Leaders in Pharmaceutical Business Intelligence (LPBI) Group

CML treated by iP stem cell conversion

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Primitive Human Leukemia Cells Grown in Lab

Rogue stem cells are at the root of all leukemias

http://www.technologynetworks.com/HTS/news.aspx?ID=185776

 

 

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Efficient generation of transgene-free induced pluripotent stem cells from normal and neoplastic bone marrow and cord blood mononuclear cells

Kejin Hu,1Junying Yu,1Kran Suknuntha,2Shulan Tian,3Karen Montgomery,4Kyung-Dal Choi,1Ron Stewart,3James A. Thomson,3 and Igor I. Slukvincorresponding author1,2

Blood. 2011 Apr 7; 117(14): e109–e119.        http://dx.doi.org:/10.1182/blood-2010-07-298331

Reprogramming blood cells to induced pluripotent stem cells (iPSCs) provides a novel tool for modeling blood diseases in vitro. However, the well-known limitations of current reprogramming technologies include low efficiency, slow kinetics, and transgene integration and residual expression. In the present study, we have demonstrated that iPSCs free of transgene and vector sequences could be generated from human BM and CB mononuclear cells using nonintegrating episomal vectors. The reprogramming described here is up to 100 times more efficient, occurs 1-3 weeks faster compared with the reprogramming of fibroblasts, and does not require isolation of progenitors or multiple rounds of transfection. Blood-derived iPSC lines lacked rearrangements of IGH and TCR, indicating that their origin is non–B- or non–T-lymphoid cells. When cocultured on OP9, blood-derived iPSCs could be differentiated back to the blood cells, albeit with lower efficiency compared to fibroblast-derived iPSCs. We also generated transgene-free iPSCs from the BM of a patient with chronic myeloid leukemia (CML). CML iPSCs showed a unique complex chromosomal translocation identified in marrow sample while displaying typical embryonic stem cell phenotype and pluripotent differentiation potential. This approach provides an opportunity to explore banked normal and diseased CB and BM samples without the limitations associated with virus-based methods

 

The advent of reprogramming technology has opened up the possibility of obtaining patient-specific induced pluripotent stem cells (iPSCs) for the study of blood diseases and for potential therapeutic applications. Although skin fibroblasts initially were used to obtain human iPSCs,1,2 several studies demonstrated successful reprogramming of CD34+ cells from CB or mobilized peripheral blood.3,4 Recently, T cells and peripheral blood mononuclear cells have also been successfully reprogrammed to iPSCs.5–7 Because genetic abnormalities are limited to hematopoietic cells in many blood diseases, successful reprogramming of blood cells represents a major advance in establishing iPSC-based models for hematologic diseases. However, because the current reprogramming methods use virus-based delivery of reprogramming factors, permanent integration of transgene and/or vector sequences into the genome, residual transgene expression, low efficiency, and slow kinetics remain the major problems surrounding this technology. To overcome these problems, several approaches have been used, including transient transfection, RNA transfection, the “PiggyBac” system, protein transduction, the Cre-LoxP excision system, minicircle vectors, and episomal plasmids.8–13 Nevertheless, limitations related to low reprogramming efficiency and/or genomic integration and complexity of genetic manipulations are still not completely resolved, and the suitability of these newest techniques for blood reprogramming remains unknown.

 

We recently developed a method for obtaining human iPSCs free of vector and transgene sequences from human fibroblasts using nonintegrating episomal vectors.14 In the present study, we have demonstrated that this technology could be applied to efficiently reprogram mononuclear cells from human BM and CB to pluripotency with up to 100 times more reprogramming efficiency compared with fibroblasts. The iPSCs generated by this method were free of transgene and vector sequences and were able to differentiate back to the blood, albeit with lower efficiency compared with fibroblast-derived iPSCs. Using the same protocol, we also efficiently reprogrammed a BM sample from a patient with chronic myeloid leukemia (CML), and were able to obtain transgene-free iPSCs with unique, patient-specific complex chromosomal translocation, which would be impossible to generate using currently available genetic-engineering methods. The elimination of genomic integration and background transgene expression, some of which are oncogenes, is a critical step toward advancing iPSC technology for the modeling of blood diseases and therapeutic applications.

