Reprogramming Normal and Cancerous Human Cells Lines into Human Induced Pluripotent Stem Cells - Research Paper Example

?Reprogramming Normal and Cancerous Human Cells Lines into Human Induced Pluripotent Stem Cells by Co-electroporation with Living Xenopus laevis FrogOocytes Sergei Paylian Bioquark, Inc., P.O. Box 622, Lake Delton, WI 53940 USA Corresponding author: Tel.:+6084322227; fax: +6082530530, e-mail: info@bioquark.com Abstract Unique information on the ability of some amphibians to regenerate severely damaged tissues though the de-differentiation and re-differentiation of cells is encoded in the genetic makeup of amphibian oocytes. Using electroporation, it is possible to activate the natural reprogramming potential of living Xenopus laevis oocytes and pass it on to donor cells placed with eggs in one electroporation chamber. We demonstrated that co-electroporation at 150 v/cm / 25 µF of mature oocytes with ~105 cells/ml of suspension of various normal and cancerous human cell lines, such as bone marrow stromal cells, CD4+ T-lymphocytes, cervical carcinoma (HeLa) cells and breast adenocarcinoma (MCF-7) cells, reprograms donor cells into iPSc-like cells, which form colonies on irradiated MEF feeders. The iPSc-like cells generated by this study resemble human embryonic stem cells in colony morphology and expression of stem cell-associated transcription factors, including Oct3/4, Nanog, SOX-2, Rex-1, TRA-1-60, SSEA-1, and SSEA-4. The efficacy of reprogramming CD4+ lymphocytes into iPSc-like cells was 23.4 ± 3.5 %. Advantageously, this new method obviates the use of retroviral or lentiviral gene delivery vectors and other “non-parental” reprogramming approaches and may hold great promise as a strategy for the rapid and inexpensive production of human autologous stem cells. Keywords: human iPS cells, reprogramming, frog oocytes, Co-electroporation 1. Introduction The low efficacy of de-differentiation of human somatic cells into human induced pluripotent stem cells (iPSc) and the non-autologous nature of the final product is one of the major problems plaguing contemporary methods of reprogramming (RP). The low rate of conversion hinders investigations of the mechanisms by which reprogramming occurs. According to (Meissner et al., 2007; Condic and Rao, 2008), RP efficacy could be as low as 0.5 % with standard, four-factor retroviral RP. Markoulaki et al., (2009) found RP efficacy to be as low as 0.98 %-2.34 % when adding two more reprogramming factors. Recently, new, non-viral approaches for improving RP efficacy were reported. These methods include the use of recombinant proteins (Zhou et al., 2009); the use of DHP-derivative (novel anti-oxidant) and low oxygen-tension conditions (Jee et al., 2010). We believe that the low efficacy of reprogramming may be explained by the fact that the RP tools currently used in studies differ considerably from the delicate reprogramming machinery naturally present in living organisms. Reprogramming events observed in mammalian somatic cells induced by Xenopus laevis egg extracts were described by Miyamoto and colleagues (Miyamoto et al., 2007). At Bioquark, Inc., we developed a novel technique that allows the utilization of the natural reprogramming potential of living Xenopus laevis oocytes. We tried to generate human iPS cells in a consistent, safe and controllable way. Our system is fast, efficient, free of ethical controversy, highly reproducible, controllable, effects real time reprogramming, segregates human and amphibian components and simultaneously activates mutual semiochemical interactions and is able to produce partially reprogrammed cells which may represent the correct transitional point for successful re-differentiation or trans-differentiation reprogramming. This new experimental protocol utilizes the electroporation of living Xenopus laevis oocytes as a powerful RP tool through co-electroporating living Xenopus laevis oocytes with human donor cells in one electroporation chamber. We refer to this new procedure as “BQ-activation.” In our studies, we demonstrated that co-electroporation, with pulses of 150 v/cm / 25 µF / 7, of living