In vitro and in vivo differentiation into B cells, T cells, and myeloid cells of primitive yolk sac hematopoietic precursor cells expanded >100-fold by coculture with a clonal yolk sac endothelial cell line

The yolk sac, first site of hematopoiesis during mammalian development, contains not only hematopoietic stem cells but also the earliest precursors of endothelial cells. We have previously shown that a nonadherent yolk sac cell population (WGA+, density <1.077, AA4.1+) can give rise to B cells, T cells, and myeloid cells both in vitro and in vivo. We now report on the ability of a yolk sac-derived cloned endothelial cell line (C166) to provide a suitable microenvironment for expansion of these early precursor cells. Single day 10 embryonic mouse yolk sac hematopoietic stem cells were expanded >100 fold within 8 days by coculture with irradiated C166 cells. Colony-forming ability was retained for at least three passages in vitro, with retention of the ability to differentiate into T-cell, B-cell, and myeloid lineages. Stem cell properties were maintained by a significant fraction of nonadherent cells in the third passage, although these stem cells expressed a somewhat more mature cell surface phenotype than the initial yolk sac stem cells. When reintroduced into adult allogeneic immunocompromised (scid) hosts, they were able to give rise to all of the leukocyte lineages, including T cells, B cells, and myeloid cells. We conclude that yolk sac endothelial cells can support the stable proliferation of multipotential hematopoietic stem cells, thus generating adequate numbers of cells for study of the mechanisms involved in their subsequent development and differentiation, for in vivo hematopoietic restitution, and for potential use as a vehicle for gene transfer.

Coculturing diverse clonal populations prevents the early-stage neoplastic progression that occurs in the separate clones

Most human cancers are of monoclonal origin and display many genetic alterations. In an effort to determine whether clonal expansion itself could account for the large number of genetic alterations, we compared spontaneous transformation in cloned and uncloned populations of NIH 3T3 cells. We have reported that progressive transformation of these cells, which is driven by the stress of prolonged contact inhibition at confluence, occurs far more frequently in cultures of recent monoclonal origin than in their uncloned progenitors. In the present work we asked how coculturing six clones at early and late stages of progression would affect the dynamics of transformation in repeated rounds of confluence. When coculture started with clones in early stages of transformation, marked by light focus formation, there was a strong inhibition of the progression to the dense focus formation that occurred in separate cultures of the individual clones. In contrast, when coculture started after the individual clones had progressed to dense focus formation, there was selection of transformants from the clone producing the largest and densest foci. Mixing the cells of a single clone with a large excess of uncloned cells from a subline that was refractory to transformation markedly decreased the size of dense foci from clones in transit from light to dense focus formation, but had much less effect on foci from clones with an established capacity for dense focus formation. The major finding of protection against progression by coculturing clones in early stages of transformation suggests that the expansion of a rogue clone in vivo increasingly isolates many of its cells from genetically stabilizing interactions with surrounding clones. Such clonal isolation might account for the increase in mutation rates associated with the dysplasia in colorectal adenomas that signifies the transition between benign and malignant growth.

The cellular ecology of progressive neoplastic transformation: A clonal analysis

A comparison was made of the competence for neoplastic transformation in three different sublines of NIH 3T3 cells and multiple clonal derivatives of each. Over 90% of the neoplastic foci produced by an uncloned transformed (t-SA′) subline on a confluent background of nontransformed cells were of the dense, multilayered type, but about half of the t-SA′ clones produced only light foci in assays without background. This asymmetry apparently arose from the failure of the light focus formers to register on a background of nontransformed cells. Comparison was made of the capacity for confluence-mediated transformation between uncloned parental cultures and their clonal derivatives by using two nontransformed sublines, one of which was highly sensitive and the other relatively refractory to confluence-mediated transformation. Transformation was more frequent in the clones than in the uncloned parental cultures for both sublines. This was dramatically so in the refractory subline, where the uncloned culture showed no overt sign of transformation in serially repeated assays but increasing numbers of its clones exhibited progressive transformation. The reason for the greater susceptibility of the pure clones is apparently the suppression of transformation among the diverse membership that makes up the uncloned parental culture. Progressive selection toward increasing degrees of transformation in confluent cultures plays a major role in the development of dense focus formers, but direct induction by the constraint of confluence may contribute by heritably damaging cells. In view of our finding of increased susceptibility to transformation in clonal versus uncloned populations, expansion of some clones at the expense of others during the aging process would contribute to the marked increase of cancer with age.

