Ruijun Su

Efficient Reprogramming of Human Cord Blood CD34+ Cells Into Induced Pluripotent Stem Cells With OCT4 and SOX2 Alone

The reprogramming of cord blood (CB) cells into induced pluripotent stem cells (iPSCs) has potential applications in regenerative medicine by converting CB banks into iPSC banks for allogeneic cell replacement therapy. Therefore, further investigation into novel approaches for efficient reprogramming is necessary. Here, we show that the lentiviral expression of OCT4 together with SOX2 (OS) driven by a strong spleen focus-forming virus (SFFV) promoter in a single vector can convert 2% of CB CD34+ cells into iPSCs without additional reprogramming factors. Reprogramming efficiency was found to be critically dependent upon expression levels of OS. To generate transgene-free iPSCs, we developed an improved episomal vector with a woodchuck post-transcriptional regulatory element (Wpre) that increases transgene expression by 50%. With this vector, we successfully generated transgene-free iPSCs using OS alone. In conclusion, high-level expression of OS alone is sufficient for efficient reprogramming of CB CD34+ cells into iPSCs. This report is the first to describe the generation of transgene-free iPSCs with the use of OCT4 and SOX2 alone. These findings have important implications for the clinical applications of iPSCs.The reprogramming of cord blood (CB) cells into induced pluripotent stem cells (iPSCs) has potential applications in regenerative medicine by converting CB banks into iPSC banks for allogeneic cell replacement therapy. Therefore, further investigation into novel approaches for efficient reprogramming is necessary. Here, we show that the lentiviral expression of OCT4 together with SOX2 (OS) driven by a strong spleen focus-forming virus (SFFV) promoter in a single vector can convert 2% of CB CD34(+) cells into iPSCs without additional reprogramming factors. Reprogramming efficiency was found to be critically dependent upon expression levels of OS. To generate transgene-free iPSCs, we developed an improved episomal vector with a woodchuck post-transcriptional regulatory element (Wpre) that increases transgene expression by 50%. With this vector, we successfully generated transgene-free iPSCs using OS alone. In conclusion, high-level expression of OS alone is sufficient for efficient reprogramming of CB CD34(+) cells into iPSCs. This report is the first to describe the generation of transgene-free iPSCs with the use of OCT4 and SOX2 alone. These findings have important implications for the clinical applications of iPSCs.

Efficient Generation of Integration-Free iPS Cells from Human Adult Peripheral Blood Using BCL-XL Together with Yamanaka Factors

The ability to efficiently generate integration-free induced pluripotent stem cells (iPSCs) from the most readily available source—peripheral blood—has the potential to expedite the advances of iPSC-based therapies. We have successfully generated integration-free iPSCs from cord blood (CB) CD34+ cells with improved oriP/EBNA1-based episomal vectors (EV) using a strong spleen focus forming virus (SFFV) long terminal repeat (LTR) promoter. Here we show that Yamanaka factors (OCT4, SOX2, MYC, and KLF4)-expressing EV can also reprogram adult peripheral blood mononuclear cells (PBMNCs) into pluripotency, yet at a very low efficiency. We found that inclusion of BCL-XL increases the reprogramming efficiency by approximately 10-fold. Furthermore, culture of CD3−/CD19− cells or T/B cell-depleted MNCs for 4–6 days led to the generation of 20–30 iPSC colonies from 1 ml PB, an efficiency that is substantially higher than previously reported. PB iPSCs express pluripotency markers, form teratomas, and can be induced to differentiate in vitro into mesenchymal stem cells, cardiomyocytes, and hepatocytes. Used together, our optimized factor combination and reprogramming strategy lead to efficient generation of integration-free iPSCs from adult PB. This discovery has potential applications in iPSC banking, disease modeling and regenerative medicine.

Rapid and efficient reprogramming of human fetal and adult blood CD34+ cells into mesenchymal stem cells with a single factor.

