Tumor suppressor gene Rb is required for self-renewal of spermatogonial stem cells in mice

The retinoblastoma tumor suppressor gene Rb is essential for maintaining the quiescence and for regulating the differentiation of somatic stem cells. Inactivation of Rb in somatic stem cells typically leads to their overexpansion, often followed by increased apoptosis, defective terminal differentiation, and tumor formation. However, Rb’s roles in germ-line stem cells have not been explored. We conditionally disrupted the Rb gene in mouse germ cells in vivo and discovered unanticipated consequences for GFRa1-protein-expressing A_single (GFRa1⁺ A_s) spermatogonia, the major source of male germ-line stem cells. Rb-deficient GFRa1⁺ A_s spermatogonia were present at normal density in testes 5 d after birth, but they lacked the capacity for self-renewal, resulting in germ cell depletion by 2 mo of age. Rb deficiency did not affect the proliferative activity of GFRa1⁺ A_s spermatogonia, but their progeny were exclusively transit-amplifying progenitor spermatogonia and did not include GFRa1⁺ A_s spermatogonia. In addition, Rb deficiency caused prolonged proliferation of progenitor spermatogonia, transiently enlarging this population. Despite these defects, Rb deficiency did not block terminal differentiation into functional sperm; offspring were readily obtained from young males whose germ cell pool was not yet depleted. We conclude that Rb is required for self-renewal of germ-line stem cells, but contrary to its critical roles in somatic stem cells, it is dispensable for their proliferative activity and terminal differentiation. Thus, this study identifies an unexpected function for Rb in maintaining the stem cell pool in the male germ line.     

Adenovirus-mediated gene delivery into mouse spermatogonial stem cells

Spermatogonial stem cells represent a self-renewing population of spermatogonia, and continuous division of these cells supports spermatogenesis throughout the life of adult male animals. Previous attempts to introduce adenovirus vectors into spermatogenic cells, including spermatogonial stem cells, have failed to yield evidence of infection, suggesting that male germ cells may be resistant to adenovirus infection. In this study we show the feasibility of transducing spermatogonial stem cells by adenovirus vectors. When testis cells from ROSA26 Cre reporter mice were incubated in vitro with a Cre-expressing adenovirus vector, Cre-mediated recombination occurred at an efficiency of 49-76%, and the infected spermatogonial stem cells could reinitiate spermatogenesis after transplantation into seminiferous tubules of infertile recipient testes. No evidence of germ-line integration of adenovirus vector could be found in offspring from infected stem cells that underwent Cre-mediated recombination, which suggests that the adenovirus vector infected the cells but did not stably integrate into the germ line. Nevertheless, these results suggest that adenovirus may inadvertently integrate into the patient's germ line and indicate that there is no barrier to adenovirus infection in spermatogonial stem cells.     

An essential amino acid residue in the protein translocation channel revealed by targeted random mutagenesis of SecY

The SecY/Sec61alpha family of membrane proteins are the central subunits of the putative protein translocation channel. We introduced random mutations into a segment of Escherichia coli SecY within its cytoplasmic domain 5, which was shown previously to be important for the SecA-dependent translocation activity. Mutations were classified into those retaining function and those gaining a dominant-interfering ability caused by a loss of function. These analyses showed that Arg-357, Pro-358, Gly-359, and Thr-362 are functionally important; Arg-357, conserved in almost all organisms, was identified as an indispensable residue.     

Generation of functional cardiomyocytes from adult mouse spermatogonial stem cells

