Study On The Generation And Characterization Of Pediatric Leukemia Patient's Specific IPSCs Like Cell Line

Background and aimsAcute leukemia is the most common hematological malignancy in children, accounting for about35%of pediatric malignancies. Due to the application in the risk stratification, advances in chemotherapy regimens and improvement in supportive care, long-term disease free survival rates for ALL and AML have reached to70%-80%and50%, respectively [1-3]. However, there are still some patients with early relapse or treatment failure. HSCT (hematopoietic stem cell transplantation) is the best alternative way to cure the disease for these patients. Successful HSCT is closely related to the HLA matching. Graft-versus host disease (GVHD) would be happen if the HLA between donors and recipients are not well matched, which would jeopardize the patients*lives. So, the treatment of choice for these patients is matched related donor HSCT. However, in this country,80%of the children lack this kind of donor, so that the pros and cons of alternative approaches for those patients must be carefully weighed on a case-by-case basis. Although autologous-HSCT (auto-HSCT) can be used in selected children with ALL or AML, the disadvantage of auto-HSCT is higher leukemia recurrence rates. One of the main reasons for leukemia recurrence after transplantation is due to the residual leukemia cells or even with rare leukemia stem cells contaminated in autografts, which would be reinfused into the patient's blood stream along with the transplant causing leukemia relapse after transplantation. Thus, looking for a new kind of auto-stem cells without leukemia cell contamination will become a very important issue in decreasing the leukemia recurrence after transplantation and holds significance in clinical application.iPSCs (induced pluripotent stem cells) are recently found to be a stem cell source somewhat similar to embryo stem cells (ESCs) in certain characteristics i.e. pluripotency, which can be induced to differentiate into not only immature hematopoietic stem or progenitor cells but also mature red blood cells, monocytes and NK cells etc[7-11]. Generation of leukemia patient's specific iPSCs may provide a new approach for leukemia treatment.In order to successfully reprogram patient's specific iPSCs, several issues should be considered:(1) appropriate vector to be used;(2)appropriate transcription factor gene(s) to be transfected;(3) appropriate target cells should be chosen;(4)appropriate culture media for stem cell maintenance and differentiation should be used.Using viral vectors to deliver the defined genes is the most commonly used method in genetic engineering and somatic cell reprogramming. However, the disadvantages of the viral vector gene delivery method are the integration of viral vector backbone sequences into the genomic DNA and reactivation of residual transgene expression causing malignancies[23]. So many studies on successfully reprogrammed somatic cells into iPSCs with non-viral or non-integrated vectors have been reported[24-27].However, non-universal vector has been accepted to reprogram somatic cells.Regarding the number of transcription factor genes needed to be transfected for effectively reprograming the somatic cells, there are many studies focusing on this issue. It has been initially reported that generation of iPSCs by using a combination of four transcription factors either with Oct4, Sox2, Klf4and cMyc or Oct4, Sox2, Lin28and Nanog[4-6]. Since then, many studies have showed that the somatic cells can be successfully reprogrammed into a pluripotent state by ectopic expression of three (Oct4, Sox2and Klf4) or two (Oct4and Sox2or K1f4) or even with only single transcription factor Oct4[28-32] Based on the above observations, it can be clearly found out that Oct4is the pivotal and irreplaceable transcription factor in somatic cell reprogramming.Using single Oct4gene transfection, the induction of iPSCs in animal systems has been successful, however, it has not been reported in cells of human origin.Selection of target cells for reprogramming is another important issue. They should be easily obtainable and easy to be cultured for sufficient period of time in vitro with proliferative capacity. They include lymphocytes, monocytes, NK cells and fibroblast cells from peripheral blood, or fibroblasts from skin, or mesanchymal stem cells from bone marrow, or endothelial cells from blood vessels. Although successfully reprogrammed