Leukemia (2008) 22, 2029–2040; doi:10.1038/leu.2008.206; published online 7 August 2008

Investigating human leukemogenesis: from cell lines to in vivo models of human leukemia

J A Kennedy1,2 and F Barabé3,4,5

  1. 1Division of Cell and Molecular Biology, University Health Network, Toronto, Ontario, Canada
  2. 2Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada
  3. 3Department of Medicine, Laval University, Québec, Canada
  4. 4Department of Hematology, Enfant-Jésus Hospital, Québec, Canada
  5. 5Research Center in Infectious Diseases, CHUQ-CHUL, Laval University, Québec, Canada

Correspondence: Dr F Barabé, Research Center in Infectious Diseases, CHUQ-CHUL, Laval University, 2705 boul Laurier, RC-709, Québec, Canada G1V 4G2. E-mail:

Received 20 April 2008; Revised 16 June 2008; Accepted 4 July 2008; Published online 7 August 2008.



The hematopoietic system produces appropriate levels of blood cells over an individual's lifetime through a careful balance of differentiation, proliferation and self-renewal. The acquisition of genetic and epigenetic alterations leads to deregulation of these processes and the development of acute leukemias. A prerequisite to targeted therapies directed against these malignancies is a thorough understanding of the processes that subvert the normal developmental program of the hematopoietic system. This involves identifying the molecular lesions responsible for malignant transformation, their mechanisms of action and the cell type(s) in which they occur. Over the last 3 decades, significant progress has been made through the identification of recurrent genetic alterations and translocations in leukemic blast populations, and their subsequent functional characterization in cell lines and/or mouse models. Recently, primary human hematopoietic cells have emerged as a complementary means to characterize leukemic oncogenes. This approach enables the process of leukemogenesis to be precisely modeled in the appropriate cellular context: from primary human hematopoietic cells to leukemic stem cells capable of initiating disease in vivo. Here we review the model systems used to study leukemogenesis, and focus particularly on recent advances provided by in vitro and in vivo studies with primary human hematopoietic cells.


acute leukemia, in vivo models, NOD/SCID, cell lines, oncogenes



Malignant transformation involves the acquisition of a series of genetic and epigenetic changes that subvert normal cellular developmental programs, resulting in the generation of a neoplastic clone with deregulated growth properties.1 A clear understanding of this process requires characterization of both the molecular events underlying malignant growth as well as the cellular context in which these genetic hits occur. In the context of acute leukemias, it is generally believed that this process involves the acquisition of a series of alterations, which ultimately convert a normal hematopoietic cell, likely a hematopoietic stem cell (HSC) or another multipotent hematopoietic progenitor, into a leukemic stem cell (LSC) capable of propagating the disease clone. The resulting leukemogenic program is characterized by a differentiation arrest, increased proliferation, enhanced self-renewal, decreased apoptosis and telomere maintenance.2 Together these alterations result in the generation of a highly proliferative clone of immature leukemic blast cells with an intrinsic survival advantage and limitless replicative potential.

Human acute leukemias are heterogeneous at the molecular level, as each is driven by some combination of genetic and epigenetic alterations. Insight into the changes that underlie leukemic transformation has been attained through the identification and characterization of chromosomal translocations and other genetic/epigenetic alterations in the blast cells of patients with acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). The translocations observed in leukemias are balanced and are found stably within the entire malignant clone, suggesting that these lesions do not arise late during leukemogenesis, but instead are early hits having a causal role in disease pathogenesis.3 In support of this notion, it has been found that specific chromosomal rearrangements are associated with biologically distinct subtypes of leukemia. For example, t(15;17)(q22;q12) generates a PML–RARalpha fusion gene, which is exclusively associated with acute promyelocytic leukemia (APL, AML–M3),4, 5 whereas inv(16)(p13q22) generates a CBFbeta–SMMHC fusion found in acute myelomonocytic leukemia with abnormal eosinophils (AML–M4Eo).6 Similarly, translocations involving the mixed-lineage leukemia gene (MLL) on chromosome 11q23 are associated with poor prognosis B-ALL and AML with a unique gene-expression profile, characterized by the overexpression of homeobox genes.7 Careful genetic analysis has led to the identification of 264 different gene fusions in hematological malignancies,8 as well as other recurrent lesions, including mutations in the nucleocytoplasmic shuttling protein nucleophosmin (NPM1) in approx25% of AMLs,9 loss of function mutations/deletions in the key B-lineage transcription factor PAX5 in approx30% of B-ALL cases10 and activating mutations in the FLT3 receptor tyrosine kinase in both AMLs and ALLs.11, 12 This extensive molecular characterization has rendered the development of specific targeted therapies a definite possibility, as shown with tyrosine kinase inhibitors in chronic myeloid leukemia. However, as a prerequisite to the development of such therapeutics, the exact roles played by these oncogenes in leukemic initiation and progression must be determined through careful functional analysis. Here we review the model systems most commonly utilized for the study of leukemogenesis—cell lines, mouse models and primary human hematopoietic cells—and focus particularly on recent progress using xenotransplant approaches.