Mutational inactivation of the proapoptotic gene BAX confers selective advantage during tumor clonal evolution

A remarkable instability at simple repeated sequences characterizes gastrointestinal cancer of the microsatellite mutator phenotype (MMP). Mutations in the DNA mismatch repair gene family underlie the MMP, a landmark for hereditary nonpolyposis colorectal cancer. These tumors define a distinctive pathway for carcinogenesis because they display a particular spectrum of mutated cancer genes containing target repeats for mismatch repair deficiency. One such gene is BAX, a proapoptotic member of the Bcl-2 family of proteins, which plays a key role in programmed cell death. More than half of colon and gastric cancers of the MMP contain BAX frameshifts in a (G)8 mononucleotide tract. However, the functional significance of these mutations in tumor progression has not been established. Here we show that inactivation of the wild-type BAX allele by de novo frameshift mutations confers a strong advantage during tumor clonal evolution. Tumor subclones with only mutant alleles frequently appeared after inoculation into nude mice of single-cell clones of colon tumor cell lines with normal alleles. In contrast, no clones of BAX-expressing cells were found after inoculation of homozygous cell clones without wild-type BAX. These results support the interpretation that BAX inactivation contributes to tumor progression by providing a survival advantage. In this context, survival analyses show that BAX mutations are indicators of poor prognosis for both colon and gastric cancer of the MMP.

Minisatellite marker analysis of Trypanosoma brucei: Reconciliation of clonal, panmictic, and epidemic population genetic structures

The African trypanosome, Trypanosoma brucei, has been shown to undergo genetic exchange in the laboratory, but controversy exists as to the role of genetic exchange in natural populations. Much of the analysis to date has been derived from isoenzyme or randomly amplified polymorphic DNA data with parasite material from a range of hosts and geographical locations. These markers fail to distinguish between the human infective (T. b. rhodesiense) and nonhuman infective (T. b. brucei) “subspecies” so that parasites derived from hosts other than humans potentially contain both subspecies. To overcome some of the inherent problems with the use of such markers and diverse populations, we have analyzed a well-defined population from a discrete geographical location (Busoga, Uganda) using three recently described minisatellite markers. The parasites were primarily isolated from humans and cattle with the latter isolates further characterized by their ability to resist lysis by human serum (equivalent to human infectivity). The minisatellite markers show high levels of polymorphism, and from the data obtained we conclude that T. b. rhodesiense is genetically isolated from T. b. brucei and can be unambiguously identified by its multilocus genotype. Analysis of the genotype frequencies in the separated T. b. brucei and T. b. rhodesiense populations shows the former has an epidemic population structure whereas the latter is clonal. This finding suggests that the strong linkage disequilibrium observed in previous analyses, where human and nonhuman infective trypanosomes were not distinguished, results from the treatment of two genetically isolated populations as a single population.

Evidence for clonal propagation in natural isolates of Plasmodium falciparum from Venezuela

We have analyzed 75 isolates of Plasmodium falciparum, collected in Venezuela during both the dry (November) and rainy (May–July) seasons, with a range of genetic markers including antigen genes and 14 random amplified polymorphic DNA (RAPD) primers. Thirteen P. falciparum stocks from Kenya and four other Plasmodium species are included in the analysis for comparison. Cross-hybridization shows that the 14 RAPD primers reveal 14 separate regions of the parasite's genome. The P. falciparum isolates are a monophyletic clade, significantly different from the other Plasmodium species. We identify three RAPD characters that could be useful as “tags” for rapid species identification. The Venezuelan genotypes fall into two discrete genetic subdivisions associated with either the dry or the rainy season; the isolates collected in the rainy season exhibit greater genetic diversity. There is significant linkage disequilibrium in each seasonal subsample and in the full sample. In contrast, no linkage disequilibrium is detected in the African sample. These results support the hypothesis that the population structure of P. falciparum in Venezuela, but not in Africa, is predominantly clonal. However, the impact of genetic recombination on Venezuelan P. falciparum seems higher than in parasitic species with long-term clonal evolution like Trypanosoma cruzi, the agent of Chagas' disease. The genetic structure of the Venezuelan samples is similar to that of Escherichia coli, a bacterium that propagates clonally, with occasional genetic recombination.

Clonal analysis of stably transduced human epidermal stem cells in culture.

We have transduced normal human keratinocytes with retroviral constructs expressing a bacterial beta-galactosidase (beta-gal) gene or a human interleukin-6 (hIL-6) cDNA under control of a long terminal repeat. Efficiency of gene transfer averaged approximately 50% and 95% of clonogenic keratinocytes for beta-gal and hIL-6, respectively. Both genes were stably integrated and expressed for more than 150 generations. Clonal analysis showed that both holoclones and their transient amplifying progeny expressed the transgene permanently. Southern blot analysis on isolated clones showed that many keratinocyte stem cells integrated multiple proviral copies in their genome and that the synthesis of the exogenous gene product in vitro was proportional to the number of proviral integrations. When cohesive epidermal sheets prepared from stem cells transduced with hIL-6 were grafted on athymic animals, the serum levels of hIL-6 were strictly proportional to the rate of secretion in vitro and therefore to the number of proviral integrations. The possibility of specifying the level of transgene expression and its permanence in a homogeneous clone of stem cell origin opens new perspectives in the long-term treatment of genetic disorders.

