Differentiation Of Human Hematopoietic Cells Research Articles

Metabolic adaptation pilots the differentiation of human hematopoietic cells.

A continuous supply of energy is an essential prerequisite for survival and represents the highest priority for the cell. We hypothesize that cell differentiation is a process of optimization of energy flow in a changing environment through phenotypic adaptation. The mechanistic basis of this hypothesis is provided by the established link between core energy metabolism and epigenetic covalent modifications of chromatin. This theory predicts that early metabolic perturbations impact subsequent differentiation. To test this, we induced transient metabolic perturbations in undifferentiated human hematopoietic cells using pharmacological inhibitors targeting key metabolic reactions. We recorded changes in chromatin structure and gene expression, as well as phenotypic alterations by single-cell ATAC and RNA sequencing, time-lapse microscopy, and flow cytometry. Our observations suggest that these metabolic perturbations are shortly followed by alterations in chromatin structure, leading to changes in gene expression. We also show that these transient fluctuations alter the differentiation potential of the cells.

Novel Engraftment and T Cell Differentiation of Human Hematopoietic Cells in ART-/-IL2RG-/Y SCID Pigs.

Pigs with severe combined immunodeficiency (SCID) are an emerging biomedical animal model. Swine are anatomically and physiologically more similar to humans than mice, making them an invaluable tool for preclinical regenerative medicine and cancer research. One essential step in further developing this model is the immunological humanization of SCID pigs. In this work we have generated T− B− NK− SCID pigs through site directed CRISPR/Cas9 mutagenesis of IL2RG within a naturally occurring DCLRE1C (ARTEMIS)−/− genetic background. We confirmed ART−/− IL2RG−/Y pigs lacked T, B, and NK cells in both peripheral blood and lymphoid tissues. Additionally, we successfully performed a bone marrow transplant on one ART−/− IL2RG−/Y male SCID pig with bone marrow from a complete swine leukocyte antigen (SLA) matched donor without conditioning to reconstitute porcine T and NK cells. Next, we performed in utero injections of cultured human CD34+ selected cord blood cells into the fetal ART−/− IL2RG−/Y SCID pigs. At birth, human CD45+ CD3ε+ cells were detected in cord and peripheral blood of in utero injected SCID piglets. Human leukocytes were also detected within the bone marrow, spleen, liver, thymus, and mesenteric lymph nodes of these animals. Taken together, we describe critical steps forwards the development of an immunologically humanized SCID pig model.

NUP98-KDM5A Fusion Induces Hematopoietic Cell Proliferation and Alters Myelo-Erythropoietic Differentiation

Chromosomal translocations are common in acute myeloid leukemia (AML), causing gene fusions that encode oncogenic proteins. The NUP98 gene participates in chromosomal rearrangements with over 30 fusion partners and comprises 6-10% of de novo cases of pediatric AML. These fusions are associated with poor prognosis in children and adults. Among them, NUP98-KDM5A is further enriched in specific subpopulations, found in 15% of non-Down syndrome acute megakaryoblastic leukemia cases and 20% of pediatric acute erythroleukemia. Despite these associations, the direct impact of NUP98-KDM5A on the growth and differentiation of human hematopoietic cells has not been systemically studied. In this study, we transduced cord blood CD34+ hematopoietic stem and progenitor cells (HSPCs) with NUP98-KDM5A or control lentiviral vectors and examined cell proliferation and differentiation. Exposure to cytokines (SCF, TPO, IL-6, FLT3L, SR-1) selected for CD34+ cell growth, and expression of NUP98-KDM5A increased proliferation rate in liquid culture by 19.4-fold (p<0.01). In addition to a growth advantage, expression of NUP98-KDM5A led to differences in differentiation. NUP98-KDM5A expressing cells showed a 4.9-fold decrease (p<0.05) in myeloid terminal differentiation (defined as the CD33+, CD11B+ population) and 2.8-fold increase (p<0.05) in early stem cell progenitors (defined as the CD117+ population). Interestingly, NUP98-KDM5A expressing cells showed a 3.3-fold increase (p<0.01) in the erythroid population (defined by the CD235a+, CD71+ population). The increase in erythroid differentiation was confirmed by RNA sequencing, which showed an enrichment in genes involved in heme metabolism (Hallmark Heme Metabolism, FDR q-value 7.33x10-8) after only 72 hours post-transduction. Furthermore, RNA sequencing demonstrated upregulation of HOXA genes, including HOXA3, HOXA5, HOXA6, HOXA7, HOXA9, HOXA10 and HOXA11 (p 3.36x10-2 - 1.16x10-7), which has been previously described in other high-risk leukemic fusions (including other NUP98 fusions) as a primary mechanism of stem-cell renewal. To further elucidate NUP98-KDM5A effects on differentiation, cytokine supplements known to drive myeloid differentiation (SCF, TPO, G-CSF, and GM-CSF) were added to culture media, which demonstrated the same disruption in myelo-erythropoiesis based on a 4.0-fold decrease (p<0.001) in the terminal myeloid population, a 3.2-fold increase in CD117+ (p<0.001), and 2.6-fold increase in erythroid markers (p<0.001). Furthermore, NUP98-KDM5A recapitulated this phenotype in methylcellulose colony forming assays, with increased BFU-E and reduced CFU-GM colonies compared to control (p<0.01), despite no difference in overall colony number. Taken together, these in vitro studies demonstrate increased proliferation and altered differentiation in HSPCs by NUP98-KDM5A, with the pattern of differentiation by NUP98-KDM5A paralleling the M6 (erythroleukemia) AML subtype observed in patient samples harboring this fusion, supporting the clinical observation that NUP98-KDM5A is often a monogenic driver. To evaluate the leukemic potential of NUP98-KDM5A, cells were injected into NRG-SGM3 mice with NUP98-KDM5A inducing at 12 weeks a rapidly lethal myeloid disease in vivo (97.8% human CD45+ cells vs. 2.3% in control, p<0.001). Furthermore, spleens of the NUP98-KDM5A mice were markedly enlarged, weighing on average 926.7g vs. 47.3g spleens seen in control, a 19.8-fold increase in weight (p<0.05). In conclusion, the expression of NUP98-KDM5A in HSPCs drives proliferation and alters differentiation to maintain stem cell markers while driving an erythroid phenotype in vitro. Furthermore, this fusion is sufficient to engraft in the bone marrow and spleen of NRG-SGM3 mice in vivo and cause marked splenomegaly. Taken together, these data suggest this model properly recapitulates the human disease phenotype seen in patients expressing NUP98-KDM5A and future functional assays and drug screens may provide important insights into understanding the pathophysiology and pharmacologic vulnerabilities of this fusion class. Disclosures Mullighan: Loxo Oncology: Research Funding; Amgen: Honoraria, Other: speaker, sponsored travel; Illumina: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored travel; AbbVie: Research Funding; Pfizer: Honoraria, Other: speaker, sponsored travel, Research Funding.