Cell lines

Cell lines are monoclonal populations of immortalized cells that are able to continually proliferate in culture without undergoing senescence. These continuous cell lines are particularly useful in the research setting as they provide a virtually unlimited supply of homogeneous cell material for genetic and biochemical studies. Rodent cell lines are relatively simple to generate, as even normal mouse tissues readily undergo spontaneous transformation in vitro. On the other hand, human cell lines are much more difficult to derive, even from cancerous tissues.13 Despite this challenge, greater than 1000 human hematopoietic cell lines have been generated since the 1960s by culturing cells from the bone marrow (BM), peripheral blood (PB) or pleural effusions of ALL, AML or lymphoma patients.14, 15 Although normal hematopoietic cells can only survive in vitro for days to weeks, these cell lines are able to proliferate continuously in culture while preserving the majority of their characteristic genetic alterations and phenotypic features.

Over the last 4 decades, human hematopoietic cell lines have been extensively used in leukemia research, fueling progress in a number of key areas (reviewed in Drexler et al.15 and Koeffler and Golde16). First, they have proven particularly useful in the identification of leukemia-associated oncogenes. The breakpoints of many chromosomal translocations were determined by utilizing cell lines carrying the particular cytogenetic abnormality as source material.17 The recent finding that approx50% of T-ALL patients harbor activating mutations in NOTCH1 came about through cell line studies; this gene became a prime candidate for sequencing after several T-ALL cell lines were found to be sensitive to gamma-secretase inhibitors, which inhibit the processing of NOTCH receptors.18 Second, leukemia cell lines have been extensively used in the development and testing of therapeutics, ranging from conventional chemotherapeutics to modern targeted agents such as kinase inhibitors and monoclonal antibodies. Some examples of these advances include the finding that L-asparaginase could inhibit the in vitro growth of human B-ALL cell lines,19 and the discovery of all-trans retinoic acid as a differentiation therapy for APL using the promyelocytic HL60 cell line.20 More recently, cell lines have provided important preclinical validation of targeted molecular therapies such as imatinib that was found to inhibit the growth of cell lines expressing BCR–ABL,21 and H90, an anti-CD44 antibody, which was found to induce the differentiation of a variety of human AML cell lines.22

Since the 1960s, the extensive work of a number of groups has led to the establishment of cell lines carrying many of the key translocations and oncogenes implicated in human leukemogenesis; for example, over 30 cell lines bearing rearrangements of MLL have been described.23 These cell lines have proven to be particularly useful as 'model systems' to gain insight into the biology of the oncogene in question. In just one of many examples of this approach, studies using the Kasumi-1 cell line, which harbors an AML1–ETO translocation, have shown that the differentiation block induced by AML1–ETO is due in part to its ability to physically bind to and inactivate the master myeloid transcription factor PU.1.24 When leukemic cell lines bearing an oncogene of interest are not readily available, an alternate approach is to introduce this gene into a cell line, then to characterize its effects on proliferation, differentiation and intracellular signaling in this setting. To give one example of this application, HOX11, an orphan homeobox gene often overexpressed in T-ALL, was found to disrupt the G2/M cell-cycle checkpoint when exogenously expressed in irradiated Jurkat T-lymphoblastic cells, providing a novel link between this oncogene and the promotion of genomic instability.25 Cell lines derived from sources other than human hematopoietic malignancies have also been used to study the functional consequences of oncogene expression. For example, Ba/F3 cells are an interleukin-3 (IL-3)-dependent pro-B cell line derived from mouse PB cells.26 Following the demonstration that BCR–ABL can confer factor independence to these cells, they have been extensively used to examine the potency and downstream signaling of this and other activated tyrosine kinases.27, 28 Leukemic oncogenes are also occasionally studied in cell lines derived from nonhematopoietic sources; however, given this inappropriate cellular context, it is crucial that any result be confirmed in the hematopoietic system. For example, the MLL–AF4 oncogene was found to downregulate the cyclin-dependent kinase inhibitor p27kip1 in epithelial 293 cells, but had the opposite effect in primary BM progenitors and in Jurkat cells.29

Despite the practical ease of utilizing cell lines to examine the functional effects of leukemic oncogenes, there are some drawbacks to this approach, highlighted to some extent by the aforementioned study. Ultimately, to accurately assess the contribution of particular oncogenes to the leukemogenic process, they must be studied in an appropriate cellular context. Though leukemic cell lines are often similar to the malignant blast population from which they were derived, the process of becoming immortalized and adapted for continual growth in vitro likely involves the acquisition of a number of genetic/epigenetic changes.30 Also, during the course of extended culture in vitro, further differences (phenotypic and/or genetic) could develop.15, 31 Together, these points emphasize that cell lines should not be considered as absolute equivalents of their corresponding primary cancers. The situation is even further complicated when human leukemic cell lines are used as a setting to characterize exogenously introduced oncogenes. In addition to immortalization-associated changes, these cell lines have a preexisting leukemogenic program due to their derivation from malignant tissue; thus, any data generated in this system is colored by the possibility that the genetic and epigenetic alterations arising from either of these processes may influence the manner in which cell lines respond to the oncogene under study. For example, expression of MLL–AF9 promoted differentiation and cell-cycle arrest in the human U937 monoblastic cell line, and induced the downregulation of Hoxa7 in the murine myeloid 32Dcl3 cell line—the opposite to what has been observed in human leukemias, mouse models and in primary human hematopoietic cells.7, 32, 33, 34, 35 Also, as cell lines are already fully transformed and immortalized, they cannot be utilized to study the early events of leukemogenic initiation and progression that lead to the generation of leukemia stem cells. Together, these limitations underscore the need to study leukemia-associated oncogenes in a more relevant cellular context, namely primary hematopoietic stem and progenitor cell populations.

