Journal Club
Journal Club
Cancer Biology & Therapy 15:6, 675–677; June 2014; © 2014 Landes Bioscience
Low pH reprograms somatic murine cells into pluripotent stem cells Jonathan S Williams, Ying Xiao, and Isaac Brownell* Dermatology Branch; Center for Cancer Research; National Cancer Institute; Bethesda, MD USA
I
Keywords: induced pluripotent stem cells, embryonic stem cells, stimulustriggered acquisition of pluripotency, blastocyst complementation assay, tumor infiltrating lymphocytes Abbreviations: iPSCs, induced pluripotent stem cells; ESCs, embryonic stem cells; STAP, stimulus-triggered acquisition of pluripotency; ACTH, adrenocorticotropic hormone; CAR, chimeric antigen receptor; TILs, tumor infiltrating lymphocytes *Correspondence to: Isaac Brownell; Email: [email protected] Submitted: 02/25/2014 Accepted: 03/03/2014 Published Online: 03/11/2014 http://dx.doi.org/10.4161/cbt.28414 Comment on: Obokata H, Wakayama T, Sasai Y, Kojima K, Vacanti MP, Niwa H, Yamato M, Vacanti CA. Stimulus-triggered fate conversion of somatic cells into pluripotency. Nature 2014; 505:641-7; PMID:24476887; http://dx.doi. org/10.1038/nature12968
www.landesbioscience.com
nduced pluripotent stem cells (iPSCs) are somatic cells that are reprogrammed into a state resembling embryonic stem cells (ESCs). iPSCs represent a promising technology with applications in cancer research, yet current methods used to generate iPSCs limit their translation to clinical use. In a recent Nature article, Obokata et al. detail a novel technique to generate pluripotent murine cells called stimulus-triggered acquisition of pluripotency (STAP). STAP eliminates the need for exogenous expression of reprogramming factors used in previous iPSC technologies, instead transforming somatic cells to pluripotency using physical and chemical stimuli. The authors found that STAP cells are generated at a 10-fold higher efficiency than prior iPSC technologies. STAP cells display several features of pluripotency, namely the expression of pluripotencyrelated genes (Oct4, Nanog, Sox2, Ecat1, Esg1, and Dax1), the ability to form teratomas in vivo, and the ability to produce viable, fertile mice in blastocyst complementation assays. Here, we review these findings on STAP and contrast it to previous iPSC technologies, while noting the potential of this method to generate autologous anti-tumor immune cells for cancer therapy. Easily obtainable pluripotent stem cells are highly sought after for use in biomedical research, including cancer research and therapy. Embryonic stem cells (ESCs), which can serve as progenitors for all somatic tissues, have been developed as a source of pluripotent stem cells. However, human ESC use is controversial
due to their derivation from embryos. In 2006, an alternative stem cell source was discovered in the form of induced pluripotent stem cells (iPSCs), which are somatic cells reprogrammed to an ESC-like state by exogenous expression of pluripotencyrelated transcription factors. iPSCs were first generated from murine fibroblasts by transfection of four transcription factors important in ESCs (Oct3/4, Klf4, Sox2, and c-Myc).1 Subsequent studies showed that iPSCs could generate tissues from each germ layer, and contribute to viable embryos when injected into murine blastocysts.2,3 Within a year, human iPSCs were discovered using similar factor-induced reprogramming methods, demonstrating that iPSCs had translational potential.4,5 Several iPSC reprogramming approaches have been developed based on these seminal studies, with differences arising in how reprogramming transcription factors are delivered to a host somatic cell (for a review, see ref. 6). However, factor-based reprogramming techniques are marred with drawbacks, such as low reprogramming efficiencies, time-consuming protocols, incomplete reprogramming of somatic cells into iPSCs, and the potential for spontaneous oncogenesis. These challenges render use of iPSCs too inefficient and risky for clinical applications. There is a need for efficient, safe, and well-characterized techniques if pluripotent reprogramming technology is to be successfully applied for regenerative medicine and cancer therapy. In a recent article published in Nature, Obokata et al. demonstrate a highly efficient reprogramming technique that does not require the use of exogenous transcription factors.7 Instead, pluripotent
Cancer Biology & Therapy 675
©2014 Landes Bioscience. Do not distribute.