Irreversibility of cellular aging and neoplastic transformation: a clonal analysis.

Prolonged incubation of NIH 3T3 cells under the growth constraint of confluence results in a persistent impairment of proliferation when the cells are subcultured at low density and a greatly increased probability of neoplastic transformation in assays for transformation. These properties, along with the large accumulation of age pigment bodies in the confluent cells, are cardinal cellular characteristics of aging in organisms and validate the system as a model of cellular aging. Two cultures labeled alpha and beta were obtained after prolonged confluence; both were dominated by cells that were both slowed in growth at low population density and enhanced in growth capacity at high density, a marker of neoplastic transformation. An experiment was designed to study the reversibility of these age-related properties by serial subculture at low density of the two uncloned cultures and their progeny clones derived from assuredly single cells. Both uncloned cultures had many transformed cells and a reduced growth rate on subculture. Serial subculture resulted in a gradual increase in growth rates of both populations, but a reversal of transformation only in the alpha population. The clones originating from both populations varied in the degree of growth impairment and neoplastic transformation. None of the alpha clones increased in growth rate on low density passage nor did the transformed clones among them revert to normal growth behavior. The fastest growing beta clone was originally slower than the control clone, but caught up to it after four weekly subcultures. The other beta clones retained their reduced growth rates. Four of the five beta clones, including the fastest grower, were transformed, and none reverted on subculture. We conclude that the apparent reversal of impaired growth and transformation in the uncloned parental alpha population resulted from the selective growth at low density of fast growing nontransformed clones. The reversal of impaired growth in the uncloned parental beta population was also the result of selective growth of fast growing clones, but in this case they were highly transformed so no apparent reversal of transformation occurred. The clonal results indicate that neither the impaired growth nor the neoplastic transformation found in aging cells is reversible. We discuss the possible contribution of epigenetic and genetic processes to these irreversible changes.

Molecular evidence that the asexual industrial fungus Trichoderma reesei is a clonal derivative of the ascomycete Hypocrea jecorina.

The relationship of the important cellulase producing asexual fungus Trichoderma reesei to its putative teleomorphic (sexual) ancestor Hypocrea jecorina and other species of the Trichoderma sect. Longibrachiatum was studied by PCR-fingerprinting and sequence analyses of the nuclear ribosomal DNA region containing the internal transcribed spacers (ITS-1 and ITS-2) and the 5.8S rRNA gene. The differences in the corresponding ITS sequences allowed a grouping of anamorphic (asexual) species of Trichoderma sect. Longibrachiatum into Trichoderma longibrachiatum, Trichoderma pseudokoningii, and Trichoderma reesei. The sexual species Hypocrea schweinitzii and H. jecorina were also clearly separated from each other. H. jecorina and T. reesei exhibited identical sequences, suggesting close relatedness or even species identity. Intraspecific and interspecific variation in the PCR-fingerprinting patterns supported the differentiation of species based on ITS sequences, the grouping of the strains, and the assignment of these strains to individual species. The variations between T. reesei and H. jecorina were at the same order of magnitude as found between all strains of H. jecorina, but much lower than the observed interspecific variations. Identical ITS sequences and the high similarity of PCR-fingerprinting patterns indicate a very close relationship between T. reesei and H. jecorina, whereas differences of the ITS sequences and the PCR-fingerprinting patterns show a clear phylogenetic distance between T. reesei/H. jecorina and T. longibrachiatum. T. reesei is considered to be an asexual, clonal line derived from a population of the tropical ascomycete H. jecorina.

Transduction of pluripotent human hematopoietic stem cells demonstrated by clonal analysis after engraftment in immune-deficient mice.

Gene transduction of pluripotent human hematopoietic stem cells (HSCs) is necessary for successful gene therapy of genetic disorders involving hematolymphoid cells. Evidence for transduction of pluripotent HSCs can be deduced from the demonstration of a retroviral vector integrated into the same cellular chromosomal DNA site in myeloid and lymphoid cells descended from a common HSC precursor. CD34+ progenitors from human bone marrow and mobilized peripheral blood were transduced by retroviral vectors and used for long-term engraftment in immune-deficient (beige/nude/XIS) mice. Human lymphoid and myeloid populations were recovered from the marrow of the mice after 7-11 months, and individual human granulocyte-macrophage and T-cell clones were isolated and expanded ex vivo. Inverse PCR from the retroviral long terminal repeat into the flanking genomic DNA was performed on each sorted cell population. The recovered cellular DNA segments that flanked proviral integrants were sequenced to confirm identity. Three mice were found (of 24 informative mice) to contain human lymphoid and myeloid populations with identical proviral integration sites, confirming that pluripotent human HSCs had been transduced.