In vivo models of murine leukemia

Cell line studies and the analysis of primary leukemia patient samples have provided significant advances in our understanding of leukemogenesis, particularly through the retrospective identification of causative genetic lesions. However, to gain insight into the early events in leukemic transformation, there is a need for prospective in vivo models. In this respect, the mouse has proven to be an exceptionally useful model system. Mice share gross physiological, anatomical and genomic similarities with humans and are relatively easy to genetically manipulate, enabling the experimental modeling of hematopoietic malignancies which, for obvious ethical reasons, is not possible in human subjects.36 Consequently, mouse models have been utilized extensively for the functional characterization of oncogenes and the prospective modeling of the sequence of hits required for full leukemic transformation. The main approaches used for these studies, the development of transgenic mice and retroviral transduction/transplantation, are discussed in turn.

The earliest transgenic mice were developed by the direct injection of exogenous DNA cassettes encoding a gene of interest and a heterologous promoter into the pronuclei of fertilized zygotes. Depending on the characteristics of the chosen promoter, expression could be limited to the hematopoietic system or even restricted to particular stages of differentiation. This approach established the leukemogenic potential of many oncogenes, including p190 BCR–ABL, c-Myc and PML–RARalpha37, 38, 39 However, a significant limitation of this methodology is that the level of expression of the transgene is heavily influenced by copy number and the random genomic integration site.36 The emergence of embryonic stem cell technology has led to the development of a second generation of transgenic mice by a 'knock-in' approach. Homologous recombination allows for activated oncogenes to be introduced into their native genomic loci and therefore remain subject to regulation by their endogenous promoters, circumventing the main disadvantages of conventional transgenics. However, as these oncogenes are present in the germline and can be expressed during development, they can, as is the case with AML1–ETO, lead to embryonic lethal phenotypes.40 This issue has been circumvented by incorporating loxP-stop cassettes into the transgenes under study and using Cre recombinase to tightly regulate the expression of transcripts that can be fully translated.41, 42 In a further refinement of this approach, 'invertor' mice have been developed, where a floxed, inverted cDNA encoding the C-terminal fusion partner is knocked into an intron within the N-terminal partner gene. Following expression of Cre, this cDNA cassette is flipped into its appropriate transcriptional orientation, enabling expression of the fusion gene transcript. This approach avoids the low levels of readthrough transcription that can occur in loxP-stop models and lead to dominant lethal effects.43 To date, the most elegant means of generating transgenic mice bearing chromosomal translocations has been developed by Rabbitts and colleagues.117 In 'translocator' mice, chromosomal translocations are generated de novo in a conditional manner by Cre-mediated interchromosomal recombination between loxP sites engineered at the breakpoint regions of both partner genes. Appropriately regulated Cre expression allows for the translocation to be generated in different cell types within the hematopoietic system, allowing one to test the effects of oncogenes in different cellular contexts.44 This approach showed that when Mll–Af9 and Mll–Enl translocations were generated in HSCs by Lmo2-regulated Cre expression, AML developed in 100% of mice by 4 months of age.44, 45 In contrast, when Mll–Af9 translocations were generated in the T-cell compartment by Lck–Cre, no disease was noted, indicating that this fusion requires a permissive environment for leukemogenicity. On the other hand, Mll–Enl was oncogenic when expressed in T-cell progenitors, as it resulted in the development of T-cell lymphomas. Interestingly, many of these mice did not succumb to lymphoid disease, but instead developed myeloid leukemias, indicating that Mll–Enl expression in T-cell progenitors can induce lineage reassignment.45 Together, this work elegantly demonstrated that both the properties of a given oncogene and its cellular context are central to the process of leukemogenesis and can specify the resulting leukemia lineage.