A novel technique with therapeutic implications
676
after low pH exposure. Together, these findings validated that Oct4-GFP+ CD45− cells can arise from differentiated CD45 + leukocytes. Obokata et al. then explored whether the Oct4-GFP+ population possessed other hallmarks of pluripotency, namely (1) the expression of pluripotency-related markers and (2) the ability to produce differentiated cells of all three germ layers. After a week in culture, acid-induced GFP+ CD45− cells were found to express six pluripotency-related genes (Oct4, Nanog, Sox2, Ecat1, Esg1, and Dax1) at levels comparable to murine ESCs. In addition, after exposure to in vitro differentiation assays, established clusters of Oct4-GFP+ cells expressed markers of all three germ layers (ectoderm, mesoderm, and endoderm), suggesting they had achieved pluripotency. To confirm this in vivo, acid-induced Oct4-GFP+ clusters were sorted for high GFP expression and injected into immunodeficient NOD/ SCID mice. Forty percent of injected mice formed teratomas, which are tumors composed of differentiated cells from multiple germ layers. Immunohistochemical analysis of these tumors showed morphologic features of epidermis, skeletal muscle, and intestinal villi, and staining for βIII-tubulin, smooth muscle actin, and α-fetoprotein, all of which correspond to tissue originating from ectoderm, mesoderm, and endoderm, respectively. These results collectively show that GFP+ CD45− cells induced by acid treatment are pluripotent, leading Obokata et al. to name this population “STAP cells”. Obokata et al. were also able to generate STAP cells from other differentiated cells. They exposed immature Oct4-GFP mouse bone marrow, brain, lung, muscle, adipose, liver, cartilage, and fibroblasts to low pH and quantified the clustered GFP+ cells that formed after one week in culture. Between 10% and 30% of surviving cells from each tissue type were STAP cells (Oct4-GFP+) and expressed Oct4, Nanog, Sox2, Ecat1, Esg1, and Dax1 at levels comparable to ESCs and STAP cells derived from splenic leukocytes. These findings suggest that STAP cells can be generated from diverse tissue types, a feature in common with iPSC reprogramming techniques.
Cancer Biology & Therapy
While teratoma formation by a cell population is a robust demonstration of pluripotency, more stringent tests require the generation of viable, fertile mice. In these assays, pluripotent cells that constitutively express a fluorescent reporter are injected into non-fluorescent blastocysts composed of either diploid or tetraploid cells. After implantation into pseudopregnant females, the embryos are allowed to develop and are monitored for incorporation of fluorescent cells. Pluripotent cells injected into diploid blastocysts result in chimeric mice, whereas tetraploid complementation generates embryos derived entirely from injected cells. Obokata et al. injected clusters of STAP cells derived from CD45 + splenic cells expressing a GFP reporter (cag-GFP) into both diploid and tetraploid embryos. In the diploid assay, the chimeric skin, lung, liver, heart, and muscle contained an average of 20–30% GFP+ cells, with brain showing the highest average contribution at approximately 65%. GFP+ mice were also generated from tetraploid embryo injections, and mice from both assays were capable of germline transmission. This demonstrated that the early embryonic microenvironment allowed STAP cells to form all somatic tissue types, including germ cells. Importantly, Obokata et al. observed no tumor formation after 18 mo in mice generated from these complementation assays. Finally, Obokata et al. were able to grow STAP cells as proliferative, ESClike cell lines. This is a desirable feature of iPSC technology, as it allows for autologous pluripotent cell lines to be derived from individual patients. Obokata et al. were able to induce STAP cells into a proliferative state by culturing them in medium containing adrenocorticotropic hormone (ACTH). They referred to these proliferative, self-renewing cells as STAP stem cells. STAP stem cells were able to be clonally passaged as single cells, and proliferate in ESGRO-2i medium, which is commonly used to grow murine iPSCs and ESCs. STAP stem cells also retained the pluripotent features seen in STAP cells, including, the expression of Oct4, Nanog, and Klf4, the ability to form teratomas, and the ability to produce viable, fertile mice after blastocyst injection.