SIX1 Transcription Factor Enhances Human Erythropoiesis Via GATA1

Erythropoiesis is orchestrated by the coordinated action of multiple transcription factors. The master erythropoietic regulator GATA1 is itself modulated via interactions with multiple co-regulatory factors, such as FOG1, KLF1, and LMO2. Though the PAX-SIX-EYA-DACH network (PSEDN) of conserved transcription factors has been well characterized in the formation of eyes, kidney, branchial structures, and skeletal muscles, a role for PSEDN members in hematopoietic systems has only recently been recognized (Liu et al., Nature 2019 PMID:30894749). Here, we studied the PSEDN member SIX1 and discovered its ability to drive erythroid differentiation of human hematopoietic cells. Enforced overexpression (OE) of SIX1 in human TF1 erythroleukemia cells or primary CD34+ hematopoietic stem-progenitor cells (HSPCs) stimulated the generation of erythroid cells, as determined by increased numbers of cells expressing erythroid-selective surface markers (CD235ahiCD71hiCD34-) and hemoglobin (HBB). Conversely, SIX1 knockout in TF1 cells or primary HSPCs reduced erythroid cell generation in response to erythropoietin (EPO). SIX1 OE could also stimulate TF1 cell erythroid differentiation in the absence of EPO. Further analysis of SIX1 OE in TF1 cells revealed that SIX1 stimulated the expression of multiple functionally important erythroid molecules including ALAS2, SLC4a1, EPOR, SPTA1, KLF1 and ANK1. By gene set enrichment analysis (GSEA) of global RNA-seq data, SIX1 OE stimulated heme metabolism genes as well as many genes known to be regulated by GATA1, including FOG1-dependent and -independent genes. SIX1 OE reduced GATA2 and increased GATA1 protein and RNA expression, resembling GATA switching downstream of EPO signaling. To determine whether GATA1 was necessary for SIX1 to stimulate erythropoiesis, we generated GATA1 knockout cells using CRISPR/Cas9 technology. In contrast to control cells, SIX1 OE in GATA1 knockout cells failed to stimulate erythropoiesis, indicating that SIX1 stimulation of erythropoiesis requires GATA1. To gain further insight into the mechanism by which SIX1 stimulates erythropoiesis, the promiscuous biotin ligase, BirA (Choi-Rhee et al., Protein Sci. 2004 PMID:15459338), was fused in-frame to SIX1 to determine the SIX1 proximal interactome. Streptavidin-enrichment of biotinylated proteins in SIX1-BirA OE lysates revealed GATA1 and FOG1 as proximal interactors of SIX1-BirA, but not of BirA alone. When co-expressed in HEK293T cells GATA1 and SIX1 were found to co-immunoprecipitate, suggesting the two proteins can physically interact in a complex. We demonstrated the functional consequence of the SIX1 interaction with GATA1 using a GATA1-dependent luciferase reporter gene harboring three copies of GATA binding sites. Cells in which SIX1 and GATA1 were co-expressed exhibited significantly higher levels of luciferase expression compared to cells expressing only GATA1, suggesting SIX1 could stimulate GATA1-dependent transcription. Introduction of mutations in SIX known to cause Branchio-Oto-Renal (BOR) (Ruf et al., PNAS 2004 PMID:15141091) syndrome did not inhibit the ability of SIX to bind GATA1 nor its ability to drive erythropoiesis. Taken together our results suggest that SIX1 can stimulate erythropoiesis via multiple mechanisms, including increased GATA1 expression and function. Our findings provide the first demonstration of a role for the PSEDN in erythropoiesis and reveal unknown physical and functional interactions between two central developmental transcriptional networks (GATA:FOG network and PSEDN). Disclosures No relevant conflicts of interest to declare.

GADD45b Plays an Essential Role in the G-CSF Triggered Granulocytic Differentiation of Human Hematopoietic Cells