Generation of iPSCs from mononuclear cells

Frozen CB mononuclear cells were obtained from AllCells. BM mononuclear cells from normal donors and from a patient with CML in the chronic phase were purchased from AllCells. Total BM cells intended for final disposition were also obtained from the University of Wisconsin Hospital and Clinics. Whole BM was cultured overnight in expansion medium consisting of StemSpan SFEM (StemCell Technologies) supplemented with Ex-Cyte (0.2%; Celliance) and recombinant human IL-3 (10 ng/mL), IL-6 (100 ng/mL), SCF (100 ng/mL), and FMS-related tyrosine kinase-3 ligand (Flt3L;100 ng/mL; all from PeproTech). The next day, Histopaque (Sigma-Aldrich) separation was performed to obtain the mononuclear cells. For reprogramming, BM mononuclear cells were cultured in expansion medium for 2 days (Figure 1A). After removing the dead cells by spinning over a 20% Percoll gradient (Sigma-Aldrich), 1 × 105 to 3.7 × 106viable cells were transfected with combination 19 of reprogramming factors (9 μg of pEP4EO2SET2K and pEP4EO2SEN2K and 6 μg of pCEP4M2L)14 using the CD34+ Nucleofector kit (Lonza). After an additional 2 days of culturing in expansion medium and removing the dead cells by Percoll density centrifugation, cells were transferred onto MEFs and cultured in iPSC medium. Starting from day 10, MEF-conditioned medium was used, and this was changed every day. The individual iPSC colonies were picked up for expansion from days 17-21. CB mononuclear cells were reprogrammed using the same conditions with or without the addition of 1μM thiazovivin (Stemgent).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3083304/figure/F1/

Figure 1

Efficient generation of transgene-free iPSCs from BM mononuclear cells. (A) Schematic diagram of reprogramming protocol. (B) Kinetics of morphologic changes after blood reprogramming. (C-D) Comparison of reprogramming efficiency between blood cells and …

High efficiency of reprogramming of mononuclear cells from human BM and CB

For the production of iPSCs, BM mononuclear cells were cultured in serum-free expansion medium supplemented with human SCF, IL-3, IL-6, and Flt3L for 2 days to expand hematopoietic progenitors, and transfected with episomal vectors (combination 19)14 by nucleofection. After an additional 2 days of culture in hematopoietic medium, floating cells were transferred onto MEF feeders (Figure 1A). Cells in coculture underwent a series of changes, including morphologic transformation from round to cuboidal shape, with eventual formation of ALP+ colonies with typical ESC morphology at approximately day 17-21 of culture (Figure 1B-C). By picking up 50 of 88 high-quality iPSC colonies, we were able to obtain 47 iPSC lines in a single reprogramming experiment, representing 352 iPSC lines per 106 transfected cells. This high reprogramming efficiency of blood cells was reproduced in another experiment (Figure 1D). In contrast, we obtained only a few iPSC lines by transfection of 106 fibroblasts with episomal plasmids expressing the same set of reprogramming factors.14 To confirm superior efficiency of BM-cell reprogramming, we performed side-by-side reprogramming experiments with BM mononuclear cells and neonatal fibroblasts and evaluated the number of ALP+ colonies after the first passage. As shown in Figure 1C, reprogrammed BM mononuclear cells generated a much higher number of ALP+ colonies compared with fibroblasts in 2 independent experiments. BM iPSCs expressed the typical ESC markers OCT4, SOX2, NANOG, LIN28, SSEA3, SSEA4, TRA-1-60, TRA-1-81, and ALP as determined by RT-PCR and flow cytometry (Figure 1E,J). We also observed up-regulation of other ESC signature genes REX1 (ZFP42), GDF3, DNMT3B, andTDGF1, which were not present in our reprogramming cocktails (Figure 1F,J). As expected, BM iPSCs lost expression of the pan-hematopoietic markers CD45 and CD43 (data not shown) and genes typically found in the BM hematopoietic cells (Figure 2C). To characterize the molecular properties of BM iPSCs, we performed a global analysis of the gene expression of blood-derived iPSCs and compared them with 5 hESC lines and 3 iPSCs derived from fibroblasts using plasmid combination 19 (DF19 iPSC lines).14 In this analysis, we also included 2 iPSC lines derived from fibroblasts using the same set of reprogramming factors but using expression vectors with different transgene arrangements (combination 6, DF6 iPSC lines).14Global analysis of gene expression confirmed the similarity of BM iPSCs to 5 hESC and 5 fibroblast iPSC lines. As shown in Figure 2A, BM iPSCs clustered together with hESCs and fibroblast-derived iPSCs, but were distant from the parental BM cells. Similarly, analysis of scatter plots shows a much tighter correlation of reprogrammed BM cells with hESCs than with parental cells (Figure 2B). The pluripotency of iPSC-derived cell lines was confirmed using a teratoma-formation assay with demonstration of derivatives of all 3 germ layers (Figure 1G). Whereas we detected an abnormal karyotype in one BM iPSC line, the majority of them maintained the normal karyotype (Figure 1H).

