilatation of the central vein of the liver lobules and fatty degeneration of the hepatocytes. In addition, 20 day‐fetuses showed a marked increase of necrotic hepatocytes associated with an increased average of megakaryocytes and periportal leukocytic infiltration. Moreover, doxycycline induced a significant increase in the percentage of DNA damage and tail length of examined samples. Conclusively, doxycycline caused certain fetal abnormalities, so it is advisable to avoid using this drug during pregnancy.     

Growth and differentiation of fetal rat small intestinal epithelium in tissue culture: Relationship to fetal age

Explants of small intestinal tissue have been cultured from fetal and young rats (from 13-day fetuses to 3-week-old rats). Growth of morphologically typical epithelial cells was obtained from explants of tissue from 14–20 day fetuses. Optimal growth was obtained using tissue from 17-day fetuses with outgrowth from the explant being observed 1-day after explant. Eighty per cent of explants developed epithelial growth by 11 days in culture. Initially, the epithelial outgrowth showed no morphological evidence of differentiation but after 5–10 days in culture differentiation into goblet or elongated cells with alkaline phosphatase activity occurred. Cells with brush borders and goblet cells were identified using electron microscopy. No differentiation occurred if the explant was removed even though growth continued.It was very difficult to culture tissue from fetuses older than 20 days' gestation, and when small intestine of 18–20-day fetuses was divided into two parts (proximal and distal) and cultured separately, growth of epithelial cells from explants of the proximal segment was less successful than that of the distal segment, indicating that the growth ability of these epithelial cells in vitro was closely related to tissue maturation in vivo. In contrast to the apparent relationship between fetal age and successful growth of intestinal epithelial cells, squamous epithelial cells of the esophagus could be grown from explants of 14-day fetus through newborn and 3-week-old rats.     

The development of adipocytes in primary stromal-vascular culture of fetal pig adipose tissue

Primary cultures of stromal-vascular cells of adipose tissue from fetuses at 70 and 110 days of gestation were evaluated as potential model systems for studies of fetal adipocyte differentiation and proliferation. In the cultures, fat cells developed as very discrete clusters. Fat cell cluster development was dependent on initial cell density and time. Histochemical analysis for NADP-dependent dehydrogenases revealed an age of donor effect. Similar levels of enzymes (malate and glucose-6-phosphate dehydrogenase) were apparent in fat cell clusters and stromal cells in cultures of cells from fetuses at 70 days of gestation. These enzymes were only present in fat cell clusters in cultures of cells from fetuses at 110 days of gestation. The distribution of histochemically detectable esterase activity was dependent on the cell density at time of analysis. In areas of high cell density, esterase was restricted to fat cell clusters whereas, both stromal cells and fat cells were esterase reactive in areas of low cell density. Omitting PMS from the dehydrogenase media revealed differences in enzyme reactions of cells grown on collagen-coated and uncoated glass surfaces. These studies demonstrate that primary cultures of stromal-vascular cells from 110-day-old fetuses would be a useful system to identify factors involved in adipocyte proliferation and differentiation.     

Effects of endogenous and exogenous glucocorticoids on liver differentiation

The effects of maternal bilateral adrenalectomy on day 1 of gestation and betamethasone treatment on fetal liver development were compared, in terms of biochemical and morphological parameters. For fetuses 20 days old (E20), absence of maternal glucocorticoids during gestation caused an increase in the number of nuclei in whole livers, and a significantly decrease of both body weight and protein content per nucleus, in comparison with the control group (C). Betamethasone injection on days 15, 16 and 17 of gestation into adrenalectomized pregnant rats (ADX + BET) did not completely prevent these effects. The electron microscopic analysis of the ADX fetal liver (E20) showed some hepatocyte lesions such as loss of cytoplasmic organelles, increase in hematopoietic cell number as well as a lower cellular maturation in comparison with the control group. The fetal liver from ADX + BET mothers 20 days after gestation displayed a noticeable involution of the hematopoietic component in spite of its relatively immature stage. However, there was no significant change in the degree of fetal hepatocyte lesions. Therefore, supply of maternal glucocorticoids from the beginning of gestation is essential for maintenance of the integral structure of the rat fetal hepatic parenchyma, for the correct maturation of the blood strains and for the beginning of involution of the hematopoietic tissue at the end of gestation.     