Volume 15 Issue 6
©2014 Landes Bioscience. Do not distribute.
murine cells are generated by subjecting differentiated cells to harsh physical and chemical stimuli. This method, which they refer to as stimulus-triggered acquisition of pluripotency (STAP), could potentially overcome critical drawbacks of previous iPSC techniques and increase the feasibility of applying cellular reprogramming to the clinic. Obokata hypothesized that somatic cells could be stressed into pluripotency after noticing that cultured cells exhibited a small, ESC-like morphology when passed through a capillary tube.8 This observation prompted Obokata et al. to query whether different types of physical and chemical stimuli could reprogram somatic cells to a pluripotent state. To do this, they monitored Oct4 expression, a marker of pluripotency, in stressed somatic cells from week-old Oct4-GFP reporter mice. Using CD45 + splenic leukocytes, Obokata et al. found that, in the right culture conditions, a 30 min exposure to a pH of 5.7 transformed CD45 + cells to GFP+ CD45− cells with the highest efficiency of all stimuli tested. Most CD45 + cells died after exposure to low pH, yet after one week in culture, ~30% of surviving cells were GFP+ CD45− (representing approximately 10% of the starting CD45 + population). CD45 + cells left untreated showed no endogenous GFP after a week in culture. Interestingly, the authors note that identical treatment of adult spleen leukocytes resulted in lower numbers of surviving cells, but do not report if mature CD45 + or other differentiated cells could effectively be reprogrammed via the STAP method. To confirm the Oct4-GFP+ cells were reprogrammed leukocytes, the authors had to rule out the possibility of a contaminating population of stem cells that were able to expand following exposure to low pH. First, they showed that this possibility was unlikely, as the splenic cells did not proliferate during the treatment protocol. Obokata et al. also demonstrated that a portion of the GFP+ CD45− cells had genomic rearrangements in the T-cell receptor β locus, suggesting they arose from a differentiated CD45 + T-lymphocyte. Finally, individual CD45 + cells were followed by video microscopy and were observed to shrink in size, lose CD45 staining, and gain GFP expression
www.landesbioscience.com
could theoretically be expanded and differentiated in vitro into T-lymphocytes or myeloid cells engineered to attack a patient’s tumor. Factor-induced iPSCs have already been used to generate chimeric antigen receptor (CAR) transduced T cells capable of producing anti-cancer activity in mouse xenografts of B-cell lymphomas.9 Another approach would be to use STAP technology to expand autologous tumor-infiltrating lymphocytes (TILs). TILs are immune cells that infiltrate solid tumors and are associated with a better prognosis. Collection and expansion of TILs for adoptive T-cell therapy has been used to treat patients with melanoma, but expanded TILs are difficult to maintain in culture.10 STAP could be used to reprogram TILs to stem cells that would retain their T-cell receptor rearrangements. These STAP stem cells could then be used to generate large numbers of autologous anti-tumor T cells for adoptive therapy. Time will tell whether STAP technology can accelerate cellular reprogramming to a practical level for cancer treatments. Regardless, this report by Obokata et al. has potentially radical implications for all applications of pluripotent stem cell technology and will likely be heavily scrutinized in the years to come. Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Acknowledgments
References 1.
Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126:66376; PMID:16904174; http://dx.doi.org/10.1016/j. cell.2006.07.024 2. Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells. Nature 2007; 448:313-7; PMID:17554338; http:// dx.doi.org/10.1038/nature05934 3. Wernig M, Meissner A, Foreman R, Brambrink T, Ku M, Hochedlinger K, Bernstein BE, Jaenisch R. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 2007; 448:31824; PMID:17554336; http://dx.doi.org/10.1038/ nature05944 4. Yu J, Vodyanik MA, Smuga-Otto K, AntosiewiczBourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 2007; 318:1917-20; PMID:18029452; http://dx.doi. org/10.1126/science.1151526 5. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007; 131:861-72; PMID:18035408; http://dx.doi.org/10.1016/j. cell.2007.11.019 6. Rajasingh J. Progress in molecular biology and translational science. Academic Press; c2012. Chapter 3, 51-82. 7. Obokata H, Wakayama T, Sasai Y, Kojima K, Vacanti MP, Niwa H, Yamato M, Vacanti CA. Stimulustriggered fate conversion of somatic cells into pluripotency. Nature 2014; 505:641-7; PMID:24476887; http://dx.doi.org/10.1038/nature12968 8. Cyranoski D. Acid bath offers easy path to stem cells. Nature 2014; 505:596; PMID:24476866; http:// dx.doi.org/10.1038/505596a 9. Themeli M, Kloss CC, Ciriello G, Fedorov VD, Perna F, Gonen M, Sadelain M. Generation of tumortargeted human T lymphocytes from induced pluripotent stem cells for cancer therapy. Nat Biotechnol 2013; 31:928-33; PMID:23934177; http://dx.doi. org/10.1038/nbt.2678 10. Wu R, Forget MA, Chacon J, Bernatchez C, Haymaker C, Chen JQ, Hwu P, Radvanyi LG. Adoptive T-cell therapy using autologous tumorinfiltrating lymphocytes for metastatic melanoma: current status and future outlook. Cancer J 2012; 18:160-75; PMID:22453018; http://dx.doi. org/10.1097/PPO.0b013e31824d4465
This publication was supported by the National Institutes of Health Intramural Research Program, Center of Cancer Research, National Cancer Institute. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.
Cancer Biology & Therapy 677
©2014 Landes Bioscience. Do not distribute.
Interestingly, Obokata et al. note that, unlike STAP cells, these proliferating cells resembled murine ESCs in morphology, chromatin ultrastructure, and absence of X-chromosome inactivation. They also found that STAP stem cells express Essrβ, an ESC marker not seen in normal STAP cells. These latter findings suggest that culturing STAP cells in ACTH transforms them to a state more closely resembling ESCs. In this study, Obokata et al. show a potentially groundbreaking somatic cell reprogramming technique with the ability to overcome common drawbacks of factorbased iPSC technologies. The next major tests facing STAP technology are replicating these findings in other laboratories and its application to adult murine cells and human cells. If human STAP stem cells can be generated with the reported ease and efficiency cited in this study, this would dramatically increase the availability of patient-derived pluripotent cells for use in research, disease treatment, and regenerative medicine. One of the notable advantages of STAP stem cells is that they eliminate the risk of spontaneous oncogenesis and genomic aberrations associated with factor-based reprogramming techniques. In addition, STAP reprogramming appears to have a 10-fold higher efficiency than factor-based iPSC protocols. Low reprogramming efficiency is a serious drawback to iPSC techniques, with the most efficient methods yielding just 1% iPSCs from the starting somatic population.6 Replicating the features of murine STAP cells in humans could have major implications for cancer treatment as well. Many have pointed out the potential application of iPSCs to generate large numbers of autologous anti-tumor immune cells. STAP stem cells from any tissue source
Journal Club
Cancer Biology & Therapy 15:6, 675–677; June 2014; © 2014 Landes Bioscience
Low pH reprograms somatic murine cells into pluripotent stem cells Jonathan S Williams, Ying Xiao, and Isaac Brownell* Dermatology Branch; Center for Cancer Research; National Cancer Institute; Bethesda, MD USA