 

 

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3083304/bin/zh89991168710002.gif

Figure 2

Global analysis of gene expression in hESCs and iPSCs generated from BM, CB, and fibroblasts and their parental cells. (A) Pearson correlation analysis of global gene expression. (B) Scatter plots comparing the global gene-expression profiles of BM9 iPSC …

 

Although we used single-cell subcloning to isolate cells that had lost episomal plasmids in our previous reprogramming studies,14 our initial subcloning experiments with BM iPSCs demonstrated that all clones obtained at passage 15 were transgene-free (Figure 1I). Based on these experiments, we concluded that episomal plasmids were cured from BM iPSCs faster than we had previously thought. To analyze the kinetics of episomal plasmid loss, we extracted episomal DNA at different passage from 10 random BM iPSC lines. We found that episomal DNA was lost progressively, and was absent in some samples as early as passage 3. By passage 7, we did not detect any transgene in 7 of 10 lines checked with multiple pairs of primers (Figure 1K).

We applied a similar approach to the reprogramming of mononuclear cells of CB. Although the efficiency of reprogramming was much lower, we were able to obtain 6 CB iPSCs from approximately 3 × 106transfected CB mononuclear cells. By adding small-molecule thiazovivin21 to reprogramming cultures, we were able to increase the reprogramming efficiency of CB cells by more than 10 times (Figure 3B). We obtained a total of 22 CB iPSC lines from 2 reprogramming experiments. All CB iPSCs displayed the typical hESC phenotype and gene-expression profile (Figure 3A,G). Six selected CB iPSC lines showed pluripotency in the teratoma assay and were free of episomal vectors and genomic integration CB iPSCs (Figure 3E-F).

 

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3083304/bin/zh89991168710003.gif

Figure 3

Reprogramming of CB mononuclear cells with nonintegrating constructs. (A) All 22 CB iPSC lines express hESC-specific surface markers as indicated, and express OCT4, NANOG, and SOX2. iPSC lines checked are: CB iPSC1 to CB iPSC6, CB iPSCT1 to CB iPSCT10, …

Hematopoietic differentiation potential of blood-derived iPSCs

To test hematopoietic differentiation potential of blood-derived iPSCs, we used iPSC cocultured with OP9.22As we showed previously, hematopoietic differentiation from hESCs proceeds through the formation of a population of CD34+ cells, which includes CD34+CD43+ hematopoietic progenitors, CD34+CD31+CD43−endothelial cells, and CD34+CD31−CD43− mesenchymal cells. The 3 major populations of CD43+hematopoietic cells include CD235a/CD41a+ erythro-megakaryocytic progenitors and lin−CD43+CD45−and CD45+ multipotent progenitors.18 Earlier, we found that fibroblast-derived iPSCs and hESCs follow a very similar pattern of hematopoietic differentiation, although significant variation in blood-forming potential was observed between different iPSC clones. In addition, we noted that the generation of 4 iPSC clones was sufficient to ensure that at least one clone showed good hematopoietic differentiation potential.23 Testing of 4 BM iPSC lines revealed a similar differentiation pattern of BM iPSCs (Figure 4A). However, opposite our expectations, all 4 BM iPSCs produced fewer CD43+ hematopoietic progenitors than H1 hESCs or transgene-free fibroblast-derived iPSCs obtained using a similar method. Screening 5 additional BM iPSCs and 6 CB iPSCs failed to reveal a clone with higher differentiation potential, indicating that our blood-derived iPSCs were somewhat resistant to differentiating back to the blood in coculture with OP9 (Figure 4B). Because recent studies have suggested that lymphoid cell–derived iPSCs differentiate into blood less efficiently than CD34+ cell–derived iPSCs,7 we evaluated the rearrangement of TCR and IGH genes in our cells to determine whether our iPSCs originated from lymphoid cells. As shown in Figure 5, all 9 tested iPSC lines lacked rearrangements of TCR and IGH, indicating that their origin was non–B- or non–T-lymphoid cells.