The mechanism of maturation arrest of bone marrow myeloid progenitors in severe congenital neutropenia (CN) patients is not fully elucidated. We found, that treatment of healthy individuals with G-CSF induces mRNA expression of GADD45b (Growth Arrest and DNA-Damage-inducible, beta) in CD33+ bone marrow myeloid progenitors. However, the expression of GADD45b was not activated in CD33+ cells of G-CSF treated CN patients. GADD45b functions as stress sensor downstream of G-CSF signaling and is essential in stress-induced murine myelopoiesis. Less is known about the function of GADD45b in the myeloid differentiation of human HSPCs. We hypothesized, that the inability of G-CSF to induce GADD45b expression might be a cause of diminished granulopoiesis in CN patients. To test this hypothesis, we inhibited GADD45b expression in CD34+ cells and iPSCs of healthy donors by introducing indels in exon 1 of the GADD45B gene using specific CRISPR/Cas9-gRNA ribonucleoprotein (RNP). We evaluated G-CSF-triggered myeloid differentiation of GADD45b-deficient iPSCs using embryoid body (EB)-based method and found that iPSCs cells present with severely diminished granulocytic differentiation upon GADD45b knockout, as assessed by FACS, CFU assay and morphological examination of cytospin slides. We also observed reduced G-CSF-mediated granulocytic differentiation of GADD45b-deficient CD34+ cells of healthy individuals in colony-forming units (CFU) assay and liquid culture differentiation followed by FACS analysis on day 7 and day 14.Importantly, rescue of GADD45b in HSPCs of one CN patient by lentivirus-based transduction of GADD45B cDNA restored defective granulocytic differentiation, as compared to control transduced cells. These data strongly support the essential role of GADD45b in G-CSF-mediated granulocytic differentiation. GADD45b rescue analysis of additional CN patients cells is ongoing.To study the mechanism of GADD45b activation upon G-CSF stimulation of hematopoietic cells, we performed in silico analysis of GADD45B promoter and found putative binding sites for G-CSF responsive hematopoietic transcription factors, including CEBPA, CEBPB, KLF4, STAT3 and STAT5. Using the dual luciferase reporter assay with 1.6 kb region of the GADD45B gene promoter, we found that KLF4, STAT5, CEBPA and CEBPB activate GADD45b expression in a dose-dependent manner. Intriguingly, CEBPA expression is severely diminished in myeloid cells of CN patients (Skokowa et al., 2006) and we assumed that G-CSF is not able to activate GADD45b expression in CN patients because of defective CEBPA. To study the mechanism by which GADD45b mediates myeloid differentiation, we performed RNA sequencing of WT or GADD45b-deficient CD34+ HSPCs treated or not with G-CSF. Interestingly, in GADD45b-deficient cells, G-CSF failed to induce mRNA expression of several genes essential for granulocytic differentiation and granulocyte functions including GLI1, CAMP/LL37, MMP8, CD16, LCN2, OLFM4, CX3CR1, SIGLEC5, as compared to WT cells. Reactome and Gene Set Enrichment Analysis (GSEA) of RNA-Seq data sets also revealed deregulation of the “myeloid CEBPA network”, “GLI proteins pathway” and “neutrophil degranulation pathway” in G-CSF-treated GADD45b-deficient CD34+ cells, as compared to control G-CSF-exposed cells. Of note, severely diminished expression of plasma CAMP/LL37 levels is a unique feature of CN patients (Y. Ye et al. 2015).In summary, our data suggest that GADD45b plays an essential role in granulocytic differentiation of human hematopoietic cells and inability of G-CSF to induce GADD45b expression in myeloid cells of CN patients may be a reason for the defective granulopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Enhanced and Accelerated Repopulation of Mutant C-Kit Immunodeficient Mice By Transplants of Primary Chronic and Acute Myeloid Leukemic Human Cells

Background: Assessment of the growth and differentiation of human hematopoietic cells transplanted into immunodeficient mice has become an important method for evaluating the numbers and properties of cells with engraftment potential and the progeny they can generate in an in vivo setting. Such xenograft models have undergone numerous modifications and have also been applied with increasing success to primary sources of many types of malignant as well as normal human hematopoietic cells. These advances include the incorporation of cDNAs encoding various human-specific growth factor genes into the genomes of the widely used and long-lived NOD-scid-IL2Rγcnull (NSG) mouse, the creation of regenerated human bone ossicles within the mice to more closely mimic the human marrow microenvironment, recognition of the superior supportive activity of female mice, and more recently of NSG mice additionally compromised by their genetic acquisition of a c-kit deficiency. Exploitation of these modifications to recipients of human hematopoietic cells has greatly increased our understanding of the complex composition of both normal and leukemic populations and their responses to various treatments. Nevertheless, many examples remain of both acute and chronic human myeloid leukemias that do not engraft or engraft very poorly, even in the most permissive mice thus far tested. These include samples of many cases of chronic phase chronic myeloid leukemia (CP-CML), myelodysplastic syndromes (MDS), and approximately one third of all cases of acute myeloid leukemia (AML). We have now created 2 new strains of immunodeficient mice that have the B, T, NK immunodeficiency determined by a SirpαNOD-Rag1-/--IL2Rγc-/- genotype (which is equivalent to that of NSG mice) and a c-kit deficiency obtained by replacement of the wild-type c-kit gene with a homozygous W41/W41genotype (SRG-W41 mice). The second strain has the same SRG-W41 genotype but is also transgenic for human IL3, GM-CSF, and SCF production (SRG-W41-3GS). NRG/SRG mice were chosen as the parental strain because they support similar levels of chimerism as NSG mice when given a radiobiologically equivalent dose of radiation, but can benefit from split or low dose rate irradiation protocols.Results: We first showed that leukemic cells from patients with AML and CP-CML produce progeny more rapidly in sub-lethally irradiated parental SRG-3GS mice than in SRG mice, and that SRG-W41 mice can support higher levels of human myeloid and B-lymphoid chimerism by normal human CD34+ cord blood (CB) and adult bone marrow (BM) cells than similarly suppressed or non-irradiated SRG controls. We then compared the chimerism obtained in different strains of mice transplanted each with 6x106 CD34+ cells from 1 CP-CML patient or ~106 CD34+ cells from 5 different AML patients pre-selected for their previously demonstrated poor or inability to engraft NSG-3GS mice. Within a few weeks, the level of the predominantly myeloid chimerism in 2 non-irradiated male SRG-W41-3GS mice and 2 sub-lethally irradiated female SRG-3GS recipients became equivalent and this equivalence was sustained for over a year. Only 3 of 5 AML samples tested engrafted either sex of sub-lethally irradiated SRG-W41-3GS or SRG-3GS mice, and in each case, the speed and level of engraftment was greatly enhanced in the SRG-W41-3GS mice as compared to sex-matched SRG-3GS hosts (with 20-30-fold and 2-3-fold higher levels at week 6 and 10 respectively in female as compared to male recipients of either strain). All the engrafted SRG-W41-3GS female hosts became moribund within 20 weeks post-transplant with a predominance of human leukemic cells in their BM and enlarged spleens filled with leukemic cells. In contrast, the SRG-3GS recipients of the same AML cells survived more than 20 weeks with a more delayed appearance of leukemic cells. Sublethally irradiated female SRG-W41-3GS recipients of 0.1-0.2x106 CD34+ cells from 3 of 3 chronic myelomonocytic leukemia (CMML) patients tested to date have also shown engraftment at either week 3 or 11 and, in one case, up to 17 weeks post-transplant.Conclusion: The introduction of a c-kit deficiency into highly immunodeficient SRG mice producing human IL3, GM-CSF and SCF transgenically offers new opportunities for examining the growth potential and sensitivity to treatment of a broader spectrum of primary human leukemias and neoplastic conditions than has been previously possible. DisclosuresDeininger:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; CTI BioPharma Corp.: Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; BMS: Consultancy, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Longterm Multilineage Human Hematopoietic Repopulation of Untreated KIT-Mutant Immunodeficient Mice