An alternate approach for functionally characterizing leukemic oncogenes is to isolate murine BM progenitors, transduce these cells ex vivo with an oncogene-carrying retrovirus, then characterize the resultant effects on proliferation, differentiation and self-renewal. One particularly useful in vitro approach involves the serial replating of methylcellulose colonies, which enables identification of oncogenes that can enhance the self-renewal of clonogenic progenitors, as first demonstrated for MLL–ENL.46 The BM progenitors can also be transplanted back into syngeneic recipients following transduction, enabling an assessment of their leukemogenic potential in vivo. This approach has been successfully used to develop in vivo models of hematopoietic malignancies with a wide variety of oncogenes, including BCR–ABL47, 48 and MLL fusions.46 Compared to transgenic mouse models, these BM transduction/transplantation (BMT) studies are less laborious, circumvent the issue of embryonic lethality, and enable assessment of disease clonality through analysis of proviral integration sites.49 Cooperation between oncogenes in leukemogenesis can also be assessed in these models by using separate or compound retroviruses to introduce multiple genetic lesions, as has been accomplished for Hoxa9 and Meis1 overexpression in the development of AML.50 In addition, the starting cell population in BMT studies can be varied to provide mechanistic insights into leukemogenesis. For example, BM from knockout mice can be used to determine the requirement of downstream signaling pathways in leukemogenesis, as has recently been confirmed for STAT5a in BCR-ABL-mediated disease.51 Also, by using sorted populations of hematopoietic stem/progenitor cells as starting material in these experiments, the susceptibility of different target populations to leukemic transformation can be determined. Using this approach it was found that HSCs, common myeloid progenitors and granulocyte-macrophage progenitors, but not common lymphoid progenitors, megakaryocyte-erythroid progenitors or c-kitneg (progenitor-depleted) cells could be transformed by MLL–ENL and result in the development of AMLs with the same latency and phenotype.52 These studies indicated that MLL–ENL could target and transform both stem cell and committed progenitor cell populations; however, fewer injected cells were required for leukemia to be initiated with HSCs compared to the other fractions, suggesting that stem cells may be more efficiently transformed than committed cells. Using a similar approach, the ability of BCR–ABL, the hallmark translocation in CML, and MOZ–TIF, an AML-associated fusion gene, to transform different mouse cell populations has been examined. Interestingly, myeloid progenitors could be transformed by MOZ–TIF2, but not BCR–ABL, resulting in immortalized cell lines in vitro and AML upon transplantation into mice.53 Biologically, BCR–ABL differs from MLL–ENL and MOZ–TIF2 in that it cannot increase the serial replating capacity of clonogenic myeloid progenitors; thus, it appears that the ability of an oncogene to endow committed progenitors with self-renewal capacity is a critical parameter in determining whether it can target these cells for transformation.54

Together, transgenic mice and murine BMT studies have been essential in the functional characterization of leukemic oncogenes and have established countless in vivo models of hematopoietic malignancies that have provided both mechanistic insights into leukemogenesis and a setting to test novel therapeutics (reviewed in Bernardi et al.36). However, the biology of mouse and human cells is not identical, particularly in terms of tumorigenesis. The spectrum of spontaneous neoplasms that develop in these two species differ significantly; laboratory mice tend to develop cancers of the mesenchymal tissues, such as lymphomas and sarcomas, whereas humans tend to develop epithelial carcinomas.55 Moreover, molecular studies have shown that the mechanism of neoplastic transformation differs between these species. Two cooperating oncogenes, which perturb the p53 and Ras pathways, can readily induce the transformation of mouse embryonic fibroblasts,56, 57 but the same combination fails to transform human cells. Instead, the perturbation of six different pathways (p53, RB, PP2A, telomerase and the downstream RAS effectors RAF and the RAL guanine exchange factors) is required.58 Of these pathways, the differences in telomere biology between mouse and human cells are the best documented. Mouse telomeres are substantially longer than those of humans (40–60 vs 10 kb) and telomerase, the enzyme responsible for maintaining and extending telomeric ends, is active in the majority of somatic cells in mice, but not in humans.59, 60 It is largely due to these differences that murine cells readily undergo spontaneous immortalization in vitro, whereas this is an extremely low frequency event with human cells as they must first actively acquire a means to prevent telomere shortening and bypass senescence.13, 61 Also, more directly related to leukemogenesis, the phenotypes generated in some transgenic mouse models and BMT studies do not match those observed in human patients with the same genetic lesion. For example, the MLL–ENL fusion gene is frequently associated with B-lymphoid disease in humans, but strictly generates AML in BMT studies and in Lmo2-Cre translocator mice.44, 46 Furthermore, it has been shown that the same oncogene can have disparate effects on mouse and human hematopoietic progenitor cells. A mutant isoform of C/EBPalpha associated with human AML blocked myeloid differentiation in CD34+ human cells, whereas its expression in mouse cells induced a decrease in clonogenicity, but no differentiation block.62

Together, these observations highlight that species-specific differences do exist in the mechanisms of malignant transformation; therefore, caution should be exercised when extrapolating results from mouse models to the human situation.61, 63 They also underscore the need for experimental models where primary human cells are used as a substrate for transformation, to gain an appreciation of the elements of the human leukemogenic process, from the functional contributions of individual oncogenes, to the sequence of molecular lesions required for the generation of LSCs.

Primary human hematopoietic cells: in vitro studies

As detailed in the preceding sections, studies in mice and in cell lines have provided invaluable insights into leukemogenesis, but a limitation of both of these model systems is that oncogenes are not assessed in the appropriate cellular context—that of primary human hematopoietic stem and progenitor cells. To address this issue, experimental protocols have been developed where genetic 'hits' are induced in normal human primitive hematopoietic cells by retroviral-mediated oncogene overexpression, allowing for the early steps of leukemogenesis to be effectively modeled (Figure 1). In terms of primary source material, human hematopoietic cells are available in the form of adult BM, mobilized PB or umbilical cord blood (CB). The last is used more frequently, as it is readily available and is naturally enriched for primitive HSCs and progenitor cells.64 To focus on the relevant cellular context for leukemogenesis, further enrichment for these primitive populations is performed before transduction, by either positive selection for CD34+ cells or lineage depletion to remove cells expressing mature lineage markers. In terms of gene delivery, extensive work, largely in parallel with the field of gene therapy, has gone into the development of replication-deficient viral particles, which can infect human cells, integrate stably into the host cell genome, and efficiently direct expression of both a gene of interest and a selectable marker. The murine stem cell retroviral vectors generated by Hawley et al.,65 which utilize the strong promoter of the viral 5'-untranslated region to drive expression of a gene of interest, were among the first vectors to be successful in this regard and remain widely used today.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact or the author

Schematic of the various in vivo and in vitro experimental protocols that can be employed when leukemic oncogenes are studied in the context of primary human hematopoietic cells.