human cord blood derived endothelial cells, mobilized peripheral blood stem/progenitor cells and T lymphocytes into iPSCs have been documented［15-17］ generation of iPSCs from leukemia patient's somatic cells has been rarely reported and reprogrammed pediatric leukemia patient's somatic cells into iPSCs has not yet been reported in the literature. LP MUELLER et al successfully isolated MSCs from chemotherapy-exposed BM in sufficient number and quality for potential clinical applications in chemotherapeutically treated patients[22] Thus, it is possible to use the BM derived MSCs from leukemia patients as target cells to reprogram patient's specific iPSCs.The aim of this study was trying to reprogram the BM derived MSCs from leukemia children into iPSCs, to analyze their biological characteristics and to explore the possibility of iPSCs differentiation into CD34+and/or CD45+hematopoietic cells. We hope that our results can provide useful information to the studies involving in the personalized treatment of leukemia in the future.Our study mainly includes the following three parts:(1) Observation of iPSCs related gene expression in leukemia cells, subsequently by cloning of iPSCs related genes and construction of recombinant vectors.(2)Isolation, culture, identification of BM derived MSCs and study on their biological characteristics.(3)Generation of iPSCs like cells from human BM-MSCs and study on their biological characteristics. Material and Methods1Expression of iPSCs related genes in leukemia cells, cloning of iPSCs related genes and construction of recombinant vectors.1.1Study on the expression profiles of iPSCs related genes in leukemia cell lines and primary leukemia samples. RT-PCR and Real-Time PCR were used to examine the expression of iPSCs related genes including Oct4, Sox2, cMyc, K1f4, Lin28and Nanog in9leukemia cell lines (Raji, Nalm-6, Molt-3, Molt-4, HL60, K562, U937, Meg-01and KGla) and53primary leukemia samples.9bone marrows from children with immune thrombocytopenia purpura (ITP) were used as controls after obtaining the informed consents from parents or guardians.1.2Cloning and sequencing analyses of iPSCs related genes. According to the results of expression profiles of iPSCs related genes, ORF sequence of iPSCs related genes were amplified by RT-PCR. The correct sequences of the target genes were cloned into pGEM?-T vector.1.3Construction of recombinant vectors of iPSCs related genes. Through restriction endonuclease reaction and ligation reaction, the ORF sequences of iPSCs related genes in pGEM?-T vector were inserted in the eukaryotic expression vector pcDNA3.1.2Isolation, culture, identification of BM derived MSCs and study on their biological characteristics.2.1Culture and identification of BM derived MSCs. Whole bone marrow culture and density gradient centrifugation methods were combined to isolate MSCs, and the surface antigen expressions of MSCs were examined by flow cytometry (FCM).2.2Analysis on the embryonic stem cells (ESCs)-like biological characteristics of hBM-MSCs. RT-PCR and Real-Time PCR were used to detect the expression of iPSCs related genes in MSCs. FCM and immunofluorescence histology technique were used to examine the expression of pluripotency markers such as SSEA4, TRA-1-60and TRA-1-81and so on. 3Generation of iPSCs like cells from BM-MSCs and study on their biological characteristics.3.1Generation of MSCs derived iPSCs like cells with stable expression of Oct4. The recombinant plasmid pcDNA3.1/Oct4was transfected into MSCs and the cells with stable expression of Oct4were identified by subcloning. RT-PCR, Real-Time PCR, FCM, immunofluorescence histology and western blot were used to detect the expression of Oct4in both RNA and protein levels.3.2Study on the biological characteristics of MSCs with stable expression of Oct4. FCM and immunofluorescence histology were used to detect the expression of pluripotency markers of ESCs. RT-PCR and Real-Time PCR were used to examine the expression of Sox2and Nanog.3.3Analysis of the transient transfection efficiency with different combination of transcription factors. Four different combinations (each combination with a green fluorescent protein (GFP)) were transfected into MSCs, respectively. After two rounds of co-transfection, the expression level of pluripotency markers of ESCs was examined by FCM. Transient transfection efficiency was compared according to the expression level of GFP.3.4Identification of MSCs derived iPSCs like cells and