Background: Assessment of the growth and differentiation of human hematopoietic cells in immunodeficient mice has become vital to much basic and translational research. Historically, the primary focus has been to quantify different types of repopulating cells, based on the diversity and longevity of their clonal outputs in transplanted mice, assuming the results would be relevant to clinical transplants in humans. With the development of mice that lack B, T and NK cells on a variety of backgrounds and that have normal life expectancies but differences in cellular DNA repair ability, an increasing application is to use mice repopulated with human cells to interrogate and perturb mechanisms controlling normal, genetically modified and malignant cell behavior. We have previously shown that C57Bl6 mice homozygous for the W41 mutation of c-kit are fertile with a normal lifespan but have a functionally compromised hematopoietic stem cell (HSC) population. This enables single transplanted syngeneic HSCs to be detected at high frequency in these mice when they have been given a sublethal irradiation dose. Importantly, the HSC-derived clones produced in these mice display the same growth, self-renewal and differentiation abilities as in myeloablated recipients that require a co-transplant of normal mouse bone marrow (BM) cells to support their survival. We now report the development and improved repopulation by human cord blood (CB) CD34+ cells of mice that have the same genetically determined B, T, NK immunodeficiency as NOD/Rag1-/--IL2Rγc-/- (NRG) into which a homozygous W41 gene has been introduced.This was achieved by crossing and backcrossing the progeny of NRG x C57Bl6-W41/W41 matings and selecting mice that were homozygous for the Sirpα allele of the NOD mouse, the null Rag1 and null IL2Rgamma chain, genes of the NRG mouse and the W41 gene to obtain all of these on an otherwise mixed NODxC57Bl/6 background (NRG-W41 mice). The NRG mouse was chosen because the Rag1 KO has no effect on the radiosensitivity of other tissues as is the case with the scid (S) gene in the NSG mouse. As a result, use of the NRG mouse allows exploitation of the radioprotective effect of a reduced irradiation dose rate and hence delivery of a selectively higher dose to the HSCs of the host.Results: Initial studies showed that parental NRG mice given 900 cGy split or spread continuously over 3 hrs show similar repopulation by human CD34+ CB cells as NSG mice given 315 cGy, but are more robust with consistent longterm survival. We then performed a pilot experiment using the same transplant design (2x104 CD34+ CB cells/mouse) to compare chimerism obtained in NRG-W41 mice given an estimated “equivalent” radiosensitizing regimen of 150 cGy. The levels of multiple lineages of human cells measured in the BM and spleen 20 weeks post-transplant revealed these were greatly increased in the NRG-W41 mice (>95% human CD45+ cells in the BM vs 40% in NRG mice). Kinetic analysis of human cells in the blood also showed an enhanced output of human myeloid and B-lymphoid cells over time (5-fold higher in the NRG-W41 mice after >3 weeks). Particularly notable was the selectively increased (20-fold) and sustained output of human glycophorin A+ (GPA+) erythroid cells in the NRG-W41 mice (5% human GPA+ cells in the BM of 20-week NRG-W41 mice given 150 cGy and 5x104 CD34+ CB cells/mouse vs 0.25% in the BM of the matched NRG mice given 900 cGy). A similar marked increase (20-fold) was seen on the level of circulating human platelets (SSClowCD41+ CD61+ cells) in comparable groups of transplanted NRG-W41 and NRG mice. We then investigated the extent of repopulation achievable in untreated NRG-W41 recipients. We therefore transplanted mice of both strains with 5x104 CD34+CB cells each and have now followed the levels of human cells in their circulation and BM for up to 20 weeks. Human cells were barely detectable at 3 weeks post-transplant in either strain, but then in the unirradiated NRG-W41 mice only, their levels (all lineages) increased to close to those attained in NRG mice given 900 cGy.Conclusion: NRG-W41 mice support robustly enhanced and long term generation in vivo of a wide range of human hematopoietic cell types including erythrocytes and platelets, with high levels of chimerism achieved even in unirradiated primary recipients transplanted with relatively low numbers of human CD34+ CB cells. DisclosuresNo relevant conflicts of interest to declare.

A New Immunodeficient Mouse Strain, NOD/SCID IL2Rγ−/− SGM3, Promotes Enhanced Human Hematopoietic Cell Xenografts with a Robust T Cell Component.