The direct conversion of skin cells into somatic stem cells has opened new therapeutic possibilities in regenerative medicine. Here, we show that human induced mesenchymal stem cells (iMSCs) can be efficiently generated from cord blood (CB)- or adult peripheral blood (PB)-CD34+ cells by direct reprogramming with a single factor, OCT4. In the presence of a GSK3 inhibitor, 16% of the OCT4-transduced CD34+ cells are converted into iMSCs within 2 weeks. Efficient direct reprogramming is achieved with both episomal vector-mediated transient OCT4 expression and lentiviral vector-mediated OCT4 transduction. The iMSCs express MSC markers, resemble bone marrow (BM)-MSCs in morphology, and possess in vitro multilineage differentiation capacity, yet have a greater proliferative capacity compared with BM-MSCs. Similar to BM-MSCs, the implanted iMSCs form bone and connective tissues, and are non-tumorigenic in mice. However, BM-MSCs do not, whereas iMSCs do form muscle fibers, indicating a potential functional advantage of iMSCs. In addition, we observed that a high level of OCT4 expression is required for the initial reprogramming and the optimal iMSC self-renewal, while a reduction of OCT4 expression is required for multilineage differentiation. Our method will contribute to the generation of patient-specific iMSCs, which could have applications in regenerative medicine. This discovery may also facilitate the development of strategies for direct conversion of blood cells into other types of cells of clinical importance.

Regulator of myeloid differentiation and function:The secret life of Ikaros

Ikaros (also known as Lyf-1) was initially described as a lymphoid-specific transcription factor. Although Ikaros has been shown to regulate hematopoietic stem cell renewal, as well as the development and function of cells from multiple hematopoietic lineages, including the myeloid lineage, Ikaros has primarily been studied in context of lymphoid development and malignancy. This review focuses on the role of Ikaros in myeloid cells. We address the importance of post-transcriptional regulation of Ikaros function; the emerging role of Ikaros in myeloid malignancy; Ikaros as a regulator of myeloid differentiation and function; and the selective expression of Ikaros isoform-x in cells with myeloid potential. We highlight the challenges of dissecting Ikaros function in lineage commitment decisions among lymphoid-myeloid progenitors that have emerged as a major myeloid differentiation pathway in recent studies, which leads to reconstruction of the traditional map of murine and human hematopoiesis.

Differences in Mouse and Human Nonmemory B Cell Pools

Identifying cross-species similarities and differences in immune development and function is critical for maximizing the translational potential of animal models. Coexpression of CD21 and CD24 distinguishes transitional and mature B cell subsets in mice. In this study, we validate these markers for identifying analogous subsets in humans and use them to compare the nonmemory B cell pools in mice and humans, across tissues, and during fetal/neonatal and adult life. Among human CD19+IgM+ B cells, the CD21/CD24 schema identifies distinct populations that correspond to transitional 1 (T1), transitional 2 (T2), follicular mature, and marginal zone subsets identified in mice. Markers specific to human B cell development validate the identity of marginal zone cells and the maturation status of human CD21/CD24 nonmemory B cell subsets. A comparison of the nonmemory B cell pools in bone marrow, blood, and spleen in mice and humans shows that transitional B cells comprise a much smaller fraction in adult humans than mice. T1 cells are a major contributor to the nonmemory B cell pool in mouse bone marrow, in which their frequency is more than twice that in humans. Conversely, in spleen, the T1:T2 ratio shows that T2 cells are proportionally ∼8-fold higher in humans than in mice. Despite the relatively small contribution of transitional B cells to the human nonmemory pool, the number of naive follicular mature cells produced per transitional B cell is 3- to 6-fold higher across tissues than in mice. These data suggest differing dynamics or mechanisms produce the nonmemory B cell compartments in mice and humans.