study on their biological characteristics. Immunofluorescence histology, FCM and western blot were used to examine the protein expression of pluripotency markers. Alkaline phosphatase staining, telomerase activity, embryoid body (EBs) and teratoma formation were used to identify the characteristics of MSCs derived iPSCs like cells.3.5Hematopoietic differentiation of MSCs derived iPSCs like cells. MSCs derived iPSCs like cells were induced to differentiate into CD34+and/or CD45+in DMEM medium supplementd with a cocktail of hematopoietic growth factors including IL-3, IL-6, Flt-3Ligand, SCF, G-CSF and BMP4. FCM was used to detect the protein expression of surface antigen CD34and/or CD45while RT-PCR was used to detect the expression of CD34and/or CD45genes. Results1Expression of iPSCs related genes in leukemia cells, cloning of iPSCs related genes and construction of vectors.1.1Study on the expression profiles of iPSCs related genes in leukemia cell lines and primary leukemia samples. RT-PCR results showed that9/9cell lines expressed cMyc and Lin28genes,3/9(U937,K562and HL60) expressed Klf4gene,2/9(Molt-3and K562) expressed Oct4gene,2/9(KG1a and Meg-01) expressed Nanog gene and3/9(KG1a,Meg-01and Raji) dimly expressed Sox2gene. In primary leukemia samples, cMyc, Klf4, lin28, Sox2, Oct4and Nanog were detected in53/53,16/53,20/53,11/53,7/53and3/53of leukemia samples, respectively.Real Time PCR showed that cMyc was over-expressed in leukemia cell lines (N=9) compared to its expression in control (N=9)(P=0.012), whereas Klf4(P=0.001) and Sox2(P=0.010) were down-regulated, respectively. No significance was found between leukemia cell lines and control in terms of the expression levels of Nanog (P=0.08), Lin28(P=0.882) and Oct4(P=0.456). The mean expression levels of cMyc, Klf4, Sox2, Nanog, Lin28and Oct4in9leukemia cell lines were4.63±8.51%,0.45±0.51%,0.014±0.023%,0.004±0.006%,0.083±0.085%and0.45±0.51%of internal reference (3-actin, respectively. No significance was found between AML and ALL cell lines in terms of expression levels of cMyc (P=0.063), Klf4(P=0.111) and Sox2(P=0.286)Compared with the control samples (N=9), cMyc was over-expressed in all primary leukemia samples tested (N=53)(P=0.001), whereas Klf4(P=0.04) was down-regulated in those samples. No significance was found between control and primary leukemia samples in terms of Sox2(P=0.266), Nanog (P=0.237), Lin28(P=0.769) and Oct4(P=0.93) expression. The mean expression levels of the six genes were3.49±3.78%,0.83±0.38%,0.33±1.29%,0.83±0.38%,0.009±0.036%and0.83±0.38%of the internal reference (3-actin, respectively. There was no statistical significance in the expression of cMyc (P=0.803) and Klf4(P=0.098) between AML and ALL leukemia samples.1.2Cloning and sequencing analyses of iPSCs related genes. cMyc gene with1320bp and Lin28gene with630bp were amplified from the cDNA of KGla, Nalm-6and Meg-01, respectively. The sequences of PCR products were found to be exact the same as the theoretical sequences of cMyc and Lin28genes. Nanog gene with918bp was amplified from the cDNA of KGla and Meg-01, however, there were six base mutations (3/6nonsense mutations and3/6sense mutations) in the Nanog sequence. Klf4gene with1413bp and Sox2gene with954bp were amplified from the cDNA of U937and teratoma, respectively. The PCR product sequences of Klf4and Sox2genes were the same as the corresponding theoretical sequences from GeneBank. Oct4gene with1083bp was amplified from the cDNA of liver and testicular tumor tissue, however, there were many base mutations in the amplified sequence. The three sense-mutation bases were reversed using SOE PCR. The correct sequence of each iPSCs related gene was cloned into pGEM?-T vector, respectively.1.3Construction of recombinant vectors of iPSCs related genes.By means of restriction endonuclease reaction and ligation reaction, the ORF sequences of iPSCs related genes being cloned into pGEM?