Abstract 3524Poster Board III-461Several strains of immunodeficient mice have been employed in the effort to model both normal and aberrant growth and differentiation of human hematopoietic cells in vivo. Despite numerous successes in this field, room remains for improvement. For example, robust models of in vivo differentiation and expansion of human erythroid and T cells are lacking or challenging at best. With the goal to further improve in vivo human hematopoietic cell study, we have crossed the NOD/SCID IL2Rγ knockout mice (NSG) with the NOD/SCID-SGM3 mice (NS-SGM; transgenic expression of human cytokines SCF, GM-CSF, and IL-3) to create a new strain of immunodeficient mice (NSG-SGM3). Uncultured umbilical cord blood CD34+ cells (UCB) were used to initiate xenografts in these three strains as well as NOD/SCID (NS) mice for comparison. Although xenografts with high human composition can be set up in NS mice, the graft, which is B-lymphoid biased without any measurable T cell component, tends to fade over time. In comparison, NSG mice offer longer sustained engraftment with a predominantly B-lymphoid bias and an inconsistent minor T cell component that is absent in NS mice. These mice also display increased levels of human cells in the periphery, allowing for easier measurement of the graft. Similar to NS grafts, NS-SGM3 grafts are initiated at high levels of chimerism, decrease significantly over time, lack T cells, and have a relatively low peripheral involvement. However, the graft consists of a more balanced myeloid/lymphoid distribution. The NSG-SGM3 combines some of the useful properties of the parent strains. Grafts remain at high, sustained levels through 16 weeks while maintaining a fairly balanced myeloid/lymphoid contribution. Chimerism in the NSG-SGM3 mice is readily detectable in the periphery as well as the BM. Remarkably, as early as 8 weeks post-transplant, we observed consistently high human T cell engraftment and expansion far superior to what we observed in NSG mice in parallel experiments using the same UCB samples. Flow cytometric analysis revealed the presence of numerous T cell sub-populations, suggesting significant recapitulation of human T cell development and function in NSG-SGM3 mice. Although it remains unclear as to why the unique combination of SGM3 cytokine expression and IL2Rγ knockout promotes enhanced human T cell engraftment and expansion, this mouse presents an improved opportunity to examine human T cell development and function in a xenograft setting as well as modeling T cell associated diseases including HIV infection, GVHD, and T cell leukemia. Disclosures:No relevant conflicts of interest to declare.

Alternative RNA Splicing Produces Multiple Forms of c-Myb with Unique Transcriptional Activities

The c-Myb transcription factor regulates the proliferation and differentiation of hematopoietic cells, and activated alleles of c-myb induce leukemias and lymphomas in animals. Relatively minor changes in the structure of c-Myb protein change the genes that it regulates and can unleash its latent transforming activities. Here, quantitative assays were used to analyze the alternative splicing of human c-myb transcripts. We identified an array of variant transcripts, expressed in highly regulated, lineage-specific patterns, that were formed through the use of alternate exons 8A, 9A, 9B, 10A, 13A, and 14A. Expression levels of the different splice variant transcripts were regulated independently of one another during human hematopoietic cell differentiation, and the alternative splicing of c-myb mRNAs was increased in primary leukemia samples. The alternatively spliced c-myb transcripts were associated with polysomes and encoded a series of c-Myb proteins with identical DNA binding domains but unique C-terminal domains. In several types of assays, the variant c-Myb proteins exhibited quantitative and qualitative differences in transcriptional activities and specificities. The results suggest that the human c-myb gene encodes a family of related proteins with different transcriptional activities. Enhanced alternative splicing may be a mechanism for unmasking the transforming activity of c-myb in human leukemias.

The Feature of TCR Vβ Subfamily T Cells Expansion in NOD/SCID Mice Transplanted with Human Cord Blood Hematopoietic Stem Cell.

Reconstitute of functional human lymphocytes in some mouse strains has been intensely explored because this approach could provide a valuable means of assessing human immunity or of preclinical testing of vaccines, pathogens, and new therapeuticstrategies. The NOD/SCID mouse strain was found to be an efficient recipient for the reconstitution of human hematopoietic cells. In these xenogenic transplantation models, growth of human lymphocytes has been demonstrated in bone marrow, peripheral blood and spleen of the mice after i.v. inoculation. These mouse models showed the multilineage differentiation of human hematopoietic cells to varying degrees. Moreover, the engraftment of human hematopoietic cells is highly variable. Complete multilineage differentiation, especially T cells, has not been achieved using this strain. Examination of T cell receptor (TCR) gene repertoire is important to analysis the immune status of models, because clonal expansion of T cells permit the identification of specific antigen response of T cells. Little is known about the feature of T-cell immunity in the humanized NOD/SCID mouse model.In order to investigate the distribution and clonal expansion of TCR Vβsubfamily T cells in NOD/SCID mice transplanted with human cord blood hematopoietic stem cell. TCR Vβ repertoire usage and clonality were analyzed. The NOD/SCID mice were sublethally irradiated (60Co, 300cGy) to eliminate residual innate immunity in the host. The experimental mice were transplanted intravenously with cord blood CD34+ cells sorted by MACS. After 6 weeks, RNA was obtained from peripheral blood, bone marrow, thymus of model. The gene expression and clonality of TCR Vβ repertoire were detected by RT-PCR and GeneScan technique. A certain range of TCR Vβ usage was exhibited in the bone marrow of mice, which included TCR Vβ1, 2, 9, 13, 19. Further, oligoclonal expression of some TCR Vβ subfamilies (Vβ9, 13, 19) were identified by GeneScan technique. To investigate the reason involving oligoclonal expansion of TCR Vβ subfamily T cells from cord blood in mouse models, the T-cell culture with tissue-antigen of NOD/SCID mouse was performed in vitro. The cells from peripheral blood mononuclear cells and bone marrow, spleen, thymus in NOD/SCID mice were wiped off by freeze thawing and served as tissue-antigen. Cord blood mononuclear cells were separately cultured with the component from those murine cells for 15 to 20 days. Oligoclonal expression or oligoclonal trend of some TCR Vβ subfamilies (Vβ10, 11 and Vβ2, 15, 16, 19) was detected in T cells after stimulation with tissue-antigen of NOD/SCID mouse. Interesting, the similar clonal expansion of TCR Vβ11 subfamily was found in T cells cultured with peripheral blood, bone marrow and spleen respectively. The results indicate that the TCR Vβ subfamily T cells could be reconstituted in humanized NOD/SCID mouse transplanted with CD34+cells from cord blood. The restricted expression and clonal expansion of some CB T cell clones might be induced by tissue-antigens of NOD/SCID mice.