Biochemical changes in the developing rat central nervous system due to hyperthermia

Rat embryos at 10 days of gestation were exposed to 43 degrees C for 8 minutes by submerging the exteriorized right uterine horn in heated saline solution and then reinserting the uterine horn into the abdominal cavity. At 15 days, the fetuses were removed, and cells from the cerebral hemispheres were dissociated and grown as primary cultures. Embryos from the left uterine horn served as controls. No morphological changes were observed between the cultures of cells from control and heat-exposed embryos at different days in culture. However, exposure of embryos to hyperthermia at 10 days significantly affected the developmental pattern of activities of acetylcholine esterase associated with cholinergic neurons and of 2',3'-cyclic nucleotide phosphohydrolase associated with oligodendrocytes and myelin membrane formation. These results suggest that hyperthermia at 10 days of gestation in the rat may lead to an impairment in the development of neurons and oligodendrocytes in the central nervous system.     

Hypothalamic influence on differentiation of the immunoreactive ACTH beta-LPH, alpha- and beta-endorphin containing cells in adenohypophysial primordia of rat fetus in organ culture (author's transl)]

Adenohypophysial primordia were isolated in rat fetuses from day 12.5 to day 15.5 of gestation. The organ culture employed for maintenance of the primordia was made up according to Watanabe et al. (1973). The fixation of primordia in Bouin Hollande's solution was performed after 9, 8, 7 or 6 days of culture when the normal duration of pregnancy was achieved. The cultivated primordia were immunologically studied using different antisera: anti-alpha(17-39)ACTH, anti-beta(1-24)ACTH, anti-beta-LPH, anti-alpha and anti-beta-endorphins, with immunoperoxidase or immunofluorescence techniques, including control experiments of the specificity of the antisera. A similar study was performed on pituitaries removed from normal rat fetuses from day 16.5 of gestation and each day up to birth, and fixated immediately. In vivo the first cells reacting with all the antisera used in this study were observed on day 16.5 of gestation; their number increased during gestation (Fig. 1 A, B and C). Immunoreactive cells with the different antisera could be detected in primordia isolated on day 12.5 of gestation after 9 days of culture. Numerous groups of cells were observed in primordia of older fetuses (Fig. 2 A and B). These data indicate that the corticotropic cells in rat fetuses could start to be differentiated without stimuli from the hypothalamus since primordia were isolated before the appearance of this cell type in normal rat fetuses and before the differentiation of the hypothalamus. The presence of ACTH and other peptides such as beta-LPH or beta-endorphin would support the hypothesis of a common precursor in this cell type existing early in gestation. Similar results were obtained in human fetuses.     

Impairment of rat fetal beta-cell development by maternal exposure to dexamethasone during different time-windows

Aim

Glucocorticoids (GCs) take part in the direct control of cell lineage during the late phase of pancreas development when endocrine and exocrine cell differentiation occurs. However, other tissues such as the vasculature exert a critical role before that phase. This study aims to investigate the consequences of overexposure to exogenous glucocorticoids during different time-windows of gestation for the development of the fetal endocrine pancreas.

Methods

Pregnant Wistar rats received dexamethasone acetate in their drinking water (1 µg/ml) during the last week or throughout gestation. Fetuses and their pancreases were analyzed at day 15 and 21 of gestation. Morphometrical analysis was performed on pancreatic sections after immunohistochemistry techniques and insulin secretion was evaluated on fetal islets collected in vitro.

Results

Dexamethasone given the last week or throughout gestation reduced the beta-cell mass in 21-day-old fetuses by respectively 18% or 62%. This was accompanied by a defect in insulin secretion. The alpha-cell mass was reduced similarly. Neither islet vascularization nor beta-cell proliferation was affected when dexamethasone was administered during the last week, which was however the case when given throughout gestation. When given from the beginning of gestation, dexamethasone reduced the number of cells expressing the early marker of endocrine lineage neurogenin-3 when analyzed at 15 days of fetal age.