Stem cell-based therapy is a promising approach for the treatment of heart failure. Adult stem cells with the pluripotency of embryonic stem cells (ESCs) would be an ideal cell source. Recently, we reported the successful establishment of multipotent adult germline stem cells (maGSCs) from mouse testis. These cultured maGSCs show phenotypic characteristics similar to ESCs and can spontaneously differentiate into cells from all 3 germ layers. In the present study, we used the hanging drop method to differentiate maGSCs into cardiomyocytes and analyzed their functional properties. Differentiation efficiency of beating cardiomyocytes from maGSCs was similar to that from ESCs. The maGSC-derived cardiomyocytes expressed cardiac-specific L-type Ca(2+) channels and responded to Ca(2+) channel-modulating drugs. Cx43 was expressed at cell-to-cell contacts in cardiac clusters, and fluorescence recovery after photobleaching assay showed the presence of functional gap junctions among cardiomyocytes. Action potential analyses demonstrated the presence of pacemaker-, ventricle-, atrial-, and Purkinje-like cardiomyocytes. Stimulation with isoproterenol resulted in a significant increase in beating frequency, whereas the addition of cadmium chloride abolished spontaneous electrical activity. Confocal microscopy analysis of intracellular Ca(2+) in maGSC-derived cardiomyocytes showed that calcium increased periodically throughout the cell in a homogenous fashion, pointing to a fine regulated Ca(2+) release from intracellular Ca(2+) stores. By using line-scan mode, we found rhythmic Ca(2+) transients. Furthermore, we transplanted maGSCs into normal hearts of mice and found that maGSCs were able to proliferate and differentiate. No tumor formation was found up to 1 month after cell transplantation. Taken together, we believe that maGSCs provide a new source of distinct types of cardiomyocytes for basic research and potential therapeutic application.     

AMD3100 synergizes with G-CSF to mobilize repopulating stem cells in Fanconi anemia knockout mice

Fanconi anemia (FA) is a heterogeneous inherited disorder characterized by a progressive bone marrow (BM) failure and susceptibility to myeloid leukemia. Genetic correction using gene-transfer technology is one potential therapy. A major hurdle in applying this technology in FA patients is the inability of granulocyte colony-stimulating factor (G-CSF) to mobilize sufficient numbers of hematopoietic stem (HSC)/progenitor cells (HPC) from the BM to the peripheral blood. Whether the low number of CD34(+) cells is a result of BM hypoplasia or an inability of G-CSF to adequately mobilize FA HSC/HPC remains incompletely understood. Here we use competitive repopulation of lethally irradiated primary and secondary recipients to show that in two murine models of FA, AMD3100 synergizes with G-CSF resulting in a mobilization of HSC, whereas G-CSF alone fails to mobilize stem cells even in the absence of hypoplasia.     

De novo testosterone production in luteinizing hormone receptor knockout mice after transplantation of leydig stem cells

Mesenchymal stem cells or Leydig cell progenitors are rare and difficult to isolate from adult testes. The property of differential efflux of Hoechst 33342 dye by the multi-drug-like transporter enriches murine hematopoietic stem cells from bone marrow. Our work on testicular cell transplantation suggests that the "Hoechst dim" side population (SP) also contains Leydig stem cells or progenitors that proliferate and differentiate into mature functional Leydig cells. We harvested testicular cells from cryptorchid ROSA26 mice, stained them with Hoechst dye, and isolated the cell population that excludes the dye using flow cytometry. Mice with targeted deletion of the LH receptor (LHR) gene were used as the recipients of the transplanted cells. These mice are hypogonadal and infertile. Both testicular SP and non-SP cells were transplanted into the interstitium of the LHR knockout recipients' testes. Serial serum testosterone assays revealed a significant increase in the circulating testosterone levels and restoration of spermatogenesis in the LHR-knockout recipients transplanted with the SP cells compared with that of those transplanted with non-SP. A SP cell concentration- and time-dependent increase in circulating testosterone was observed. This demonstrates the successful transplantation of functional putative Leydig stem cells into a hypogonadal recipient. The increase in testosterone concentration indicates the de novo synthesis of androgen by the transplanted SP cells. This method offers a novel technique to isolate Leydig stem cells and to study Leydig cell development.     

Testosterone inhibits spermatogonial differentiation in juvenile spermatogonial depletion mice.