Xenopus laevis oocytes with different normal and cancerous human cells lines, such as bone marrow stromal cells, CD4+ lymphocytes, cervical carcinoma (HeLa) cells and breast adenocarcinoma (MCF-7) cells, reprograms donor cells into iPSc-like cells, which form colonies on irradiated MEF feeders. The iPSc-like cells produced with this protocol resemble human embryonic stem cells in colony morphology and the expression of stem-cell associated transcription factors Oct3/4, Nanog, SOX-2, Rex-1, TRA-1-60, SSEA-1, and SSEA4. Importantly, this new method obviates the use of retroviral or lentiviral gene-delivery vectors and other “non-parental” reprogramming approaches and may hold great promise as a means of rapid and inexpensive production of human autologous stem cells. 2. Materials and Methods 2.1. Cell Lines Human Bone Marrow Stromal Cells (BMSCs) were provided by Tulane University Center of Gene Therapy. GFP-expressing BMSCs (BMSCGFP) were stably transfected at the same facility. Prior to release, two trials of frozen, passage-1 cells were analyzed over three passages for colony forming units, cell growth, and differentiation into fat, bone, and chondrocytes. Human Peripheral Blood CD4+ T-lymphocytes (CD4TLs). Pathogen-free poietics® CD4TLs were purchased. T-Lymphocytes were maintained as a cell suspension in T25 culture flasks at 37 °C and 5 % CO2 in 5 ml of lymphocyte growth medium-3 which was specially developed for the growth and support of human lymphocytes and dendritic cells. LGM-3® was supplemented with 10 % FBS, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 1 % pen/strep, and 50 ng/ml recombinant human Interleukin-4. Human cervical carcinoma (HeLa) cells were grown at 37 °C and 5 % CO2 in T25 flasks filled with 5 ml of Eagle’s essential medium supplemented with 10 % FBS, 1 mM sodium pyruvate , 0.1 mM NEAA, and 1 % pen/strep. Human breast adenocarcinoma (MCF-7) cells were maintained in Eagle’s Essential Medium supplemented with 10 % FBS, 1 mM sodium pyruvate, 0.1 mM NEAA, 1 % pen/strep, and 0.01 mg/ml recombinant human insulin. 2.2. Preparation and Maintenance of Xenopus laevis Oocytes All experiments were carried out in accordance with Institutional Animal Care and Use Committee (IACUC) policies. South African clawed, egg-bearing frogs (Xenopus laevis) were adopted to the new environment for two weeks at ?18 oC using a 12 / 12-hour light/dark cycle and were kept in carbon-filtered water supplemented with 13.3 g/gallon sodium monochloride (Rand and Kalishman, 2001). The bags of the ovaries were surgically removed from the frog and placed into an oocyte washing (OW) solution containing 82.5 mM NaCl, 5.0 mM HEPES, 2.5 mM KCl, 1 mM MgCl2, 1.0 mM Na2HPO4, and 0.5 % pen/strep titrated to pH 7.4. Bags containing oocytes were disrupted with fine forceps, followed by multiple rinses in OW. After a final rinse, the remaining follicular cell layers were digested by placing material into a 0.2 % collagenase type II solution for one hour or more at room temperature. Defolliculated oocytes were rinsed in the OW solution and then placed for overnight incubation in a fresh holding buffer (HB) containing 5 mM NaCl, 5.0 mM HEPES, 2.5 mM KCl, 1 mM MgCl2, 1.0 mM Na2HPO4, 0.5 % pen/strep, 1.0 mM CaCl2, 2.5 mM pyruvate, and 5 % heat-inactivated horse serum titrated to pH 7.4. Recovered oocytes in the final stage of maturity were collected in sterile 6-well cell culture clusters prefilled with an HB solution and then incubated at 17 ?C in a low-temperature incubator for 24 hours before they were collected for electroporation experiments. 2.3. Co-electroporation of Xenopus laevis Oocytes with Donor Cells. Forty to fifty fresh Xenopus Laevis oocytes were placed in sterile Gene Pulser electroporation cuvettes prefilled with 400 ?l of serum-free DMEM containing 1.0 x105 - 1.5x105 cells/ml of specimen cells in suspension. Only cells with a viability of above 90 % were used for the experiments. Cuvettes were filled to 800 ul with serum-free DMEM and then placed into the shocking chamber. Co-electroporation of frog oocytes with the suspension of human donor cells was conducted using the following electroporation parameters: 150 v/cm / 25 µF / 7 pulses, with time constant at 0.5 - 0.7 msec. After electroporation, cuvettes containing oocytes and donor cells were placed in a low-temperature incubator at 17 oC for three hours to recover. Subsequently, donor cells were removed from the electroporation cuvette and transferred to T25 culture flasks containing iMEF feeder cells and ES-cell medium. Activated cells were left undisturbed for two days, and then the medium was refreshed. 