I
Keywords: induced pluripotent stem cells, embryonic stem cells, stimulustriggered acquisition of pluripotency, blastocyst complementation assay, tumor infiltrating lymphocytes Abbreviations: iPSCs, induced pluripotent stem cells; ESCs, embryonic stem cells; STAP, stimulus-triggered acquisition of pluripotency; ACTH, adrenocorticotropic hormone; CAR, chimeric antigen receptor; TILs, tumor infiltrating lymphocytes *Correspondence to: Isaac Brownell; Email: [email protected] Submitted: 02/25/2014 Accepted: 03/03/2014 Published Online: 03/11/2014 http://dx.doi.org/10.4161/cbt.28414 Comment on: Obokata H, Wakayama T, Sasai Y, Kojima K, Vacanti MP, Niwa H, Yamato M, Vacanti CA. Stimulus-triggered fate conversion of somatic cells into pluripotency. Nature 2014; 505:641-7; PMID:24476887; http://dx.doi. org/10.1038/nature12968
www.landesbioscience.com
nduced pluripotent stem cells (iPSCs) are somatic cells that are reprogrammed into a state resembling embryonic stem cells (ESCs). iPSCs represent a promising technology with applications in cancer research, yet current methods used to generate iPSCs limit their translation to clinical use. In a recent Nature article, Obokata et al. detail a novel technique to generate pluripotent murine cells called stimulus-triggered acquisition of pluripotency (STAP). STAP eliminates the need for exogenous expression of reprogramming factors used in previous iPSC technologies, instead transforming somatic cells to pluripotency using physical and chemical stimuli. The authors found that STAP cells are generated at a 10-fold higher efficiency than prior iPSC technologies. STAP cells display several features of pluripotency, namely the expression of pluripotencyrelated genes (Oct4, Nanog, Sox2, Ecat1, Esg1, and Dax1), the ability to form teratomas in vivo, and the ability to produce viable, fertile mice in blastocyst complementation assays. Here, we review these findings on STAP and contrast it to previous iPSC technologies, while noting the potential of this method to generate autologous anti-tumor immune cells for cancer therapy. Easily obtainable pluripotent stem cells are highly sought after for use in biomedical research, including cancer research and therapy. Embryonic stem cells (ESCs), which can serve as progenitors for all somatic tissues, have been developed as a source of pluripotent stem cells. However, human ESC use is controversial
due to their derivation from embryos. In 2006, an alternative stem cell source was discovered in the form of induced pluripotent stem cells (iPSCs), which are somatic cells reprogrammed to an ESC-like state by exogenous expression of pluripotencyrelated transcription factors. iPSCs were first generated from murine fibroblasts by transfection of four transcription factors important in ESCs (Oct3/4, Klf4, Sox2, and c-Myc).1 Subsequent studies showed that iPSCs could generate tissues from each germ layer, and contribute to viable embryos when injected into murine blastocysts.2,3 Within a year, human iPSCs were discovered using similar factor-induced reprogramming methods, demonstrating that iPSCs had translational potential.4,5 Several iPSC reprogramming approaches have been developed based on these seminal studies, with differences arising in how reprogramming transcription factors are delivered to a host somatic cell (for a review, see ref. 6). However, factor-based reprogramming techniques are marred with drawbacks, such as low reprogramming efficiencies, time-consuming protocols, incomplete reprogramming of somatic cells into iPSCs, and the potential for spontaneous oncogenesis. These challenges render use of iPSCs too inefficient and risky for clinical applications. There is a need for efficient, safe, and well-characterized techniques if pluripotent reprogramming technology is to be successfully applied for regenerative medicine and cancer therapy. In a recent article published in Nature, Obokata et al. demonstrate a highly efficient reprogramming technique that does not require the use of exogenous transcription factors.7 Instead, pluripotent
Cancer Biology & Therapy 675
©2014 Landes Bioscience. Do not distribute.