The juvenile spermatogonial depletion (jsd) mutation results in spermatogonial arrest after the first wave of spermatogenesis. In homozygous jsd mice in a hybrid background (C3HxB6) that were identified with microsatellite markers, the percentage of tubules showing differentiating germ cells [tubule differentiation index (TDI)] rapidly decreased after 7 weeks of age with a correlative increase in the intratesticular testosterone (ITT) levels. Treatment with a GnRH antagonist, Cetrorelix, suppressed ITT and stimulated spermatogonial differentiation at the end of treatment. When treated mice were killed 5-13.3 weeks after the end of treatment, the ITT progressively increased, and the TDI progressively declined, but there was a transient appearance of tubules with mature spermatids. To delineate the role of testosterone (T) in spermatogonial arrest, we gave 7.6-week-old jsd mice exogenous T and/or the androgen receptor antagonist flutamide with or without GnRH antagonist for 4 weeks. Flutamide alone moderately stimulated spermatogonial differentiation (TDI = 30%). GnRH antagonist increased the TDI to 73%, and the addition of flutamide to the GnRH antagonist treatment further increased it to 95%. When T was combined with GnRH antagonist treatment, ITT was increased, and the TDI was reduced to 7%. Addition of flutamide to this combination reversed the T inhibition of GnRH antagonist stimulation of spermatogonial differentiation to a TDI of 57%. ITT levels showed a good negative correlation to the TDI obtained with various treatments, but no such correlation was observed for FSH or LH levels. The results indicate that T inhibits the ability of spermatogonia to differentiate in jsd mice through an androgen receptor-mediated process.     

Partial rescue of PTH/PTHrP receptor knockout mice by targeted expression of the Jansen transgene.

The homozygous ablation of the gene encoding the PTH/PTHrP receptor (PPR(-/-)) leads to early lethality and limited developmental defects, including an acceleration of chondrocyte differentiation. In contrast to the findings in homozygous PTHrP-ablated (PTHrP(-/-)) animals, these PPR(-/-) mice show an increase in cortical bone, a decrease in trabecular bone, and a defect in bone mineralization. Opposite observations are made in Jansen's metaphyseal chondrodysplasia, a disorder caused by constitutively active PPR mutants, and in transgenic animals expressing one of these receptor mutants (HKrk-H223R) under control of the type alpha1(I) collagen promoter. Expression of the Jansen transgene under the control of the type alpha1(II) collagen promoter was, furthermore, shown to delay chondrocyte differentiation and to prevent the dramatic acceleration of chondrocyte differentiation in PTHrP(-/-) mice, thus rescuing the early lethality of these animals. In the present study we demonstrated that the type alpha1(II) collagen promoter Jansen transgene restored most of the bone abnormalities in PPR(-/-) mice, but did not prevent their perinatal lethality. These findings suggested that factors other than impaired gas exchange due to an abnormal rib cage contribute to the early death of PPR(-/-) mice.     

Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells

Spermatogonial stem cells (SSCs) self-renew and produce large numbers of committed progenitors that are destined to differentiate into spermatozoa throughout life. However, the growth factors essential for self-renewal of SSCs remain unclear. In this study, a serum-free culture system and a transplantation assay for SSCs were used to identify exogenous soluble factors that promote proliferation of SSCs. Mouse pup testis cells were enriched for SSCs by selection with an anti-Thy-1 antibody and cultured on STO (SIM mouse embryo-derived thioguanine and ouabain resistant) feeders in a serum-free defined medium. In the presence of glial cell line-derived neurotrophic factor (GDNF), SSCs from DBA/2J strain mice formed densely packed clumps of cells and continuously proliferated. However, other strains of mice required the addition of soluble GDNF-family receptor alpha-1 and basic fibroblast growth factor to support replication. The functional transplantation assay proved that the clump-forming cells are indeed SSCs. Thus, GDNF-induced cell signaling plays a central role in SSC self-renewal. The number of SSCs in culture doubled every 5.6 days, and the clump-forming cells strongly expressed Oct-4. Under these conditions, SSCs proliferated over 6 months, reconstituted long-term spermatogenesis after transplantation into recipient testes, and restored fertility to infertile recipients. The identification of exogenous factors that allow continuous proliferation of SSCs in vitro establishes the foundation to study the basic biology of SSCs and makes possible germ-line modification by sophisticated technologies. Moreover, the ability to recover, culture indefinitely, and transplant SSCs will make the germ-line of individual males available for periods extending beyond a normal lifetime.     