2.4. Culturing of Primary iPS Cells BQ-activated donor cells were cultured on iMEF feeder cells in 0.1 % gelatin-coated T25 culture flasks containing 5 ml of specially formulated Embryomax® DMEM culture medium. Medium was supplemented with 15 % FBS, 1 mM sodium pyruvate, 0.1 mM NEAA, 1 % pen/strep, 100 ?M beta-mercaptoethanol, and 1000 U/mL ESGRO®. We found that 1000 U of ESGRO® per 1.0 mL of tissue culture media is required to maintain embryonic stem (ES) cells with a stem-cell phenotype. After formation of clusters, iPS cells were separated from the feeder cells using the differential sedimentation technique previously described by Doetschman (Doetschman, 2002). The Doetschman sedimentation method results in the removal of more than 99 % of contaminating feeder cells from the iPS cell suspension. 2.5. Subculturing of Primary iPS Cells on Feeder Cells and Feeder-Free Substrates Primary human iPS cells were plated on T25 culture flasks containing either iMEF feeder cells or feeder-free StemAdhere™ pluripotency substrate. Subcultured iPS cell were cultured in a NutriStem™ medium. 2.6. Cryopreservation of Human iPS Cells For the cryopreservation of human iPSc-like cells, we used the standard slow-cooling freezing method. One ml of iPSc-like cells was gently resuspended in 1.5 ml containing 0.5 mL of 2X hES cell freezing medium (60 % FBS, 20 % hES cell culture medium, and 20 % DMSO). Cryovials were transferred to 5100 Cryo 1 °C Freezing Container, refrigerated at -80 °C overnight and then rapidly transferred to liquid nitrogen. 2.7. Alkaline Phosphatase Staining and Immunocytochemistry An alkaline phosphatase (AP) substrate solution was prepared using Vector® Blue Alkaline Phosphatase Substrate Kit III as per the manufacturer’s instructions. All immunocytochemistry studies were carried out at room temperature. All populations of iPS cells in T25 culture flasks were stained and immunochemistry done. The 2.5 µg/ml concentrations of primary and secondary antibodies and normal sera used in each staining were Oct3/4, NANOG, Sox-2, TRA-1-60, SSEA-1, Rex-1, goat-anti mouse lgM-TR, donkey-anti-mouse lgG-FITC, donkey anti-goat IgG-FITC, donkey anti-goat IgG-TR, normal donkey serum, and normal goat serum. DNA staining was performed using DAPI. Calculation of the Efficacy of Reprogramming of Human CD4+ Lymphocytes We calculated the efficacy of reprogramming only human CD4+ lymphocytes. We demonstrated that, at early stages (12 h – 24 h) of reprogramming, the expression of the Nanog gene in BQ-activated human CD4+ lymphocytes precedes the formation of accomplished CD4+ LiPSc-like clusters. This observation suggested the possible evaluation of the efficacy of reprogramming by scoring the number of reprogrammed cells expressing Nanog gene. This approach gave us the opportunity to calculate three to four times, with some proximity, the total number of glowing cells per specimen (GCS) in each T25 flask. Subtracting the number of nonspecific binding sites (NSB) in the control flasks from the GCS and knowing the original number of cells taken into BQ-activation (BQC), we calculated the mean and standard deviation for each treatment. 