Full figure and legend (163K)

The first studies using retroviruses to introduce leukemic oncogenes into primitive human hematopoietic progenitors were reported approximately 10 years ago, and utilized in vitro assays to gauge the resultant effects upon proliferation, differentiation and self-renewal. Proof-of-principle was provided by a series of experiments where N-RAS was successfully expressed in CD34+ CB cells and blocked erythroid differentiation in suspension cultures.66 This approach has since been utilized for the functional characterization of a large series of oncogenes (summarized in Table 1). In the majority of these studies, phenotypes highly reminiscent of those observed in patients that bear these oncogenes have been noted. For example, expression of the APL-associated oncogene PML–RARalpha resulted in an accumulation of promyelocytes in vitro.70 Similarly, CBFbeta–SMMHC that is specifically associated with AML–M4Eo, a subtype of leukemia associated with myelomonocytic blasts and accompanying eosinophilia, generated a combination of these cell types in long-term cultures.78 Interestingly, though CBFbeta–SMMHC had previously been studied using murine BMT and transgenic approaches,83, 84 this was the first model where the eosinophilia characteristic of this translocation was recapitulated, again highlighting the importance of studying oncogenes in primary human hematopoietic cells. However, studies in this setting have occasionally yielded unexpected results, as in the case with activated tyrosine kinases. Expression of TEL–JAK2, BCR–ABL or FLT3–ITD all result in extensive erythropoietin-independent erythropoiesis in vitro, a finding that had not been noted in previous work with mouse models or patient samples.79, 72, 76 Together, these reports suggest that activated tyrosine kinases, when expressed at high levels in human hematopoietic progenitor cells by viral vectors, drive EPO-independent erythropoiesis at the expense of myelopoiesis, an effect which appears to be mediated at least in part by the constitutive activation of STAT5 and the downregulation of C/EBPalpha.85

Xenotransplantation assays for studying human leukemia

For many years, a fundamental limitation of utilizing primary human hematopoietic cells in the study of leukemogenesis was an inability to extend this approach beyond in vitro studies and into an in vivo setting, where the initiation and progression of human hematopoietic malignancies can be fully modeled. However, this approach was rendered feasible following the development of xenotransplantation models, which use immunodeficient mice as a setting to study normal and malignant human hematopoiesis. In a series of pioneering studies beginning in the late 1980s Dick and colleagues injected human hematopoietic cells intravenously (i.v.) into sublethally irradiated immunodeficient mice, an approach modeled after clinical transplantation protocols. In their initial studies, either bg/nu/xid or severe-combined immunodeficient (SCID) mice were transplanted i.v. with human BM and developed human macrophage colony-forming units in their BM 5 weeks later, indicating that primitive human hematopoietic cells could successfully engraft, proliferate and undergo myeloid differentiation in the murine BM microenvironment.86 In follow-up studies where mice were either treated with a wider range of human cytokines or transplanted with CB mononuclear cells, more robust human engraftment was achieved and primitive cells as well as differentiated myeloid and B-lymphoid cells were detected in the recipient BM.87, 88 The cells capable of initiating the human grafts in this assay were operationally defined as SCID-repopulating cells (SRCs). Purification studies have shown that SRCs are enriched in the CD34+CD38- fraction of lineage-depleted CB (Lin-CB) and BM, but are not present in the more prevalent CD34+CD38hi population.89 Since these early studies, significant improvements have been made to these models in an effort to increase the achievable levels of human chimerism in transplanted mice. The development of mouse strains with further defects in their innate and adaptive immune systems, such as NOD/LtSz-scid/scid (NOD/SCID)90, 91 and others (see later), have allowed for high-level, multilineage human cell engraftment to be reproducibly achieved. Aside from immunological barriers, the successful engraftment of HSCs injected by the standard i.v. route requires efficient homing to the BM, a complex process involving circulation through blood, recognition and extravasation through the marrow vasculature and migration to a supportive niche. Recently, protocols have been developed where human hematopoietic cells are directly delivered into BM cavities, bypassing the obstacles associated with homing from the bloodstream and have resulted in more sensitive detection of repopulating cells.92, 93, 94 Another highly useful approach is intrahepatic transplantation into newborn immunodeficient mice. The liver of neonates is a site that is naturally primed for extensive hematopoietic expansion and consequently allows for the efficient engraftment and differentiation of human HSCs.95 Using this transplantation approach in BALB/c Rag2-/-Il2rg-/- mice, a highly immunodeficient strain, which lacks T, B and natural killer (NK) cells, Manz and colleagues successfully generated mice engrafted with a functional immune system consisting of human B, T and dendritic cells capable of acting in concert to mount adaptive immune responses.95