 

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3083304/bin/zh89991168710004.gif

Figure 4

Hematopoietic differentiation potential of BM- and CB-derived iPSCs. (A) In coculture with OP9, blood-derived iPSCs generate a CD34+ population of cells with typical subsets including CD43+hematopoietic progenitors, CD31+CD43− endothelial cells, …

 

Figure 5

Analyses of TCR and IGH rearrangement in BM and CB iPSC lines. (A) PCR analyses of TCRB rearrangements. (B) PCR analyses of TCRG rearrangement. (C) PCR analyses of IGH rearrangements. FR indicates framework. (D) Specimen controls. M indicates the 50-bp…

 

Reprogramming of BM samples with CML

Reprogramming of neoplastic BM cells provides an opportunity to address the effect of oncogenes and patient-specific chromosomal abnormalities on the development of the leukemia phenotype in vitro. However the virus-based approach for reprogramming leukemic cells is highly undesirable because of genomic integration and background expression of reprogramming factors, some of which are oncogenes. Therefore, we applied episomal vectors to generate transgene-free iPSCs from a patient with CML in the chronic phase. We picked, expanded, and froze 50 CML iPSC lines from a single reprogramming. As with normal BM, we were able to generate multiple transgene-free CML iPSC lines with typical features of pluripotent stem cells. Two transgene-free CML iPSC lines were selected and characterized (Figure 6). RT-PCR analysis revealed that both CML iPSCs retained typical BCR-ABL fusion (Figure 6H). Moreover, the CML iPSCs were found to have a complex karyotype with a 4-way translocation between chromosomes 1, 9, 22, and 11 that was present in the patient BM (Figure 7). CML iPSC lines lacked rearrangement of TCR or IGH, indicating derivation from nonlymphoid cells (Figure 5). After hematopoietic differentiation, these cell lines generated CD43+ hematopoietic progenitors, which included typical subsets of CD235a/CD41a+ erythro-megakaryocytic and lin−CD34+CD43+CD45+/− multipotent progenitors (Figure 6E). In a colony-forming assay, these differentiated CML iPSCs formed all types of hematopoietic colonies, including granulocyte, erythrocyte, monocyte, megakaryocyte and giant granulocyte-macrophage colonies (Figure 6F).

 

Figure 6

Generation of iPSCs from BM samples from a patient in the chronic phase of CML. (A) Flow cytometric analysis of hESC-specific marker expression in CML iPSC15 and CML iPSC17. (B) Bright-field image demonstrating typical hESC morphology of CML iPSCs growing …

 

Figure 7

Karyograms of BM cells from a patient with CML and the 2 iPSCs derived from these cells. Top left panel shows spectral karyogram of CML iPSC15. SKY analysis demonstrates the 4-way translocation between chromosomes 1, 9, 11, and 22, shown here by classification-colored …

 

Current methods for blood reprogramming rely on use of genome-integrating viruses and require several rounds of viral infection. Our data show that iPSC lines free of any transgene or vector sequence could be obtained using EBV-based episomal vectors. The efficiency of reprogramming blood cells by this method was at least 100 times higher than that of fibroblasts and was similar or higher to reported reprogramming efficiency using virus-based methods. Although previous studies have demonstrated the generation of iPSCs from blood using CD34+ cells3,4 or T cells,5–7 these methods require the isolation of progenitors or mature blood cells before reprogramming. We demonstrated that successful reprogramming could be achieved using just 106-107 mononuclear cells from CB or BM without any additional purification steps. Moreover, iPSCs with rearranged TCR or IGH may be undesirable for potential therapeutic applications and modeling of lymphoid development, because prearranged antigen-receptor genes are expressed precociously in early hematopoietic progenitors, leading to abnormal hematopoietic and lymphoid development and predisposition for lymphomas.24 A selective reprogramming of nonlymphoid cells using our method makes it possible to obtain iPSCs lacking TCR and IGH rearrangements using nonseparated mononuclear cells. Reprogramming of blood cells with episomal vectors occurs more rapidly than fibroblasts and is associated with a loss of episomal DNA in the majority of iPSC lines after 7 passages, thus eliminating the requirement for extensive additional subcloning steps. Human BM and CB represent the most accessible sources of somatic cells, with extensive and diverse archived samples available. Successful reprogramming of frozen blood samples containing less than 107 mononuclear cells in the present study clearly demonstrates the applicability of the described method for the generation of transgene-free iPSCs without rearranged antigen-receptor genes from archived samples of normal and diseased blood cells for studies of hematopoietic development, blood disease pathogenesis, and drug screening, and potentially for therapeutic purposes.