Genetic heterogeneity in isogenic homozygous clonal zebrafish.

The C32 isogenic homozygous diploid (IHD) strain of the zebrafish (Danio rerio) was found to be polyallelic at a malate dehydrogenase locus (sMdh-A). A variant allele is thought to have arisen via mutation within the past 10 bisexual generations that have maintained the strain since its last gynogenetic cloning event; this unique allele now predominates at the sMdh-A locus. The estimated mutation rate in this species is sufficiently high that long-term genetic homogeneity of its IHD clones cannot be assumed. Researchers using such bisexually maintained clones should be aware that they are not necessarily using genetically uniform subjects. Genetic uniformity of cloned IHD zebrafish will be maximized if experimental subjects are obtained soon after a cloning event.

Identification and characterization of an immunophilin expressed during the clonal expansion phase of adipocyte differentiation.

Mouse 3T3-L1 cells differentiate into fat-laden adipocytes in response to a cocktail of adipogenic hormones. This conversion process occurs in two discrete steps. During an early clonal expansion phase, confluent 3T3-L1 cells proliferate and express the products of the beta and delta members of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors. The cells subsequently arrest mitotic growth, induce the expression of the alpha form of C/EBP, and acquire the morphology of fully differentiated adipocytes. Many of the genes induced during the terminal phase of adipocyte conversion are directly activated by C/EBP alpha, and gratuitous expression of this transcription factor is capable of catalyzing adipose conversion in a number of different cultured cell lines. The genetic program undertaken during the clonal expansion phase of 3T3-L1 differentiation, controlled in part by C/EBP beta and C/EBP delta, is less clearly understood. To study the molecular events occurring during clonal expansion, we have identified mRNAs that selectively accumulate during this phase of adipocyte conversion. One such mRNA encodes an immunophilin hereby designated FKBP51. In this report we provide the initial molecular characterization of FKBP51.

Rapamycin inhibits clonal expansion and adipogenic differentiation of 3T3-L1 cells.

Differentiating 3T3-L1 cells express an immunophilin early during the adipocyte conversion program as described in this issue [Yeh, W.-C., Li, T.-K., Bierer, B. E. & McKnight, S. L. (1995) Proc. Natl. Acad. Sci. USA 92, 11081-11085]. The temporal expression profile of this protein, designated FK506-binding protein (FKBP) 51, is concordant with the clonal-expansion period undertaken by 3T3-L1 cells after exposure to adipogenic hormones. Having observed FKBP51 synthesis early during adipogenesis, we tested the effects of three immunosuppressive drugs--cyclosporin A, FK506, and rapamycin--on the terminal-differentiation process. Adipocyte conversion was not affected by either cyclosporin A or FK506 and yet was significantly reduced by rapamycin at drug concentrations as low as 10 nM. Clonal expansion was impeded in drug-treated cultures, as was the accumulation of cytoplasmic lipid droplets normally seen late during differentiation. Rapamycin treatment likewise inhibited the expression of CCAAT/enhancer binding protein alpha, a transcription factor required for 3T3-L1 cell differentiation. All three of these effects were reversed by high FK506 concentrations, indicating that the operative inhibitory event was mediated by an immunophilin-rapamycin complex.

Extreme clonal uniformity of Phoxinus eos/neogaeus gynogens (pisces: Cyprinidae) among variable habitats in northern Minnesota beaver ponds.

Genetic surveys of parthenogenetic vertebrate populations have demonstrated a common pattern of relatively high degrees of clonal variation and the coexistence of numerous clones. In striking contrast, the Phoxinus eos/Phoxinus neogaeus/hybrid gynogen complex of cyprinid fishes exhibits no clonal variation within a northern Minnesota drainage characterized by successional beaver ponds. Gynogens were sampled from three habitats in each of four different pond types in a single drainage in Voyageurs National Park, Minnesota. The abundance of gynogens relative to sexual dace varied with pond type, being least common in deep upland ponds and most common in shallow, collapsed, lowland ponds (13.4% and 48.6%, respectively). Simple-sequence multilocus DNA fingerprinting of 464 individual gynogens detected one, and only one, clone. DNA fingerprints, generated sequentially by using three oligonucleotide probes, (CAC)5, (GACA)4, and the Jeffreys' 33.15 probe, all revealed the same unprecedented lack of variation. The extreme lack of clonal diversity in these gynogens across a range of habitat types does not fit the general pattern of high clonal diversity found within populations of other vertebrate parthenogens.