Since their development, the aforementioned xenotransplantation assays have been adapted for the study of human hematopoietic malignancies. Following injection into immunodeficient mice, PB and BM cells from primary human ALLs and AMLs are capable of growing in vivo and generating grafts which phenotypically resemble the parent leukemia.96, 97 Interestingly, it has been found that poor prognosis leukemias tend to robustly engraft immunodeficient mice, suggesting that a correlation exists between xeno-engraftment potential and disease aggressiveness in humans.98, 99, 100 This model has also been used preclinically as a means to evaluate the efficacy of candidate therapeutics. For example, a monoclonal antibody directed against CD44, a cell-surface marker highly expressed on myeloid blasts, has been shown to reduce leukemic engraftment in vivo by interfering with homing to the BM.101, 102 Another potential target for treatment, the SDF-1/CXCR4 axis, has also been validated in this model, as administration of neutralizing anti-CXCR4 antibodies to NOD/SCID mice with established AML grafts induced a significant decrease in the level of leukemic blasts in vivo.103

Xenotransplantation assays have also been extensively used to characterize the cell types responsible for initiating leukemic growth in vivo. The injection of limiting numbers of leukemic blasts into recipient mice has shown that only 1 in 104–107 bulk blasts in AML samples have the capacity to initiate human disease in vivo.97, 104, 105 These cells, operationally defined as SCID-leukemia initiating cells (SL-ICs), were subsequently found to reside only in the CD34+CD38- fraction of AML, providing evidence for the existence of a phenotypically distinct population of LSCs, responsible for initiating and propagating disease in vivo.97, 104 Building on these early studies, recent work by Ishikawa and colleagues has shown that the CD34+CD38- AML LSCs are quiescent, and reside in the osteoblast-rich endosteal region of the bones of repopulated mice where they are protected from chemotherapy-induced apoptosis.106 Similar experimental approaches have also been utilized to identify LSC populations in ALLs. Transplantation of subfractions of pediatric T-ALL has shown that only the primitive CD34+CD7- or CD34+CD4- populations and not the more differentiated CD7+ or CD4+ fractions are capable of NOD/SCID engraftment.107 However, a different pattern has been noted in primary B-ALL samples. NOD/SCID transplantation studies using TEL-AML1 and BCR-ABL-positive B-ALLs demonstrated that leukemic grafts were exclusively generated by the CD19+ population, whereas CD19- cells could only form normal hematopoietic grafts, presumably due to the presence of normal HSCs within this fraction.108 Consistent with this, a recent report has shown that CD34+CD38+CD19+ and CD34+CD38-CD19+ cells but not the CD34+CD38-CD19-CD10- cells in primary B-ALL samples could generate disease upon transplantation into immunodeficient mice.109 Together, these studies have clearly established that phenotypic differences exist between LSCs in AML and B-ALL.

It is important to note that the growth of human leukemic cells in xenotransplant recipients is undoubtedly limited by residual elements of the recipient immune system, the absence of cross-species reactivity of some cytokines, and differences between the murine and human microenvironment. Together, these factors may limit the ability of some human leukemia samples to successfully engraft, and also lead to an underestimation of the absolute frequency of LSC.110, 111 Thus, a reassessment of leukemic engraftment potential and LSC frequencies using updated xenotransplantation protocols which minimize these barriers (by intrafemoral transplantation, human cytokine administration and the usage of recipients with further immunodeficiencies) is warranted.

In vivo models of human leukemogenesis

To develop an accurate experimental model of human leukemogenesis — the process whereby a primary human hematopoietic cell is converted by a series of genetic/epigenetic hits into a LSC—there are two absolute prerequisites. First, as stressed in the preceding sections, leukemic oncogenes must be studied in the appropriate cellular context, namely primary human hematopoietic cells. Second, these genetically modified cells must be assayed for their ability to initiate disease in vivo, a requirement which has been made possible through the development of xenotransplantation assays. However, the successful realization of this experimental approach has proven to be technically challenging. The first, albeit limited, successes were achieved by transplanting the blast cell populations generated during in vitro propagation of transduced human hematopoietic cells. Using this approach, AML1–ETO, TLS–ERG and CBFbeta–SMMHC–expressing cells generated low level human grafts (maximum 10% of the mouse BM), that did not progress to a full leukemia.68, 74, 78

An alternate method for developing in vivo models of human leukemogenesis, akin to the mouse BMT approach, involves transplantation of transduced cells immediately following the infection period. Due to the inherent difficulty in transducing and expressing exogenous genes in SRCs,91 the initial experiments along these cell lines were unsuccessful.69 However, taking advantage of recent technological advances in retroviral gene transfer protocols and immunodeficient mouse models, a number of recent reports have successfully utilized this technology to directly assay the effects of oncogenes on primary human cells in vivo (Table 2). However, in these studies, the resulting growth disruptions have generally been limited to shifts in the lineage distribution of transduced cells and did not result in overt hematological disease. For example, the expression of AML1–ETO did not affect the multilineage differentiation of human cells in NOD/SCID mice but was associated with an increased proportion of CD34+ cells.75 In another study, the expression of HOXA10 promoted human myelopoiesis while significantly reducing B lymphopoiesis in repopulated mice.71 Similarly, p210 BCR–ABL transduced human CB cells assayed in immunodeficient mice produced an altered spectrum of progeny, with an increased ratio of myeloid to B-lymphoid cells and an increase in erythroid and megakaryocytic cells. Of note, after 5–6 months, a minor proportion of transplanted mice (4 of 44) developed signs of myeloproliferative disease, namely an increased white blood cell count and/or splenomegaly.112