Androgen suppression-induced stimulation of spermatogonial differentiation in juvenile spermatogonial depletion mice acts by elevating the testicular temperature

Why both testosterone (T) suppression and cryptorchidism reverse the block in spermatogonial differentiation in adult mice homozygous for the juvenile spermatogonial depletion (jsd) mutation has been a conundrum. To resolve this conundrum, we analyzed interrelations between T suppression, testicular temperature, and spermatogonial differentiation and used in vitro techniques to separate the effects of the two treatments on the spermatogonial differentiation block in jsd mice. Temporal analysis revealed that surgical cryptorchidism rapidly stimulated spermatogonial differentiation whereas androgen ablation treatment produced a delayed and gradual differentiation. The androgen suppression caused scrotal shrinkage, significantly increasing the intrascrotal temperature. When serum T or intratesticular T (ITT) levels were modulated separately in GnRH antagonist-treated mice by exogenous delivery of T or LH, respectively, the inhibition of spermatogonial differentiation correlated with the serum T and not with ITT levels. Thus, the block must be caused by peripheral androgen action. When testicular explants from jsd mice were cultured in vitro at 32.5 C, spermatogonial differentiation was not observed, but at 37 C significant differentiation was evident. In contrast, addition of T to the culture medium did not block the stimulation of spermatogonial differentiation at 37 C, and androgen ablation with aminoglutethimide and hydroxyflutamide did not stimulate differentiation at 32.5 C, suggesting that T had no direct effect on spermatogonial differentiation in jsd mice. These data show that elevation of temperature directly overcomes the spermatogonial differentiation block in adult jsd mice and that T suppression acts indirectly in vivo by causing scrotal regression and thereby elevating the testicular temperature.     

Cryptorchidism rescues spermatogonial differentiation in juvenile spermatogonial depletion (jsd) mice

Male mice homozygous for jsd mutation undergo an initial wave of spermatogenesis, but spermatogonial differentiation ceases a few weeks after birth; at that point the tubules show only type A spermatogonia and Sertoli cells. To test whether testicular descent into the scrotum contributes to the block in spermatogonial differentiation, jsd mutant (jsd/jsd) mice were bilaterally cryptorchidized at the age of 4 wk. Surprisingly, 8 wk later, germ cell differentiation was maintained in 98% of the tubules, a rate that fell to 13.5% in mice without surgery. The testis weight and the degree of spermatogenesis in cryptorchidized normal (jsd/+) and jsd mutant mice were almost identical. Furthermore, germ cell differentiation was also restored in almost all the tubules in 20-wk- and 70-wk-old jsd mutant testis unilaterally cryptorchidized 8 wk earlier, whereas the contralateral scrotal testis in these mice showed differentiation in only 6% of tubules. In irradiated LBNF1 rats, which have a block in spermatogonial differentiation similar to that in jsd mutant mice, unilateral cryptorchidism produced a small but significant increase in the percentage of differentiated tubules. In both of these models, the intratesticular levels of testosterone in the cryptorchidized testes were still above the physiological range, and the serum testosterone and LH levels were unchanged after bilateral or unilateral cryptorchidization. Cryptorchidism also did not alter serum FSH levels after bilateral and unilateral cryptorchidism in jsd mutant mice and irradiated rats, respectively. We conclude that cryptorchidism reverses the phenotype in jsd mutant mice. The findings show for the first time that spermatogenesis in rodents, and spermatogonial differentiation in particular, is sensitive to reduced scrotal temperature. Furthermore, we conclude that in jsd mutant mice spermatogonial differentiation is inhibited by testosterone only at the normal scrotal temperature.     

Calcium dependence of aequorin bioluminescence dissected by random mutagenesis

Aequorin bioluminescence is emitted as a rapidly decaying flash upon calcium binding. Random mutagenesis and functional screening were used to isolate aequorin mutants showing slow decay rate of luminescence. Calcium sensitivity curves were shifted in all mutants, and an intrinsic link between calcium sensitivity and decay rate was suggested by the position of all mutations in or near EF-hand calcium-binding sites. From these results, a low calcium affinity was assigned to the N-terminal EF hand and a high affinity to the C-terminal EF-hand pair. In WT aequorin, the increase of the decay rate with calcium occurred at constant total photon yield and thus determined a corresponding increase of light intensity. Increase of the decay rate was underlain by variations of a fast and a slow component and required the contribution of all three EF hands. Conversely, analyses of double EF-hand mutants suggested that single EF hands are sufficient to trigger luminescence at a slow rate. Finally, a model postulating that proportions of a fast and a slow light-emitting state depend on calcium concentration adequately described the calcium dependence of aequorin bioluminescence. Our results suggest that variations of luminescence kinetics, which depend on three EF hands endowed with different calcium affinities, critically determine the amplitude of aequorin responses to biological calcium signals.     