Conclusions

GCs reduce the beta- and alpha-cell mass by different mechanisms according to the stage of development during which the treatment was applied. In fetuses exposed to glucocorticoids the last week of gestation only, beta-cell mass is reduced due to impairment of beta-cell commitment, whereas in fetuses exposed throughout gestation, islet vascularization and lower beta-cell proliferation are involved as well, amplifying the reduction of the endocrine mass.     

Fetal growth in the baboon during the second half of pregnancy

The normal growth profile of critical fetal organs through the last third of gestation has not been documented in detail in human fetuses or the fetus of any nonhuman primate species. Recent epidemiological studies in human pregnancy suggest that fetal growth plays a major role in the programming of life-long health by modifying cardiovascular, pancreatic, brain, and liver growth. The present study aimed to produce a detailed database of individual organ growth in the fetal baboon in late gestation. Fetal organ weights were obtained from 43 baboon fetuses between 121 and 177 days of gestation. Various organs (brain, heart, kidney, femur, intestines, and spinal cord) showed no sign of slowed growth in late gestation while growth of others (lung, liver, stomach, and bladder) accelerated in late gestation. The fetal adrenal and thymus showed a decrease in growth rate over the final 20 and 10 days of gestation respectively. These observations provide a database that will permit analysis of factors responsible for regulation of normal and altered fetal organ development in this important experimental species.     

Morphological and biochemical integrity of human liver slices in long-term culture: effects of oxygen tension

We tested the effects of low 20% O₂) and high (70% O₂) oxygen tension on the morphological and biochemical integrity of human liver slices incubated for up to 72 h in supplemented Williams' E medium in a dynamic rotating culture system. High oxygen tension was more effective than low oxygen tension for preserving morphological integrity in long-term culture 48–72 h). After 72 h of culture with 70% O₂, the lobular pattern was well preserved, and the survival of hepatocytes approximately 80%) and other cell types was good. Immunohistochemical studies showed good preservation of the region-specific expression of CYP2E1 and CYP3A4 isoenzymes for up to 72 h of incubation in 70% O₂. As compared to 20% O₂, the oxidized glutathione content and reactive oxygen species production were slightly increased in 70% O₂, suggesting that minimal oxidative stress occurred with the high oxygen tension. In conclusion, despite slight oxidative stress associated with high oxygen tension, 70% O₂ appeared more appropriate than 20% O₂ for preserving the morphological and biochemical integrity of human liver slices cultured in a dynamic organ culture system for up to 72 h. This revised version was published online in August 2006 with corrections to the Cover Date.     

Immunohistochemical evidence of lactoferrin in human embryo–fetal bone and cartilage tissues

Lf (lactoferrin) is an 80‐kDa iron‐binding protein, which has been suggested to promote bone growth in murine models. In view of this, we aimed to analyse the immunohistochemical distribution of Lf in human embryonal and fetal bone and cartilaginous tissues at different gestational weeks in order to evaluate whether a role for this protein might be proposed also in human osteogenesis. Bone and cartilaginous specimens were taken at autopsy from 25 fetuses (8–34 weeks of gestation). Ten samples of human adult bone and cartilage were also submitted to the immunohistochemical procedures. Sections, 4‐μm thick, were cut from formalin‐fixed paraffin‐embedded tissue blocks and stained with a monoclonal antibody against human Lf, following antigen retrieval procedures. Lf immunoreactivity was maily localized in the mesenchymal cells forming the periosteum as well as in chondroblasts at the eighth gestational week; a strong Lf immunoexpression in immature osteocytes and osteoblasts was noted up to the 18th gestation week, with a considerable decrease by the 24th week. No Lf expression was found in any bone area after the 30th and up to the 34th week of gestation. Our data seem to suggest an important role for Lf as a bone growth regulator in the early phases of the human endochondral ossification, with an anabolic action similar to that previously reported in cell culture lines and in animal models.     