While these studies provided proof-of-principle that xenotransplantion approaches could be utilized to study the effects of oncogenes in an in vivo setting, they were limited in that they did not generate reproducible human hematopoietic malignancies. The full potential of this model system has been realized only recently, by combining an optimized transduction/transplantation protocol with the expression of potent human oncogenes. Activation of the JAK2 kinase by chromosomal translocation or point mutation is a recurrent event in hematopoietic malignancies, including acute leukemias and myeloproliferative disorders.116 When TEL–JAK2, a constitutively active variant of the JAK2 kinase, was expressed in Lin-CB cells by a lentiviral vector and transduced cells were injected intrafemorally into NOD/SCID mice depleted of NK cells by an antibody directed against CD122,94 recipients developed myelofibrosis by 9 weeks post transplant. This disease was characterized by marrow hypocellularity, anemia and BM changes including extensive reticulin deposition and an accumulation of atypical murine megakaryocytes, illustrating that activated JAK2 signaling in the context of primitive human hematopoietic cells is sufficient to drive key processes implicated in the pathophysiology of idiopathic myelofibrosis.79

Using a similar approach, MLL fusion genes have been shown to be capable of initiating human leukemogenesis in vivo. These translocations are found in aggressive cases of AML and ALL, and are particularly prevalent in infant leukemias, with a concordance rate near 100% in monozygotic twins with a shared placenta, significantly higher than that noted with other leukemia-associated translocations, emphasizing their potency.117, 118 When Lin-CB cells were infected with a retrovirus expressing either MLL–AF9 or MLL–ENL and transplanted intrafemorally or i.v. into immunodeficient mice, recipients reproducibly developed aggressive acute leukemias. The majority of mice developed a B-ALL consisting of CD19+CD20-sIg- blast cells which frequently co-expressed myeloid surface antigens and were capable of infiltrating the spleen, liver, thymus, brain and testes, all features noted in patients bearing these translocations.35 Subsequently, this model has been used to gain insight into the biology of B-ALL LSCs, and important differences from their counterparts in AML have become apparent. Limiting dilution studies have shown that these cells are frequent, consisting approximately 0.1–0.5% of the bulk leukemic blast population and, consistent with work using primary patient samples (as discussed above by Castor et al.108 and Kong et al.109), can have a CD19+ B-lymphoid phenotype.35, 111 These findings contrast with those from studies of primary human AML, where LSC frequencies ranged between 1 in 104 and 1 in 107 and these cells were found to have an HSC-like CD34+CD38- phenotype.97, 104, 105 These studies have also shown that LSCs can undergo evolution during disease progression. Analysis of serially transplanted mice revealed that LSCs evolved over time from a primitive cell type with a germline immunoglobulin heavy chain (IgH) gene configuration to a cell type containing rearranged IgH genes.35 Moreover, CD19+ cells, which were depleted from the initial Lin-CB population, could initiate leukemic grafts when transplanted from primary leukemic mice into secondary recipients.111 Together, these results highlight that the analysis of LSC properties at single time points, such as those provided by collected clinical samples, may not provide a complete picture of LSC biology. Thus, it is essential to utilize a prospective setting to study human leukemogenesis—commencing with initiation of this program in normal human hematopoietic cells and following disease progression over time, through either secondary transplantation or periodic BM aspirations.119

Future directions

Since the initial development of xenotransplant assays for the study of human hematopoiesis, significant improvements have been made to these models to increase the achievable levels of human chimerism. However, even with these adaptations, limitations continue to exist. Of note, NOD/SCID mice and their related derivatives have a relatively short lifespan (approx37 weeks) due to the development of lethal thymic lymphomas.90 This precludes the utilization of these strains for long-term studies of both normal hematopoiesis and leukemogenesis. Though MLL fusion genes successfully generate leukemia in primary xenograft recipients with high penetrance,35 these oncogenes are unique with respect to their potency, as demonstrated by their frequent association with short latency infant disease118 and the finding that MLL leukemias have a limited number of copy number alterations compared to other B-ALLs.10 Thus, human cells transduced with other oncogenes will likely require a longer period of time to accumulate the series of additional genetic/epigenetic lesions required to generate overt disease. Although serial transplantation is one option that enables long-term studies in NOD/SCID mice, a more physiologically representative approach is now available, as a result of the development of new strains of immunodeficient mice (reviewed in Shultz et al.120). NOD/SCID mice with targeted mutations of the Il-2 receptor gamma chain (Il2rg) locus lack T, B and NK cells and can be followed for extended periods of time post transplantation, as, due to their defective Il2r signaling, they do not develop thymic lymphomas.120 Two separate strains of these mice have been developed: NODShi.Cg-PrkdcscidIl2rgtm1Sug (NOG) mice, which have a truncated Il2rg chain,121 and NOD.Cg-PrkdcscidIl2rgtm1Wj1 (NOD/SCID/Il2rgnull) mice, which completely lack Il2rg chain expression.122 Though these mice differ in terms of their Il2rg-targeted mutation and their inbred strain background, both are long lived and support robust human hematopoietic grafts, which include de novo T cells, as well as circulating platelets and erythocytes, all of which did not develop reproducibly in previous models.121, 122, 123, 124, 125 Thus, an additional advantage of these mice is the potential to model human hematopoietic malignancies involving a range of blood lineages, such as polycythemia vera (PV), essential thrombocythemia and T-ALL. Another recently developed, long-lived immunodeficient mouse strain that is able to support multilineage human hematopoeisis is BALB/c Rag2-/-Il2g-/- (described above). Their utility has been illustrated in a recent study, which showed that JAK2V617F, an oncogene associated with PV, promoted erythroid engraftment when expressed in human hematopoietic progenitor cells.115 Subsequently, it was shown that this growth could be inhibited by a selective JAK2 inhibitor, highlighting the utility of CB transduction-xenotransplantation models for the assessment of novel therapeutics.