Stimulation of spermatogonial differentiation in juvenile spermatogonial depletion (jsd) mutant mice by gonadotropin-releasing hormone antagonist treatment.

Male juvenile spermatogonial depletion (jsd) mutant mice are sterile because of spermatogenic failure and so may provide a model for genetically caused human male infertility. To test the effects of testosterone suppression therapy on spermatogenesis in jsd/jsd mice, we treated them with Nal-Glu, a GnRH antagonist. Treatment with Nal-Glu at 2500 microg/kg/day was started at 5.5 or 8 weeks of age and continued for 4 or 8 weeks. Differentiation of spermatogonia was evaluated by the percentage of tubules containing two or more spermatocytes (% of differentiating tubules). Nal-Glu treatment caused a marked decrease in the weights of the testes and seminal vesicles and intratesticular testosterone concentrations. However, in contrast to a value of 1.3% in untreated jsd/jsd mice, the mean % of differentiating tubules was 59.9% and 25.1% in treatment groups started at 5.5 and 8 weeks of age, respectively. We propose that spermatogonial differentiation in jsd/jsd mutant mice is sensitive to the high intratesticular levels of testosterone and can only proceed when testosterone production is suppressed.     

肝癌干细胞靶向治疗的研究进展

肝癌是一种恶性肿瘤,目前的手术、放化疗均不能取得满意疗效,复发、转移一直是肝癌治疗的难题。根据近些年研究的肝癌干细胞学说,肝癌的发生、发展、复发、转移、预后都与肝癌干细胞有关。如果靶向作用于肝癌的干细胞,使其数目减少或者被消灭掉,就会使肿瘤切除术后的复发、转移性及耐药性降低或被清除。对肝癌干细胞的靶向治疗进展情况作一综述,认为肝癌干细胞的靶向治疗方法虽然有很多种,但是靶向性不强仍然是亟需解决的关键问题,是否可以通过双靶向或者多靶向联合解决靶向性的问题还需要进一步的实验研究证实。     

Rats produced by interspecies spermatogonial transplantation in mice and in vitro microinsemination

Spermatogonial transplantation has demonstrated a unique opportunity for studying spermatogenesis and provided an assay for spermatogonial stem cells. However, it has remained unknown whether germ cells that matured in a xenogeneic environment are functionally normal. In this investigation, we demonstrate the successful production of xenogeneic offspring by using spermatogonial transplantation. Rat spermatogonial stem cells were collected from immature testis and transplanted into the seminiferous tubules of busulfan-treated nude mouse testis. Using rat spermatids or spermatozoa that developed in xenogeneic surrogate mice, rat offspring were born from fresh and cryopreserved donor cells after microinsemination with rat oocytes. These offspring were fertile and had a normal imprinting pattern. The xenogeneic offspring production by interspecies germ cell transplantation and in vitro microinsemination will become a powerful tool in animal transgenesis and species conservation.     

Somatic stem cells and the kinetics of mutagenesis and carcinogenesis

There is now strong experimental evidence that epithelial stem cells arrange their sister chromatids at mitosis such that the same template DNA strands stay together through successive divisions; DNA labeled with tritiated thymidine in infancy is still present in the stem cells of adult mice even though these cells are incorporating (and later losing) bromodeoxyuridine [Potten, C. S., Owen, G., Booth, D. & Booth, C. (2002) J. Cell Sci.115, 2381-2388]. But a cell that preserves "immortal strands" will avoid the accumulation of replication errors only if it inhibits those pathways for DNA repair that involve potentially error-prone resynthesis of damaged strands, and this appears to be a property of intestinal stem cells because they are extremely sensitive to the lethal effects of agents that damage DNA. It seems that the combination, in the stem cell, of immortal strands and the choice of death rather than error-prone repair makes epithelial stem cell systems resistant to short exposures to DNA-damaging agents, because the stem cell accumulates few if any errors, and any errors made by the daughters are destined to be discarded. This paper discusses these issues and shows that they lead to a model that explains the strange kinetics of mutagenesis and carcinogenesis in adult mammalian tissues. Coincidentally, the model also can explain why cancers arise even though the spontaneous mutation rate of differentiated mammalian cells is not high enough to generate the multiple mutations needed to form a cancer and why loss of nucleotide-excision repair does not significantly increase the frequency of the common internal cancers.     