Expression of Exogenous Cytokine in Patient-derived Xenografts via Injection with a Cytokine-transduced Stromal Cell Line

Patient-derived xenograft (PDX) mice are produced by transplanting human cells into immune deficient mice. These models are an important tool for studying the mechanisms of normal and malignant hematopoiesis and are the gold standard for identifying effective chemotherapies for many malignancies. PDX models are possible because many of the mouse cytokines also act on human cells. However, this is not the case for all cytokines, including many that are critical for studying normal and malignant hematopoiesis in human cells. Techniques that engineer mice to produce human cytokines (transgenic and knock-in models) require significant expense before the usefulness of the model has been demonstrated. Other techniques are labor intensive (injection of recombinant cytokine or lentivirus) and in some cases require high levels of technical expertise (hydrodynamic injection of DNA). This report describes a simple method for generating PDX mice that have exogenous human cytokine (TSLP, thymic stromal lymphopoietin) via weekly intraperitoneal injection of stroma that have been transduced to overexpress this cytokine. Use of this method provides an in vivo source of continuous cytokine production that achieves physiological levels of circulating human cytokine in the mouse. Plasma levels of human cytokine can be varied based on the number of stromal cells injected, and cytokine production can be initiated at any point in the experiment. This method also includes cytokine-negative control mice that are similarly produced, but through intraperitoneal injection of stroma transduced with a control vector. We have previously demonstrated that leukemia cells harvested from TSLP-expressing PDX, as compared to control PDX, exhibit a gene expression pattern more like the original patient sample. Together the cytokine-producing and cytokine-negative PDX mice produced by this method provide a model system that we have used successfully to study the role of TSLP in normal and malignant hematopoiesis.

Abstract A07: A novel patient-derived xenograft model to define the role of TSLP-induced CRLF2 signals and identify therapies for Ph-like B-ALL

A subset of high-risk B cell acute lymphoblastic leukemia (ALL) shows a gene expression profile similar to Philadelphia chromosome positive (Ph+) ALL and has been described as Ph-like ALL. Approximately 50% of Ph-like B-ALL is characterized by genetic alterations leading to overexpression of CRLF2 (CRLF2 B-ALL). CRLF2 B-ALL occurs 5 times more often in Hispanic and Native American children than others and is prevalent in adolescents and young adults. Biologically, CRLF2 acts as a receptor component for the cytokine, TSLP, which induces JAK2-STAT5 and PI3/AKT/mTOR pathway activation downstream of binding to CRLF2. While activating JAK mutations are associated with CRLF2 B-ALL, over half of CRLF2 B-ALL lack such mutations. Our data show that primary human bone marrow (BM) stromal cells express TSLP. Thus TSLP is present in the tumor microenvironment to provide TSLP-induced CRLF2 signals that could play a role in the initiation, maintenance and/or progression of CRLF2 B-ALL. Consistent with this, TSLP has been reported to increase in vitro production of human fetal B cell precursors. However studies of TSLP in B lymphopoiesis have been conducted almost exclusively in mice which show low homology (~40%) with respect to human TSLP and CRLF2. Further, phospho flow cytometry assays show that human, but not mouse TSLP activates CRLF2 signals in primary human CRLF2 B-ALL cells and cell lines as indicated by increased pSTAT5, pAKT and pS6. These data indicate that the mouse TSLP present in classic patient derived xenograft models (PDX) does not produce the TSLP-induced CRLF2 signals present in the patient. To address this challenge we engineered PDX mice to produce human TSLP (hTSLP) by transplanting them with stromal cells transduced to express hTSLP (+T mice). Control (T) mice were produced by transplantation with stroma transduced with a control vector. Supernatant from engineered +T stroma, but not T stroma, induced JAK/STAT5 and PI3K/AKT/mTOR pathway activation in human CRLF2 B-ALL cells. ELISA assays showed that serum levels of hTSLP in mice was proportional to numbers of stromal cells injected at weekly time points. Normal human serum levels of hTSLP (12-32 pg/ml) could be achieved in +T mice, while hTSLP was undetectable in T mice. Because TSLP has been shown to increase in vitro production of human B cell precursors, we evaluated the in vivo functionality of our model by comparing the production of normal B cell precursors in the BM of +T and T PDX mice generated with human umbilical cord blood CD34+ cells. Data from 3 different cord blood donors showed that production of B cell precursors is 3-5 fold increased in +T as compared to T mice. TSLP-induced increases were specific to B lineage cells, initiated in the earliest CD19+ B cell precursors, and maintained through later stages of B cell development. Next we evaluate the in vivo functionality of our model using primary CRLF2 B-ALL leukemia cells. Human CRLF2 B-ALL cells were isolated from the BM of PDX mice and whole genome microarray was performed. Evaluation of microarray data by Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis showed that genes downstream of mTOR pathway activation were upregulated in +T as compared to T PDX mice, confirming hTSLP activity in the +T PDX mice. To determine whether +T PDX mice provide a preclinical model of B-ALL that more closely mirrors patients than T PDX mice, we compared RNAseq gene expression profiles of leukemia cells from +T and T PDX mice to that from original patient sample. The gene expression pattern in +T mice was significantly closer to primary patient sample than that from T mice. The +T and T PDX mice described here provide a novel preclinical model for studying the role of TSLP in the initiation, progression and maintenance of CRLF2 B-ALL and for evaluating drug efficacy in an in vivo model that more closely mirrors the in vivo environment present in patients. Citation Format: Olivia L. Francis, Terry-Ann Milford, Ineavely Baez, Jacqueline S. Coats, Christopher L. Morris, Ross Fisher, Ben Van Handel, Ruijun Su, Batul Suterwala, Muhammad Kamal, Shadi Farzin Gohar, Sinisa Dovat, Kimberly J. Payne. A novel patient-derived xenograft model to define the role of TSLP-induced CRLF2 signals and identify therapies for Ph-like B-ALL. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A07.