Deregulated Expression of a Novel Oncogene, Ahi-1, in Murine Hematopoietic Cells Confers a Proliferative Advantage In Vitro and Leukemogenic Activity In Vivo and Enhances the Effects of BCR-ABL.

Ahi-1 (Abelson helper integration site-1) is a novel gene that we recently identified based on its common activation in v-abl or myc-induced murine leukemias and lymphomas. It encodes a unique protein with known signaling features, including SH3 and WD40-repeat domains, but its function is largely unknown. We have recently demonstrated that Ahi-1/AHI-1 transcript levels are normally down-regulated during both early murine and human hematopoietic cell differentiation and are highly increased in human leukemic cells, particularly in highly enriched populations of primitive BCR-ABL+ leukemic stem cells (lin−CD34+CD38−) in patients with chronic myeloid leukemia (CML). To investigate the potential cooperative activity of Ahi-1 in BCR-ABL-mediated signal transduction and leukemogenesis, we transduced IL-3-dependent BaF3 cells with MSCV-Ahi-1-IRES-YFP and/or MSCV-BCR-ABL-IRES-GFP retroviruses and compared the biological behavior of these cells in vitro and in vivo. All Ahi-1-transduced clonal cell lines showed increased proliferative activity and reduced apoptosis in the absence of IL-3, compared to parental BaF3 cells or control GFP-transduced cells. Interestingly, overexpression of both Ahi-1 and BCR-ABL caused more enhanced perturbations when compared to cells transduced with either Ahi-1 or BCR-ABL alone. Strikingly, intravenous injection of NOD/SCID-β2microglobulin−/−mice with BaF3 cells induced to overexpress Ahi-1 alone induces a lethal leukemia within 70 days. These leukemogenic activities were further increased by introduction of co-transduced Ahi-1 and BCR-ABL cells, producing a shorter latency of 30 days. A disease latency of 40 days was revealed by introduction of BCR-ABL-transduced cells alone. Western blot analysis showed that both protein expression and the tyrosine kinase activity of p210BCR-ABL were highly increased in cells co-transduced with Ahi-1 and BCR-ABL compared to BaF3 cells transduced with BCR-ABL alone. Similarly, we also observed higher levels of Ahi-1 protein expression in the same dually transduced cells (Ahi-1+BCR-ABL+) than in those transduced with Ahi-1 alone. We further demonstrated a similarly perturbed proliferative activity, growth factor independence and colony-forming cell (CFC) output in long-term culture initiated cell (LTC-IC) assays of 5-fluorouracil (5-FU)-treated primitive murine BM cells (Lin−Sca1+ cells) transduced with Ahi-1 and BCR-ABL, either alone or in combination. To investigate directly the cooperating oncogenic role of AHI-1 in BCR-ABL-mediated malignant transformation of CML cells, knockdown of AHI-1 expression in K562 cells, a cell line that was derived from a patient with CML and that is characterized by highly increased expression of AHI-1, was performed using retroviral-mediated RNA interference. Retroviral-mediated suppression specifically inhibited endogenous AHI-1 expression in transduced cells by 70% as evaluated by Q-RT-PCR and Western blot analyses. It further caused a significant reduction in their growth factor independence in semi-solid cultures (up to 5-fold) and in single cell cultures (2-fold) by comparison to cells transduced with a control vector. Taken together, these findings provide strong evidence of the transforming potential of Ahi-1/AHI-1 in primitive hematopoietic cells. This effect is additive with those of BCR-ABL, suggesting that AHI-1 and BCR-ABL can play a cooperate role in the development of BCR-ABL-associated diseases like CML.

Clusterin/Apolipoprotein J up-regulation after zinc exposure, replicative senescence or differentiation of human haematopoietic cells

Clusterin/Apolipoprotein J (CLU) is a cellular senescence biomarker implicated in several physiological processes. In this work we have investigated CLU expression and function in human haematopoietic cells. We found that early passage human T cell clones (TCC) express minimal endogenous amounts of CLU, which are significantly elevated in late passage cells. Moreover, exposure of TCC to increased levels of the essential micronutrient zinc in culture resulted in intense induction of CLU. Because haematopoietic cells cease proliferation following induction of terminal differentiation, we also studied the expression profile of CLU in the leukemic progenitor cell lines K562 and HL-60. We found that, like TCC, both cell lines express minimal endogenous levels of CLU in their actively proliferating state. However, when induced to differentiate into their distinct cell types, CLU was found to be up-regulated specifically in those cells expressing the main differentiation markers. Enforced stable over-expression of CLU in K562 cells inhibited the expression of the CD14 differentiation marker and blocked differentiation to either monocytes/megacaryoblasts or to erythrocytes. Overall, our results suggest that CLU is actively involved in both replicative senescence and terminal differentiation in different types of human haematopoietic cells.

Development of a Model for the Study of PU.1 Function in Primary Human Hematopoietic Cells.