Variations of chromosome radiation sensitivity in fetal and adult mice during gestation

Mice were exposed to 2 Gy of γ-rays at various days of pregnancy, and just before and after gestation. Chromosomes were analyzed 4 h after irradiation in spontaneously dividing hematopoietic cells from liver for fetuses and bone marrow for mothers. On average, there was significantly less chromosome damage in fetuses than in mothers. A very strong increase of chromosome breakage was observed in mothers at days 16–19 of gestation. This increase parallels that of gestation hormones, suggesting a direct relationship. The differences between fetuses and mothers in relation to gestation age result from the increase in the rate of chromatid and chromosome breaks but not of chromatid exchanges, which remained stable. This suggests that a DNA repair step involved in joining broken extremities is the cause. More experiments are needed to understand the origin of these variations of radiation sensitivity and the possible extrapolation of these observations to other species.     

Influence of Thyroxine In Vivo on Preadipocyte Development and Insulin-Like Growth Factor-I and IGF Binding Protein Secretion in Fetal Stromal Vascular Cell Cultures

Studies were conducted to determine the influence of thyroxine (T₄) in vivo on preadipocyte development and insulin-like growth factor-I (IGF-I) and IGF binding proteins (IGFBPs) secretion in stromal-vas-cular (S-V) cultures. Fetal pigs were hypophysectomized (hypox) at 70 days of gestation, implanted with T₄ pellets, and fetuses from the dam at 75 days of gestation. In a second experiment, hypox and T₄ implantation were performed at 75 days and fetal pigs removed at 95 days of gestation. Primary cultures of stromal vascular (S-V) cells derived from fetal adipose tissue were established. Cultures were stained for morphological analysis and conditioned media were collected for IGF-I determination by radioimmunoassay (RIA) and IGFBP analysis by Western blotting. After only 5 days of T₄ treatment, fat cell cluster number and size and lipid deposition in cultures were significantly increased compared to cultures from untreated hypox fetuses. Fetal hypox reduced IGF-I secretion by preadipocytes at both ages and T₄ treatment completely normalized IGF-I secretion (p < 0.05). Four IGFBPs (BP-1, BP-2, BP-3 & BP-4) detected in S-V cultures derived from 95-day fetuses were decreased in concentration by hypox by 44 ± 9.4%, 32 ± 9.7%, 42 ± 12% and 53 ± 6.9%. In cultures derived from T₄ treated hypox fetuses, the levels of these four IGFBPs were increased by 187 ± 25%, 239 ± 38%, 190 ± 5% and 347 ± 43% over control values, respectively. In cultures from 75-day fetuses, only IGFBP-2 (major one) and BP-1 (minor one) were detected and their secretion was also decreased by hypox and elevated by T₄ treatment (190 ± 49.5%, 156 ± 30%, respectively, of controls). The results provide direct evidence that T₄ has a major influence on fetal preadipocyte development. T₄ stimulated production of IGF-I and IGFBP in fetal S-V cultures, which in turn, may have mediated the capability of T₄ to enhance fetal adipose tissue development.     

Microenvironmental studies of pre-B and B cell development in human and mouse fetuses

Immunofluorescence techniques were used to trace the development of cells expressing mu heavy chains in humans and mice. IgM B cells were distinguished from pre-B cells by their additional expression of kappa or lambda light chains. Generation of pre-B and progeny B cells was evident in hemopoietic fetal liver and bone marrow, but not in thymus, heart, lung, spleen, kidney, and placental tissues. Pre-B and B cells, in a ratio of 2 to 1, were abundant in sections of hemopoietic liver and in bone marrow from 12- to 15-wk-old human fetuses, whereas these cells were rare in nonhemopoietic liver samples obtained beyond the 34th week. In mouse fetal liver mu+ cells appeared first around the 12th day of gestation and increased in frequency throughout the third trimester. On day 17 of gestation, kappa light chain expression by 1% of mu+ cells was noted, and the percentage of kappa+/mu+ cells increased progressively to more than 80% by 5 days after birth. Pre-B and B cells were interspersed among myeloid and more abundant erythropoietic cellular elements in the extrasinusoidal areas adjacent to hepatic cords. A loose clustering or "starburst" distribution pattern of pre-B cells became evident around day 17. These observations suggest a model for in situ generation of pre-B and progeny B cells in the hemopoietic fetal liver. In the midst of more numerous erythropoietic elements, immunoglobulin-negative precursors divide to generate a loose colony of mu+ pre-B cells that divide again before giving rise to a wave of IgM B cells.