Abstract B25: A human-mouse xenograft model to evaluate therapies and study the role of TSLP-induced signals in Ph-like ALL

While the overall survival rate for children with B cell precursor acute lymphoblastic leukemia (B-ALL) is high, a subset of children with this disease are at high risk for relapse and death. Genome-wide analysis has shown that gene expression profiles in these high-risk B-ALLs is similar to that of Philadelphia chromosome–positive ALL and these are designated Ph-like ALL. Approximately half of Ph-like ALL are characterized by genetic defects resulting in overexpression of CRLF2. CRLF2, together with the IL-7Rα, forms a receptor complex that is activated by the cytokine, TSLP. The JAK-STAT5 pathway is phosphorylated downstream of this receptor complex activation. The activating JAK mutations found in some CRLF2 B-ALL have led to speculation that TSLP stimulation is not a factor in CRLF B-ALL. In preliminary studies to address this question we evaluated the effect of TSLP on a CRLF2 B-ALL cell lines with JAK defects and which have been reported to exhibit constitutive JAK-STAT5 activation. Our data show that TSLP increases STAT5 phosphorylation in these cell lines and also in primary CRLF2 B-ALL cells. Our next step was to evaluate the role of TSLP-CRLF2 interactions in vivo in the human-mouse xenograft model. However, mouse TSLP is different from most other cytokines produced in the xenograft in that it is species-specific and does not activate the human TSLP receptor complex that includes CRLF2. Thus, traditional xenograft models do not provide the TSLP-CRLF2 interactions that we believe to be a major factor in CRLF2 B-ALL. To overcome this obstacle we engineered immune-deficient NOD/SCID IL-2Rγ null (NSG) mice to express human TSLP (hTSLP+ mice) as well as control mice that lack the TSLP cytokine (hTSLP– mice). ELISA assays show serum hTSLP levels in the hTSLP+ mice that approximate the normal range in human serum. We used this hTSLP+/- xenograft model system to study the in vivo effects of TSLP on mice transplanted with a CRLF2 B-ALL. We used this hTSLP+/– xenograft model system to evaluate the in vivo effects of TSLP on survival and proliferation of transplanted CRLF2 B-ALL cells harboring a JAK defect (MUTZ5 cell line). Mice were euthanized at 5 weeks and BM was harvested. Evaluation of BM disease by flow cytometry showed that the percentage of viable human leukemia cells in hTSLP+ mice was twice that observed in hTSLP– mice. Evaluation of cell cycle progression in human CRLF2 B-ALL cells isolated from xenograft BM showed that the percentage of cycling cells in hTSLP+ mice was 2.5 fold higher than in hTSLP– mice. When primary Ph-like ALL cells were transplanted to produce hTSLP+/– xenografts, the viable pre-B ALL cells present in the BM of hTSLP+ mice showed higher expression levels of the TSLPR components (CRLF2 and IL-7Rα) than those in the hTSLP- mice. These data provide evidence that the TSLP produced in this model is active and that it impacts primary pre-B ALL cells. Preliminary data obtained from this model suggests that TSLP provides a signal that promotes in vivo survival of CRLF2 B-ALL cells and that it may play a role in selection of leukemia clones during in vivo leukemogenesis. Microarray analysis comparing gene expression in primary CRLF2 B-ALL cells isolated from hTSLP+ and hTSLP– xenograft mice identified 565 that genes are differentially regulated (> 2 fold up or downregulated; p Citation Format: Ruijun Su, Francis L. Olivia, Shannalee R. Martinez, Ineavely Baez, Terry Ann Milford, Terrence Bennett, Ross Fisher, Christopher L. Morris, Sinisa Dovat, Kimberly J. Payne. A human-mouse xenograft model to evaluate therapies and study the role of TSLP-induced signals in Ph-like ALL. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B25.