PU.1 is one of the best-studied transcription factors governing hematopoiesis and has been shown to regulate positively differentiation of B-lymphocytes and granulocytes. PU.1 is also expressed in early erythroid progenitors and its interaction with GATA-1 was described to directly inhibit erythroid differentiation, since GATA-1 is the key regulator of erythropoiesis. In addition, the binding of GATA-1 to PU.1 was found to repress PU.1 dependent myeloid gene expression. In order to study more in detail the effect of PU.1 in primary human hematopoietic cell differentiation, we designed lentiviral vectors which allow PU.1 overexpression and PU.1 inhibition.For PU.1 overexpression, we cloned the PU.1 cDNA into the pWPIR-ires-GFP bicistronic plasmid and verified by transient transfection in 293T cells the production of PU.1 mRNA and of right sized protein. We analyzed PU.1 function by co-transfection assays into 293T cells using the pWPIR-PU.1 vector and CAT reporter genes governed by PU.1 responsive elements. By CAT ELISA assay we observed a dramatic increase in OD. The production of PU.1 mRNA and protein in Hela cells was verified by stable transduction with complete lentivectors.For PU.1 inhibition, we constructed a lentiviral vector encoding a siRNA specific for PU.1. After transduction of the K562 erythroleukemic cell line, both PU.1 mRNA and protein became undetectable, as verified by RT-PCR and Western blot, respectively, whereas GATA-1 mRNA and protein expression remained unchanged.We tested both viral constructs in an in vitro culture system, in which CD34+ hematopoietic precursors obtained from bone marrow aspirates, differentiate into mature red cells under the influence of SCF, IL-3 and Epo or into mature granulocytes by stimulation with thrombopoietin, SCF and Flt-3L. Results for cultures with PU.1 transduced cells showed inhibition of erythroid cell differentiation by 40% ± 10% (mean of three experiments) and increased myeloid proliferation, whereas cultures with siPU.1 transduced cells showed no influence on erythroid cells and strong decrease of myeloid cell proliferation (50 – 60x) and differentiation (90 % decrease of CD13+ cells).In conclusion, our model gives us the opportunity to test the function of PU.1 overexpression and/or inhibition in primary hematopoietic cells, to test the effect on target genes in various stages of differentiating precursors and the interaction with other transcription factors like GATA-1, and to analyze pathologic conditions like some forms of acute myeloid leukemia, where PU.1 was described to be mutated or downregulated.

The Wilms tumor suppressor WT1 directs stage-specific quiescence and differentiation of human hematopoietic progenitor cells.

WT1, a transcription factor implicated in both normal kidney differentiation and tumorigenesis, is also expressed in differentiating hematopoietic progenitors. Most human acute leukemias contain high levels of the wild-type transcript, while a minority have point mutations, raising the possibility that this tumor suppressor might have a paradoxical oncogenic effect in some hematopoietic cells. Using high titer retroviral infection, we demonstrate that WT1 triggers rapid growth arrest and lineage-specific differentiation in primary hematopoietic progenitors and differentiation-competent leukemia cell lines, while it induces cellular quiescence in a primitive subset of primary precursors. Growth arrest by WT1 is associated with induction of p21(CIP1), but expression of this cyclin-dependent kinase inhibitor alone is insufficient for either cellular differentiation or primitive cell preservation. The effects of WT1 are enhanced by co-expression of its naturally occurring isoforms, and are correlated with the physiological expression pattern of WT1 in vivo. Our observations suggest a role for WT1 in the differentiation of human hematopoietic cells, and provide a functional model that supports its capacity as a tumor suppressor in human acute leukemia.

The Role of Inducible 70-kDa Heat Shock Protein in Cell Cycle Control, Differentiation, and Apoptotic Cell Death of the Human Myeloid Leukemic HL-60 Cells

Several studies have suggested a role for heat shock proteins (hsps) during development and differentiation. However, relatively little is known about the role of hsp70 in controlling human hematopoietic cell differentiation and death. Here, we show that constitutive expression of human inducible 70-kDa heat shock protein (hsp70) promotes differentiation of HL-60 cells and prevents apoptosis that occurred after terminal differentiation or directly by apoptotic agents. After treatment with phorbol 12-myristate 13-acetate (PMA), hsp70-overexpressing cells (HL-60/hsp70) underwent rapid growth arrest and plastic adherence and expressed more CD14 than parental HL-60 or empty vector-transformed cells (HL-60/puro). HL-60/hsp70 cells also rapidly differentiated into granulocytes by addition of all-trans-retinoic acid, as assessed by phenotypic changes after staining with Wright-Giemsa. After differentiation into monocyte/macrophage-like cells or granulocytes, hsp70-overexpressing cells showed little evidence for apoptosis and had a prolonged survival, indicating that the survival-enhancing properties of hsp70 counteract programmed cell death that accompanies terminal differentiation. HL-60/hsp70 cells also showed more resistance than parental cells against apoptotic agents such as sodium nitroprusside, a NO-generating agent, or Taxol, a microtubule stabilizing agent. Further, heat shock of parental HL-60 cells at 42°C for 3 h increased hsp70 levels, promoted plastic adherence (<6 h) of the cells in respond to PMA, and protected cells from SNP or Taxol. Taken together, these studies demonstrate that hsp70 plays a crucial role in the differentiation of myeloid cells, participating in cell cycle controls and phenotypic changes, with protecting effects on apoptosis induced by different pathways.

Long-term cytokine production from engineered primary human stromal cells influences human hematopoiesis in an in vivo xenograft model.

Human hematopoiesis can be supported in beige/nude/ XID (bnx) mice by coinjection of human bone marrow stromal cells engineered to secrete human interleukin 3 (HuIL-3). The major limitation is a total absence of human B cell development in the mice, which could be due to supraphysiological levels of HuIL-3 in the circulation. In an effort to obtain human B lymphoid, as well as T lymphoid and myeloid cell development in the mice, CD34+ cells were coinjected with human marrow stromal cells engineered to secrete human IL-2, IL-7, stem cell factor or FLT3 ligand, +/- IL-3. No single factor other than IL-3 supported sustained human hematopoiesis in the mice, although cytokines were expressed for four to six months post-transplantation. Production of both HuIL-3 and IL-7 in the mice supported extrathymic development of human T lymphocytes, but no B cells, myeloid cells, or clonogenic progenitors were detected. Human B cells were not produced from CD34+ cells in the bnx mice under any condition tested. Another limitation to the bnx/Hu system is a lack of maturation of human red blood cells, although BFU-E are maintained. Stromal cells secreting human erythropoietin and IL-3 were cotransplanted into mice with HuCD34+ cells and an increase in hematocrit from 40%-45% to 80%-85% resulted, with production of human and murine red blood cells. Unfortunately, all mice (n = 9) suffered strokes, displayed paralysis and died within three weeks. The bnx/Hu cotransplantation model provides an interesting system in which to study human hematopoietic cell differentiation under the influence of various cytokines.