2.9. Controls We used the following controls: a) ?105 donor cells were placed in an electroporation cuvette without oocytes, electroporated and incubated for 3 hr at 17 oC b) Oocytes were placed with ?105 donor cells into the same electroporation chamber, but no electrical stimulation was applied. Cuvette containing oocytes and donor cells were incubated for 3 hr at 17 oC c) ?105 donor cells were placed an electroporation cuvette without oocytes and and incubated for 3 hr at 17 oC before transferred to ES cell media (no electroporation applied) d) Oocytes were electrically stimulated in electroporation cuvette in the absence of donor cells, then ?105 donor cells were transferred to 800 µl of extra- oocyte solution (electroporate) containing no oocytes and incubated for 3 hr at 17 oC e) Oocytes were electrically stimulated in electroporation cuvette in the absence of donor cells, then ?105 of electroporated donor cells were transferred to 800 µl of electroporate containing no oocytes and incubated for 3 hr at 17 oC f) ?105 of iMEF cells were co-electroporated with 40-50 oocytes, incubated for 3 hr at 17 oC , then separated from oocytes, and transferred to T25 flask containing complete ES growth media for culturing. 3. Results 3.1. Results of Control Experiments Controls: “a”, “b”, “c”, and “f” were demonstrated to be RP-negative. In control “d” where non-electroporated donor cells were exposed for 3 hr to electroporate we detected ?0.4% RP efficacy (calculated only for CD4TLs, data not shown). In control “e” where electroporated donor cells were exposed to electroporate for 3 hr RP efficacy was elevated in comparison with control “e” and was ?0.9% (calculated only for CD4TLs, data not shown). 3.2. De-differentiation of BMSCs and BMSCGFP into Human iPSc-Like Cells We demonstrated that BQ-activated human bone marrow stromal cells can de-differentiate into iPSc-like cells, which appeared to be indistinguishable from human embryonic stem cells in colony morphology. BMSCs strongly expressed the pluripotency-associated transcription factors Oct3/4, SOX-2, Nanog, Rex-1, and SSEA-1 (Figure 1). DAPI Oct 3/4 Sox-2 colors combined DAPI Oct 3/4 Nanog colors combined DAPI Rex-1 SSEA-1 colors combined Figure 1. De-differentiation of human BMSCs Into human iPSc - like cells (7 days after BQ-activation, 20 x). In separate studies, we used BMSCGFP to show a direct link between activated donor cells and cells that form iPSc-like clusters (Figure 2). control phase contrast fluorescent image No activation 7 days after BQ- activation 7 days after BQ- activation Figure 2. De-differentiation BMSCGFP Into human iPSc - like cells ((7 days after BQ-activation, 40 x). SSEA-1 Phase SSEA-1 Fluoro SSEA-4 Phase SSEA 3.3. De-differentiation of Human CD4+ T-Lymphocytes into Human iPSc-like Cells We also demonstrated that BQ-activated human CD4+ T-lymphocytes, when transferred directly to feeder cells, rapidly (on the third to fifth day) form iPSc-like colonies. Our results unambiguously indicate that human CD4+ T-lymphocytes de-differentiated into iPSc-like cells. They also displayed high alkaline phosphatase activity. Expression of major stem cell markers, such as Oct3/4, Nanog, SOX-2, TRA-1-60, Rex-1, and SSEA-1 were also strongly expressed in these developing human CD4+ L-iPSc-like cell colonies (Figure 3A - Figure 3X). A B C D E F G H I J K L M N K M N O P O V P X Figure 3. De-differentiation of CD4+ T-Lymphocytes Into human iPSc - like cells. (A): human CD4TL, control, 20 x, (B): CD4TL co-cultured with iMEF feeder cells, no activation, 20 x. (C-D): CD4TL -iPSc,-like cluster 5 days, 10 x-20 x. (E-F): AP-staining, 9 days, 20 x-40 x. (G-H): Two adjoin clusters of CD4TL- iPS cells, Oct 3/ 4, 10 days, 20 x. (I-J): Nanog. 10 days, 10 x. (K): DAPI staining corresponding to Sox-2 and Rex-1 staining. (L): Colors combined. (M): SOX-2, (10 days), 40 x. (N): TRA-1-60 (9 days), 40 x. (O): Rex-1 (10 days), 20 x. (P): SSEA-1 (10 days, 20 x). 3.4. De-differentiation of Human Cervical Carcinoma and Breast Adenocarcinoma Cells into iPSc-like Cells This set of experiments was designed to investigate if selected cancer cell lines (HeLa and MCF-7) are RP-responsive to BQ-activation. We demonstrated that human cervical carcinoma and breast adenocarcinoma cells both de-differentiate and partially de-differentiate into iPSc-like clusters positively expressing the Oct 3/4 and Nanog genes (Figure 4 and Figure 5). A B C D E Figure 4. De-differentiation of human cervical carcinoma (HeLa) cells Into human iPSc - like cells. (A): HeLa cells control, 20 x. (B): HeLa cells cultured on iMEF feeders (no BQ-activation), 20 x. (C): HeLa-iPSc-like cluster, 5days, 10 