Abstract B21: A human-mouse xenograft model to evaluate therapies and study the role of TSLP-induced signals in Ph-like ALL

While the overall survival rate for children with B cell precursor acute lymphoblastic leukemia (B-ALL) is high, a subset of children with this disease are at high risk for relapse and death. Genome-wide analysis has shown that gene expression profiles in these high-risk B-ALLs is similar to that of Philadelphia chromosome–positive ALL and these are designated Ph-like ALL. Approximately half of Ph-like ALL are characterized by genetic defects resulting in overexpression of CRLF2. CRLF2, together with the IL-7Rα, forms a receptor complex that is activated by the cytokine, TSLP. The JAK-STAT5 pathway is phosphorylated downstream of this receptor complex activation. The activating JAK mutations found in some CRLF2 B-ALL have led to speculation that TSLP stimulation is not a factor in CRLF B-ALL. In preliminary studies to address this question we evaluated the effect of TSLP on a CRLF2 B-ALL cell lines with JAK defects and which have been reported to exhibit constitutive JAK-STAT5 activation. Our data show that TSLP increases STAT5 phosphorylation in these cell lines and also in primary CRLF2 B-ALL cells. Our next step was to evaluate the role of TSLP-CRLF2 interactions in vivo in the human-mouse xenograft model. However, mouse TSLP is different from most other cytokines produced in the xenograft in that it is species-specific and does not activate the human TSLP receptor complex that includes CRLF2. Thus, traditional xenograft models do not provide the TSLP-CRLF2 interactions that we believe to be a major factor in CRLF2 B-ALL. To overcome this obstacle we engineered immune-deficient NOD/SCID IL-2Rγ null (NSG) mice to express human TSLP (hTSLP+ mice) as well as control mice that lack the TSLP cytokine (hTSLP– mice). ELISA assays show serum hTSLP levels in the hTSLP+ mice that approximate the normal range in human serum. We used this hTSLP+/- xenograft model system to study the in vivo effects of TSLP on mice transplanted with Ph-like B-ALL. First, we used the hTSLP+/– xenograft model system to evaluate the in vivo effects of TSLP on survival and proliferation of transplanted CRLF2 B-ALL cells harboring a JAK defect (MUTZ5 cell line). Mice were euthanized at 5 weeks and BM was harvested. Evaluation of BM disease by flow cytometry showed that the percentage of viable human leukemia cells in hTSLP+ mice was twice that observed in hTSLP– mice. Evaluation of cell cycle progression in human CRLF2 B-ALL cells isolated from xenograft BM showed that the percentage of cycling cells in hTSLP+ mice was 2.5 fold higher than in hTSLP– mice. When primary Ph-like ALL cells were transplanted to produce hTSLP+/– xenografts, the viable pre-B ALL cells present in the BM of hTSLP+ mice showed higher expression levels of the TSLPR components (CRLF2 and IL-7Rα) than those in the hTSLP- mice. These data provide evidence that the TSLP produced in this model is active and that it impacts primary pre-B ALL cells. Preliminary data obtained from this model suggests that TSLP provides a signal that promotes in vivo survival of CRLF2 B-ALL cells and that it may play a role in selection of leukemia clones during in vivo leukemogenesis. Microarray analysis comparing gene expression in primary CRLF2 B-ALL cells isolated from hTSLP+ and hTSLP– xenograft mice identified 565 that genes are differentially regulated (> 2 fold up or downregulated; p Citation Format: Olivia Francis, Ruijun Su, Shannalee Martinez, Ineavely Baez, Terry-Ann Milford, Terrence Bennett, Ross Fisher, Christopher L. Morris, Sinisa Dovat, Kimberly J. Payne. A human-mouse xenograft model to evaluate therapies and study the role of TSLP-induced signals in Ph-like ALL. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B21.