Improved engraftment of human hematopoietic cells in severe combined immunodeficient (SCID) mice carrying human cytokine transgenes.

We have generated immunodeficient scid-/scid- (SCID)-transgenic mice expressing the genes for human interleukin 3, granulocyte/macrophage-colony stimulating factor, and stem cell factor. We have compared engraftment and differentiation of human hematopoietic cells in transgenic SCID mice with two strains of nontransgenic SCID mice. Human bone marrow cells carrying the CD34 antigen or human umbilical cord blood were injected into sublethally irradiated recipients. Human DNA was detected by polymerase chain reaction in peripheral blood and bone marrow of 14 of 28 transgenic SCID mice after transplantation, but in only 2 of 15 nontransgenic SCID littermates at a 10-fold lower level. Bone marrow cultures 8 wk after transplantation of cord blood gave rise to human burst-forming unit erythroid, colony-forming unit granulocyte/macrophage, or granulocyte/erythroid/macrophage/megakaryocyte colonies. Engraftment was observed for up to 6 mo in transgenic SCID mice, twice as long as nontransgenic littermates or previous studies in which transplanted SCID mice were given daily injections of growth factors. We conclude that the level and duration of engraftment of human cells in SCID mice can be improved by expression of human cytokine transgenes and that transgenic SCID mice are an efficient model system for the study of human hematopoiesis.

Wild-Type p53 Gene Expression Induces Granulocytic Differentiation of HL-60 Cells

AbstractOverexpression of wild-type p53 gene in malignant cell tines has been shown to inhibit cell proliferation in a number of cases. However, endogenous p53 protein seems to play little role in normal cell-cycle control as suggested by the normal development of p53 null mice, and by the low p53 protein levels expressed in most cell types. Recently, increased expression of endogenous p53 protein has been observed during the cellular response to DNA damage, as well as during differentiation of human hematopoietic cells. To study the role of the p53 gene in hematopoietic differentiation, we introduced the wild-type p53 gene or the temperature-sensitive p53(Val135) mutant into p53-deficient HL-60 promyelocyte leukemia cells. Morphological analysis, flow-cytome-tric determination of granulocytic or monocytic surface markers, and ability to reduce nitroblue tetrazolium (NBT) demonstrated that expression of exogenous wild-type p53 gene in HL-60 cells induces differentiation through the granulocytic pathway. Proliferation and cell-cycle analysis performed early after expression of wild-type p53 showed that induction of differentiation is not coupled with growth arrest, which suggests that p53 is involved in differentiation independently of its activity on the cell cycle.

Wild-type p53 gene expression induces granulocytic differentiation of HL-60 cells

Overexpression of wild-type p53 gene in malignant cell lines has been shown to inhibit cell proliferation in a number of cases. However, endogenous p53 protein seems to play little role in normal cell-cycle control as suggested by the normal development of p53 null mice, and by the low p53 protein levels expressed in most cell types. Recently, increased expression of endogenous p53 protein has been observed during the cellular response to DNA damage, as well as during differentiation of human hematopoietic cells. To study the role of the p53 gene in hematopoietic differentiation, we introduced the wild-type p53 gene or the temperature-sensitive p53(Val135) mutant into p53-deficient HL-60 promyelocytic leukemia cells. Morphological analysis, flow-cytometric determination of granulocytic or monocytic surface markers, and ability to reduce nitroblue tetrazolium (NBT) demonstrated that expression of exogenous wild-type p53 gene in HL-60 cells induces differentiation through the granulocytic pathway. Proliferation and cell-cycle analysis performed early after expression of wild-type p53 showed that induction of differentiation is not coupled with growth arrest, which suggests that p53 is involved in differentiation independently of its activity on the cell cycle.

Expansion and differentiation of human hematopoietic cells from static cultures through small-scale bioreactors.

Maintenance of the progenitor cells responsible for hematopoiesis has generally been accomplished using a feeder layer of stromal cells in stationary culture. Here, we compared the expansion of the total cell and progenitor cell populations using low-density mononuclear cells (LDMCs) obtained from human bone marrow in static culture (T-flasks) and in different cell culture bioreactors designed for the scale-up of mammalian cells. Static cultures were performed without the presence of a previously established stromal cell layer. Expansion of marrow in all cases was accomplished through the use of added cytokines such as IL-3, GM-CSF, and c-kit ligand. The results for the total cell expansion in static culture ranged from 4.4- to 32-fold. The cell number increase was affected by such factors as patient to patient variability, freeze-thawing, and the combination of cytokines used. Due to widespread use and the small amount of marrow needed, static cultures were used as a basis for comparison with other expansion systems. The cell culture systems used to evaluate the scale-up of marrow cultures included suspension, microcarrier, airlift, and hollow fiber bioreactors. Using identical media, cytokines, and feed schedules, LDMCs in the suspension bioreactor expanded to a value of 1.6 compared to a normalized value of 1.0 for static cultures for the two runs investigated. Expansion results for microcarrier cultures averaged 0.75 when compared to static cultures. A cell number increase in the airlift bioreactor resulted in an expansion which was 0.70 of the control static culture. Granulocyte-macrophage and erythroid progenitor assay data were also evaluated for the suspension, microcarrier, and airlift bioreactors.(ABSTRACT TRUNCATED AT 250 WORDS)