x. (D): Expression of Oct 3/4 gene in fully de-differentiated cluster of HeLa- iPSc-like cells, 11 days, 20 x. A B C D D F Figure 5. De-differentiation of human breast adenocarcinoma (MCF-7) cells Into human iPSc - like cells (A): MCF-7 control, 10 x. (B): MCF-7 cells cultured on iMEF feeders (no BQ-activation), 40 x. (C): Expression of Oct 3/4 genes in fully de-differentiated cluster of MCF-7 iPSc-like cells, 11 days, 40 x. (D): Nanog gene expression in partially de-differentiated cluster of MCF-7–iPSfc-like cells, 11 days,40 x. 3.9. Efficacy of Reprogramming of Human CD4+ T-Lymphocytes Shortly after BQ activation (12 h – 24 h), CD4+ lymphocytes start to express the Nanog gene. By that time, single activated cells, as well as developing iPSc-like clusters can be clearly observed (Figures 11A - B), and, thus, the total number of activated cells can be calculated by the method described above. The findings on the efficacy of reprogramming of human CD4TL are present in Table 1. Table 1 Efficacy of reprogramming of human CD4+ T lymphocytes Cells BQC NSB GCS -1 GCS -2 GCS -3 GCS -4 Mean RP % ± SD CD4+L 105 201±15 25,321 22,256 27,355 19,285 23.4±3.5 Figure 6. Early expression of Nanog gene in human CD4+ lymphocytes used for the calculation of the efficacy of reprogramming of hCD4TL. (A): 20 x image of human hCD4TL in the field of phase contrast microscope, 24h after BQ-activation. (B): Same area in the field of fluorescent microscope (lymphocytes strongly express Nanog gene­). 4. Discussion In this study, we introduced a new methodology for the non-viral induction of human pluripotent stem cells derived from various human cancer cell lines and normal cell lines. The method we employed used a natural reprogramming inductor, such as Xenopus laevis oocytes and electrical stimulation, as appropriate reprogramming tools. We demonstrated that using 150 v/cm / 25µF / 7pulses to co-electroporate living amphibian oocytes with different human normal and cancer cell lines transforms human donor cells into cells resembling human induced pluripotent stem cells in colony morphology and in the expression of stem cell markers that are characteristic for human ES cells. The results of control experiments strongly suggest that co-electroporation of living Xenopus laevis oocytes with human donor cells is a vital factor in the successful reprogramming of selected human cell lines into human iPSc-like cells. This supported by low efficacy of reprogrammimg detected in controls “d” and “e” which is very interesting, because indicate that BQ-activated oocytes definitely release yet unknown RP messengers into extra-oocyte environment and that such “semiochemical emission” can be picked up by both non-electroporated and electroporated donor cells. Identification, purification, and amplification of active components of RP signaling during BQ-activation may open wide opportunity for their commercialization. Although the use of human CD4+ T-lymphocytes in RP is limited due to the difficulty of culturing them, we also studied them and demonstrated that BQ-activated human CD4+ T-lymphocytes form iPSc-like clusters on iMEF feeder cells. Newly obtained TL-iPSc-like cells displayed high alkaline phosphatase activity. The pluripotency-associated transcription factors, such as Oct3/4, Nanog, SOX-2, TRA-1-60, Rex-1, and SSEA-1, were also strongly expressed in these developing CD4TL-iPSc-like colonies. The possibility of converting cancer cells into normal cells using a reprogramming approach, which can alter cell’s transcription program, is exciting and is widely discussed in the scientific literature. This approach includes reverting adult neoplasms (Frenster and Hovsepian, 2007), epigenetic reprogramming of breast cancer cells by valproic acid (Travaglini et al., 2009), and reprogramming of human cancer cells in the mouse mammary gland (Bussard et al., 2010). We designed a set of experiments to examine if selected cancer cell lines exposed to BQ- activation may be converted into cells with new, non-cancerous patterns. We demonstrated that human cervical carcinoma and breast adenocarcinoma cells both de-differentiated and partially