Objective: The present study aimed to establish an induced pluripotent stem cell (iPSC) line from acute myelogenous leukemia (AML) cells in vitro and identify their biological characteristics. Methods: Cells from the AML-infiltrated skin from an M6 patient were infected with a lentivirus carrying OCT4, SOX2, KLF4 and C-MYC to induce iPSCs. The characteristics of the iPSCs were confirmed by alkaline phosphatase (ALP) staining. The proliferation ability of iPSCs was detected with a CCK-8 assay. The expression of pluripotency markers was measured by immunostaining, and the expression of stem cell-related genes was detected by qRT-PCR; distortion during the induction process was detected by karyotype analysis; the differentiation potential of iPSCs was determined by embryoid body-formation and teratoma-formation assays. ALP staining confirmed that these cells exhibited positive staining and had the characteristics of iPSCs. Results: The CCK-8 assay showed that the iPSCs had the ability to proliferate. Immunostaining demonstrated that iPSC clones showed positive expression of NANOG, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. qRT-PCR results revealed that the mRNA expression of Nanog, Lin28, Cripto, FOX3, DNMT3b, DPPA2, and DPPA4 significantly increased in iPSCs. Karyotype analysis found no chromosome aberration in the iPSCs. The results of the embryoid body-formation and teratoma-formation assays indicated that the iPSCs had the potential to differentiate into all three germ layers. Conclusion: Our study provided evidence that an iPSC line derived from AML cells was successfully established.
The reprogramming factors could increase the expressions of pluripotent genes and CD gene in HL-60 cells.
Background The hematopoietic potential from induced pluripotent stem cells (iPS) has been explored for some years. However, the direct reprogramming from the blood cells to hematopoietic progenitors bypass the pluripotency is even more attractive for the clinical practice. Methods The lentiviral vector encoding human reprogramming factors (Oct-4, Sox2, c-Myc, Klf4FOSCK) were transfected along with packaging plasmid pCMV-dR8.91 and envelope plasmid pCMV-VSV-G in 293T package cells to produce lentiviral particles. The construction was then confirmed by RT-PCR and restriction enzyme digestion anaysis, and the lentivirus titer was determined using GFP/DAPI based on cell count by Image J software. Primary mouse embryo fibroblast (PMEF) feeder cells were isolated and prepared from CF-1 inbred mouse strain to help maintain pluripotency and to provide a cellular matrix for stem cells growing. Lastly, the lentivirus carrying OSCK factors was used totransduce HL-60 cells in three consecutive rounds of spin-infection on the prepared feeder layers with an interval of 12 hours, while the lentivirus carrying GFP served as a negative control. The cell clusters were picked based on morphology and alkaline phosphatase(AP) staining. The stem cell properties were tested by the expression of cell surface antigens using Flow cytometry. and the mRNA expression of OCT4, SOX2, C-MYC,KLF4,etc. by QT-PCR. Results The lentiviral particles were successful packaged and used to infect HL-60 cells. We occasionally observed some Human embryonic stem cells (hES) cell-like colonies in between the cells around 14 days after infection. These cell colonies also showed similarity to hES cells in feeder dependency. Detection of cell surface CD34 by FCM showed that HL-60 cells were switched to be a hematopoietic fate, expression of CD34 from 1.77% to 98.42%. Simultaneously, expressions of the myeloid antigen CD13, CD117 decreased. The gene expressions of OCT4, SOX2 indicated that the exogenous gene were down-regulation or silence while the endogenous gene were up-regulation. However, the cell colonies can survive only for a short time which might due to the absence of the survival factors they require or the first hematopoietic microenvironment, like the yolk sac. Conclusion After being reprogrammed, the HL-60 cell derived colonies showed the similarity to hES cells in morphology, feeder dependency and the expression of stem cell antigens. However, they may need an appropriate microenvironment at different time points to be programmed into primitive hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.
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