Studies of normal human hematopoiesis in xenograft models have clearly established that sublethally irradiated immunodeficient mice are particularly supportive of human B lymphopoiesis; beyond 6 weeks post transplant, a high ratio of human B-lymphoid to myeloid cells is detected the marrow of repopulated mice, contrasting with normal adult BM.79, 119, 126 This bias toward B lymphopoiesis also appears to impact upon leukemogenesis. In vitroin vivo crossover studies with Lin-CB cells expressing MLL–ENL have shown that cells with the potential to initiate B-lymphoid and/or myeloid leukemogenic programs are clearly present immediately following transduction, but invariably generate B-ALL when directly transplanted into mice.35 Thus, insufficient human myeloid differentiation in xenograft models currently poses a significant barrier to the development of experimental models of AML in this setting. This deficiency likely arises in part from a lack of cross-reactivity of some mouse myeloid cytokines, such as IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF) and M-CSF, with human blood cells.125 Moreover, recipient mice have an intact myeloid compartment, thus, unlike lymphopoiesis, there is no inherent deficiency to be compensated, and the mouse myeloid cells likely outcompete their human counterparts following sublethal irradiation. In attempts to address this issue, transgenic NOD/SCID mice expressing human SCF, IL-3 and GM-CSF (NOD/SCID-S/3/GM) have been developed. These mice promoted terminal myelopoiesis with a slight reduction in B lymphopoiesis upon injection of normal human BM,127 and also enabled the in vivo growth of AML samples that failed to engraft standard NOD/SCID recipients.128 On the basis of these studies it appears that NOD/SCID-S/3/GM mice could provide a more supportive environment for the development of models of human myeloid malignancies, a notion supported by a recent report where MLL–AF9 was studied in these recipients and its expression in CB resulted in the reproducible generation of human AML.129 An alternate approach could involve development of an immunodeficient mouse strain where non-crossreactive mouse cytokines could be genetically deleted and replaced by their respective human counterparts that ideally would not stimulate mouse cells.125 Though it is currently unknown whether a strain with an inherent myeloid deficiency could be developed and be maintained viably, the possibility of a xenograft recipient with a more supportive, less competitive environment for human myelopoiesis remains intriguing. An alternate approach to render xenotransplant recipients more 'humanized' could involve the co-transplantation of human mesenchymal stem cells (hMSCs) and human hematopoietic cells into immunodeficient mice. Following intraosseous injection, hMSCs can contribute to the hematopoietic microenvironment within the mouse marrow, generating stromal cells, osteoblasts, endothelial cells and other BM cell types.130 Moreover, the co-transplantation of hMSCs with CB cells has been shown to increase human hematopoietic engraftment in xenograft recipients,130, 131, 132 and even shift the resulting lineage distribution from a B-lymphoid to a myeloid predominance.133 Though hMSCs have not been shown to enhance the engraftment of primary AML samples,134 this approach may nonetheless prove to be useful as a means to promote myeloid leukemogenesis in retroviral transduction-transplantation studies.



A detailed understanding of leukemogenesis requires the development of experimental models that can accurately model this process. Thus, as a complement to work in cell lines and in mice, there is a need for oncogenes and chromosomal translocations to be studied in the appropriate cellular context, that of primary human hematopoietic cells. Retroviral-mediated transduction of primary human hematopoietic cells followed by their transplantation in vivo has emerged as a feasible approach to study the process of human leukemogenesis. Continuation of this work will enable a detailed understanding of LSC biology, providing insights into their cellular origin and the molecular pathways that lead to their generation. These advancements have the potential to facilitate the development of rationally designed molecular therapeutics with the ultimate goal of improving treatment and decreasing the mortality associated with hematopoietic malignancies.



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This work was supported by the Canadian Institute of Health Research (CIHR) Clinician Scientist award (to FB), an MD/PhD studentship (to JAK) and grants from the CIHR and the Fonds de la recherche en santé du Québec (FRSQ). We thank Jean Wang, Sergei Doulatov and Faiyaz Notta for critical comments on the manuscript.