 

 

397 Induced Pluripotent Stem Cell Model of Chronic Myeloid Leukemia Revealed Olfactomedin 4 As a Novel Survival Factor for Primitive Leukemia Cells

Program: Oral and Poster Abstracts
Type: Oral

Session: 631. Chronic Myeloid Leukemia: Biology and Pathophysiology, excluding Therapy: Strategies to Circumvent Therapy Resistance

Kran Suknuntha, MD, PhD1*, Yuki Ishii, PhD2*, Kejin Hu, PhD3*, Mcintosch Brayan, PhD4*, David T. Yang, MD5,…, Jean YJ Wang, PhD2*, James Thomson, PhD, DVM6* and Igor Slukvin, MD, PhD

56th ASH Meeting 2014   https://ash.confex.com/ash/2014/webprogram/Paper70688.html

CML is a myeloproliferative disorder characterized by unregulated growth of predominantly myeloid cells, and their subsequent accumulation in the bone marrow and peripheral blood. CML originates in hematopoietic stem cells (HSCs) with t(9;22)(q34;q11.2) translocation, which causes the constitutively expression of the BCR-ABL kinase driving the expansion of leukemic progeny. Ex vivo cultures of CML-derived cell lines and primary CML cells, ectopic expression ofBCR-ABL in CD34+ cells and mouse models have provided important insights into CML pathogenesis and led to the development of targeted therapy for this neoplastic disease with BCR-ABL thyrosine kinase inhibitor (TKI), imatinib. Despite these achievements, in many cases CML remains incurable because of innate resistance of CML leukemia stem cells (LSCs) to TKI. Thus, a definitive cure for leukemia requires identifying novel therapeutic targets to eradicate LSCs. However, the rarity of LSCs within the pool of malignant cells remains a major limiting factor for their study in humans.  Recently we generated transgene-free iPSCs from the bone marrow mononuclear cells of a patient in the chronic phase of CML (CML15 iPSCs and CML17 iPSCs) and showed that these iPSCs capture the entire genome of neoplastic cells, including the unique 4-way translocation between chromosomes 1, 9, 22, and 11 that was present in the patient bone marrow (BM) (Hu et al., Blood 2011). By differentiating CML iPSCs back to the blood we were able to generate iCD34+primitive hematopoietic cells with typical LSC properties, including HSC phenotype (lin-CD34+CD45+CD90+CD117+CD45RA-RholowALDHhigh), adhesion defect, increased long-term survival and proliferation, and innate resistance to TKI imatinib. By analyzing transcriptome of CML and normal BM iCD34+ cells treated or non-treated with imatinib we discovered OLFM4 as top-ranking gene, which is selectively upregulated by imatinib in CML, but not normal BM iCD34+ cells. Using siRNA, we demonstrated that OLFM4 knockdown potentiate imatinib-induced apoptosis and suppression of CFCs in iCD34+ cells, thereby indicating that OLFM4 is involved in regulation of imatinib resistance and survival of de novo generated primitive CML cells. To find out whether findings obtained using iCD34+ cells can be translated to somatic cells, we evaluated the expression and functional role of OLFM4 in CD34+ cells obtained from parental bone marrow and bone marrow from the several other CML patients in the chronic phase. Using immunohistochemistry and RT-PCR we confirmed OLFM4 expression in lin-CD34+ and CD34- bone marrow cells from patients. Knockdown OLFM4 with siRNA in somatic CML lin-CD34+ potentiated imatininb-induced CFC suppression, abrogated LTC-ICs and engraftment of lin-CD34+ cells in NSGW41 mice,  thereby indicating that OLFM4 is critical for survival of CML LSCs.  In summary, we showed that reprogramming leukemia cells to pluripotency and then differentiating them back into blood cells can be used as a novel approach to produce an unlimited number of primitive hematopoietic cells with LSC properties and identify of novel LSC survival factors and drug targets. We validated this approach by demonstrating the successful application of the iPSC-based platform to discover OLFM4 as a novel LSC survival factor in patients in the chronic phase of CML.