-T vector were inserted in the eukaryotic expression vector pcDNA3.1. The synthetic Oct4gene sequence was inserted in the eukaryotic expression vector pcDNA3.1and peGFP-N1. The correct recombinants were identified by sequencing.2Isolation, culture, identification of BM derived MSCs and study on their biological characteristics.2.1Culture and identification of BM derived MSCs.FCM results showed that the first nine generations of hBM-MSCs highly expressed CD29, CD105, CD166and CD44, the mean positive rates (n=3) were95.89±2.20%,97.40±1.56%,96.94±1.45%and95.67±0.89%, respectively, while only the first four generations of hBM-MSCs highly expressed CD90with a mean positive rate of93.88±5.08%. After the fifth generation, CD90was only dimly expressed on hBM-MSCs. On the other hand, hBM-MSCs did not express CD34, CD38, CD14, CD19and HLA-DR, and the positive rates of the five surface antigens were all less than5%.2.2Analysis on the embryonic stem cells (ESCs)-like biological characteristics of hBM-MSCs.PCR and immunofluorescence histology results showed that the MSCs endogenously expressed Sox2. The mean positive rate of Sox2was16.70%by FCM. MSCs rarely expressed other ESCs pluripotent markers such as Oct4Nanog,SSEA4,TRA-1-60and TRA-1-81with the positive rates of all less than5%.3Generation of iPSCs like cells from BM-MSCs and study on their biological characteristics.3.1Generation of MSCs derived iPSCs like cells with stable expression of Oct4. The positive rates of Oct4in three subcloned MSCs were49.99%,70.75%and94.66%, respectively, while that in the control MSCs without Oct4transfection was only1.72%. Immunofluorescence histology and Western Blot analyses showed positive Oct4protein expression with its molecule wight of approxiamately45kDa. Real-Time PCR analysis showed that the relative mRNA expression of endogenous and total Oct4were up-regulated6.23and7.20folds, respectively, compared to its expression in control MSCs.3.2Study on the biological characteristics of MSCs with stable expression of Oct4. FCM analysis showed that the expressions of Nanog and Sox2in MSCs-Oct4cells were increased from0.61%to40.61%and from12.33%to96.21%, respectively. Whereas, the positive rates of SSEA-4, TRA-1-60and TRA-1-81were increased from8.21%to90.77%, from1.72%to64.62%and from2.53%to71.70%, respectively. Compared with the expression in control MSCs, the relative expressions of Nanog and Sox2mRNA were increased by8.68and2.13folds, respectively. Immunofluorescence histology analysis confirmed the protein expression of Nanog and Sox2in MSCs-Oct4cells.3.3Analyses of the transient transfection efficiency with different combinations of transcription factors. Transfection efficiency was decreased along with the increase of the numbers of transcription factors. Single Oct4transcription transfection had the strongest green fluorescence while co-transfection with6transcription factors (6TF) presented the weakest fluorescence. The positive rates of SSEA-4with6TF,5TF,3TF and1TF transfection were86.10%,74.16%,75.79%and89.41%, respectively while those of TRA-1-60after transfection with the same four types of combinations were32.66%,33.20%,38.55%and42.77%, respectively, and the positive rates of TRA-1-81after transfection with6TF,5TF,3TF and1TF were17.24%,39.25%,34.07%and64.97%, respectively.3.4Identification of MSCs derived iPSCs like cells and study on their biological characteristics. The MSCs derived iPSCs like cells had a clear boundary and with a high nucleus to cytoplasm ratio, which was positive both for alkaline phosphatase and telomerase activity. The MSCs derived iPSCs like cells could form EB ex vivo and expressed three germ layer genes such as SPARC, Brachyury, TBX20, TUBB3and WNT1. The positive rates of SSEA-4, TRA-1-60and TRA-1-81on day21post-differentiation were decreased from90.77%,64.62%and71.70%to4.25%,3.07%and1.69%, respectively. The MSCs derived iPSCs like cells could form tumor in null mouse, which were recognized to be in the undifferentiated stem cell state after HE staining.3.5Hematopoietic differentiation of MSCs derived iPSCs like cells.The MSCs derived iPSCs like cells could differentiate into CD34positive and CD45positive cells after co-culture with a cocktail of hematopoietic growth factors including IL-3, IL-6, Flt-3Ligand, SCF, G-CSF and BMP4. The positive rates of CD34and CD45on day20post-differentiation culture were26.22%and26.03%, respectively. Furthermore, the expression of CD34and CD45genes could be detected by RT-PCR in mRNA level. CFU-GM like colony formation was observed on day14in methyl cellulose semisolid culture.Conclusions1. Pediatric leukemia patient's bone marrow derived MSCs can be successfully reprogrammed into iPSCs like cells. 2. Single Oct4gene introduced into human bone-marrow derived MSCs can induce the expression of other pluripotency-related genes such as Sox2and Nanog. Moreover, single Oct4can reprogrammed human BM derived MSCs into iPSCs like cells.3. The MSCs derived iPSCs like cells could differentiate into CD34+and CD45+cells after co-culture with a cocktail of6growth factors ex vivo.