de-differentiated into iPSc-like clusters positively expressing the Oct 3/4 and Nanog genes. We also observed partially reprogrammed cancer cells, which may represent the correct transitional point wherein malignant cells can be “rebooted” and re-differentiated back into normalized cells. Partially reprogrammed cells can be a prelude to successful trans-differentiational reprogramming, too (Sareen and Svendsen, 2010). As opposed to cancer stem cells, BQ activated HeLa-iPSc-like cells and MCF-7-iPSc-like cells revealed an extremely low pattern of proliferation (clusters were growing extremely slowly), which may reflect on possible normalization of their proliferation activity (Figure 4 and Figure 5 ). As the main task of our studies was to demonstrate if BQ-activation can be effectively applied to multiple human cell lines, we limited the evaluation of the efficacy of reprogramming to human CD4+ T-lymphocytes. CD4+ lymphocytes start to express the Nanog gene at 12h-24h after BQ activation, when single activated cells, as well as developing clusters, can be clearly observed and, thus, the total number of activated cells can be calculated. The efficacy of reprogramming for human CD4+ lymphocytes, based on ?105 cells originally taken into the electroporation chamber, was 23.4 ± 3.5 %. This is a significant result, considering that new RP technology we are presenting can still be modified and improved. For example, we conducted some preliminary studies, which clearly indicate that BQ-activation can be modulated by fluctuations in barometric pressure and environmental temperature (data not shown). 5. Conclusion We successfully fulfilled most of our expectations with regard to the new RP system we created; specifically: it is free of ethical controversy; reprogramming is fast (taking only four to five days), efficient (?23.4 % for CD4TL) and able to produce partially reprogrammed cells; it segregates oocytes and donor cells and simultaneously opens communication gates between two RP components via electroporation; semiochemical signaling occurs in real-time; and it opens the possibility of controlled reprogramming. Our system conducts de-differentiation in a reproducible, standardized fashion when applied to different cell lines. Indeed, successful reprogramming of four different human cell lines was achieved using same natural RP inductors, which are electrically stimulated living frog oocytes. There is need for further studies to be conducted in the future in order to develop a broad understanding of the technology and to gain wide recognition and acceptance. Presently, we are conducting experiments in which several protocols aimed to prove the iPSc nature of BQ-activated cells will be undertaken. These studies will include teratoma formation, re-differentiation and trans-differentiation, molecular karyotyping, and DNA fingerprinting. We will also work in the direction of deciphering intrinsic molecular mechanisms underlying the Xenopus oocyte-mediated RP phenomena. Acknowledgments We thank Ira Pastor, VP at Phytomedics, Inc. (Jamesburg, NJ) and CEO of Bioquark, Inc. (Wisconsin Dells, WI), for his fundraising efforts and outstanding help in all aspects of this project. Also, we would like to thank Dr. Nikolai Strelchenko, Gatekeeper of the hESC Research Lab at the Reproductive Genetics Institute (Chicago, IL), and Dr. Arshak Alexanian, Associate Professor at the Department of Neurosurgery, Neuroscience Research Laboratories, Zablocki Veterans Affairs Medical Center and at the Medical College of Wisconsin (Milwaukee, WI), for their scientific advice and professional help. Disclosure of Potential Conflicts of Interest The author indicates no potential conflicts of interest. References Bussard K.M., Boulanger C.A., Booth B.W., BrunoR.D., SmithG.H., 2010.Reprogramming Human Cancer Cells in the Mouse Mammary Gland. Cancer Res; 70(15): 6336–43. Doetschman T. , 2002. Gene Targeting in Embryonic Stem Cells. A Lab. Handbook. Acad. Press; San Diego: Frenster J.H., Hovsepian J.A., 2007.Models of Embryonic Gene-Induced Initiation and Reversion of Adult Neoplasms. 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