Abstract 3097: TSLP regulates expression of genes involved in cell survival in a preclinical xenograft model of CRLF2 B-ALL

A subset of children with B cell precursor acute lymphoblastic leukemia (B-ALL) are at high risk for relapse and death. Gene expression profiles in these high-risk B-ALLs is similar to that of Philadelphia chromosome-positive ALL. Approximately half of these Ph-like B-ALL are characterized by genetic defects resulting in overexpression of CRLF2. This defect occurs 5 times more commonly in Hispanic children than others and thus is a significant biological component of pediatric cancer health disparities. CRLF2, together with the IL-7Rα, forms a receptor complex that is activated by the cytokine, TSLP. Receptor complex activation leads to JAK-STAT5 phosphorylation. The activating JAK mutations found in some CRLF2 B-ALL led to speculation that TSLP stimulation is not a factor in CRLF B-ALL. However, we and others have found that TSLP increases STAT5 phosphorylation in CRLF2 B-ALL cells, including those with JAK defects. Our next step was to evaluate the role of TSLP-CRLF2 interactions in vivo in the human-mouse xenograft model. However, mouse TSLP is species-specific and does not activate the human TSLP receptor complex that includes CRLF2. Thus, traditional xenograft models do not provide the TSLP-CRLF2 interactions that may contribute to high risk CRLF2 B-ALL. We engineered immune-deficient NOD/SCID IL-2Rγ null (NSG) mice to express human TSLP (hTSLP+ mice), as well as control mice that lack the TSLP cytokine (hTSLP- mice). Then we used this hTSLP+/- xenograft model system to evaluate the in vivo effects of TSLP on transplanted CRLF2 B-ALL cells harboring a JAK defect (MUTZ5 cell line). Transplanted mice were euthanized at 5 weeks and bone marrow (BM) was harvested. Evaluation of BM by flow cytometry showed that approximately half of the human leukemia cells were apoptotic in mice without TSLP while apoptosis was virtually absent in CRLF2 B-ALL cells harvested from hTSLP+ mice. Next we used Ingenuity Pathway Analysis to identify functions and pathways regulated by TSLP. Primary CRLF2 B-ALL cells were transplanted into hTSLP+ and hTSLP- mice. Whole genome microarray performed on primary human leukemia cells isolated from BM of xenograft mice identified 280 genes that were upregulated and 281 genes that were downregulated (> 2 fold up or downregulated; p Citation Format: Olivia L. Francis, Ruijun Su, Shannalee R. Martinez, Ineavely Baez, Terry-Ann Milford, Ross Fisher, Christopher L. Morris, Xiaobing Zhang, Valeri Filippov, Sinisa Dovat, Kimberly J. Payne. TSLP regulates expression of genes involved in cell survival in a preclinical xenograft model of CRLF2 B-ALL. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3097. doi:10.1158/1538-7445.AM2014-3097