Engineering of targeted megabase-scale deletions in human induced pluripotent stem cells

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Transposon mutagenesis identifies genes that transform neural stem cells into glioma-initiating cells

Neural stem cells (NSCs) are considered to be the cell of origin of glioblastoma multiforme (GBM). However, the genetic alterations that transform NSCs into glioma-initiating cells remain elusive. Using a unique transposon mutagenesis strategy that mutagenizes NSCs in culture, followed by additional rounds of mutagenesis to generate tumors in vivo, we have identified genes and signaling pathways that can transform NSCs into glioma-initiating cells. Mobilization of Sleeping Beauty transposons in NSCs induced the immortalization of astroglial-like cells, which were then able to generate tumors with characteristics of the mesenchymal subtype of GBM on transplantation, consistent with a potential astroglial origin for mesenchymal GBM. Sequence analysis of transposon insertion sites from tumors and immortalized cells identified more than 200 frequently mutated genes, including human GBM-associated genes, such as Met and Nf1, and made it possible to discriminate between genes that function during astroglial immortalization vs. later stages of tumor development. We also functionally validated five GBM candidate genes using a previously undescribed high-throughput method. Finally, we show that even clonally related tumors derived from the same immortalized line have acquired distinct combinations of genetic alterations during tumor development, suggesting that tumor formation in this model system involves competition among genetically variant cells, which is similar to the Darwinian evolutionary processes now thought to generate many human cancers. This mutagenesis strategy is faster and simpler than conventional transposon screens and can potentially be applied to any tissue stem/progenitor cells that can be grown and differentiated in vitro.Glioblastoma multiforme (GBM) is the most common form of malignant brain cancer in adults. Patients with GBM have a uniformly poor prognosis, with a mean survival of 1 y (1). Thus, advances on all fronts, both basic and applied, are needed to combat this deadly disease better. Recent studies have provided evidence for self-renewing, stem-like cells within human gliomas (2). These glioma-initiating cells constitute a small minority of neoplastic cells within a tumor and are defined operationally by their ability to seed new tumors (3). To target these rare glioma-initiating cells, a better understanding of the molecular mechanisms that regulate their formation is essential.Considerable progress has been made in understanding the mutations responsible for GBM. The Cancer Genome Atlas network has cataloged the recurrent genomic abnormalities in GBM by genome-wide DNA copy number events and sequence-based mutation detection for 601 genes (4). Gene expression-based molecular classification has also defined four subtypes of GBM termed proneural, neural, classical, and mesenchymal (5). Proneural GBM is enriched for the oligodendrocyte gene signature, whereas the classical group is associated with the astrocytic signature. The neural class is enriched for genes differentially expressed by neurons, whereas the mesenchymal class is associated with the cultured astroglial signature (5). Several recurrent mutations, such as PDGFRA, IDH1, EGFR, and NF1, also correlate with these GBM subtypes, providing additional support for their existence. Numerous other, often rare, mutations have also been identified in GBM. Although these datasets are valuable for understanding the molecular pathogenesis of GBM, it is still difficult to distinguish between mutations that contributed to tumor initiation and those acquired later during tumor progression.The cell of origin (COO) of GBM is still controversial. Neural stem cells (NSCs) are good candidates because the adult brain has very few proliferating cells capable of accumulating the numerous mutations required for gliomagenesis. NSCs are also more susceptible to malignant transformation than differentiated cells in the adult brain (6, 7). However, the genetic pathways that can transform NSCs into glioma-initiating cells still remain elusive. Transposon-based mutagenesis provides an unbiased, high-throughput method for identifying genes important for GBM (8). Here, we describe a unique two-step insertional mutagenesis strategy that makes it possible to identify genes and signaling pathways that are able to transform a NSC into a cancer-initiating cell for the mesenchymal subtype of GBM. In this two-step approach, NSCs are first mutagenized in vitro and the mutagenized cells are then transplanted into immunocompromised mice for subsequent tumor development following additional rounds of transposon-based mutagenesis. This makes it possible to discriminate between the genetic changes that occur early in tumor initiation and those required for tumor progression. In addition to identifying several previously undescribed GBM candidate cancer genes, our studies suggest that transposon-induced tumors mimic the evolutionary processes now thought to generate many human cancers, in which tumors have a branched cellular and genetic architecture reminiscent of Darwin’s iconic evolutionary tree.