A novel technique with therapeutic implications
676
after low pH exposure. Together, these findings validated that Oct4-GFP+ CD45− cells can arise from differentiated CD45 + leukocytes. Obokata et al. then explored whether the Oct4-GFP+ population possessed other hallmarks of pluripotency, namely (1) the expression of pluripotency-related markers and (2) the ability to produce differentiated cells of all three germ layers. After a week in culture, acid-induced GFP+ CD45− cells were found to express six pluripotency-related genes (Oct4, Nanog, Sox2, Ecat1, Esg1, and Dax1) at levels comparable to murine ESCs. In addition, after exposure to in vitro differentiation assays, established clusters of Oct4-GFP+ cells expressed markers of all three germ layers (ectoderm, mesoderm, and endoderm), suggesting they had achieved pluripotency. To confirm this in vivo, acid-induced Oct4-GFP+ clusters were sorted for high GFP expression and injected into immunodeficient NOD/ SCID mice. Forty percent of injected mice formed teratomas, which are tumors composed of differentiated cells from multiple germ layers. Immunohistochemical analysis of these tumors showed morphologic features of epidermis, skeletal muscle, and intestinal villi, and staining for βIII-tubulin, smooth muscle actin, and α-fetoprotein, all of which correspond to tissue originating from ectoderm, mesoderm, and endoderm, respectively. These results collectively show that GFP+ CD45− cells induced by acid treatment are pluripotent, leading Obokata et al. to name this population “STAP cells”. Obokata et al. were also able to generate STAP cells from other differentiated cells. They exposed immature Oct4-GFP mouse bone marrow, brain, lung, muscle, adipose, liver, cartilage, and fibroblasts to low pH and quantified the clustered GFP+ cells that formed after one week in culture. Between 10% and 30% of surviving cells from each tissue type were STAP cells (Oct4-GFP+) and expressed Oct4, Nanog, Sox2, Ecat1, Esg1, and Dax1 at levels comparable to ESCs and STAP cells derived from splenic leukocytes. These findings suggest that STAP cells can be generated from diverse tissue types, a feature in common with iPSC reprogramming techniques.
Cancer Biology & Therapy
While teratoma formation by a cell population is a robust demonstration of pluripotency, more stringent tests require the generation of viable, fertile mice. In these assays, pluripotent cells that constitutively express a fluorescent reporter are injected into non-fluorescent blastocysts composed of either diploid or tetraploid cells. After implantation into pseudopregnant females, the embryos are allowed to develop and are monitored for incorporation of fluorescent cells. Pluripotent cells injected into diploid blastocysts result in chimeric mice, whereas tetraploid complementation generates embryos derived entirely from injected cells. Obokata et al. injected clusters of STAP cells derived from CD45 + splenic cells expressing a GFP reporter (cag-GFP) into both diploid and tetraploid embryos. In the diploid assay, the chimeric skin, lung, liver, heart, and muscle contained an average of 20–30% GFP+ cells, with brain showing the highest average contribution at approximately 65%. GFP+ mice were also generated from tetraploid embryo injections, and mice from both assays were capable of germline transmission. This demonstrated that the early embryonic microenvironment allowed STAP cells to form all somatic tissue types, including germ cells. Importantly, Obokata et al. observed no tumor formation after 18 mo in mice generated from these complementation assays. Finally, Obokata et al. were able to grow STAP cells as proliferative, ESClike cell lines. This is a desirable feature of iPSC technology, as it allows for autologous pluripotent cell lines to be derived from individual patients. Obokata et al. were able to induce STAP cells into a proliferative state by culturing them in medium containing adrenocorticotropic hormone (ACTH). They referred to these proliferative, self-renewing cells as STAP stem cells. STAP stem cells were able to be clonally passaged as single cells, and proliferate in ESGRO-2i medium, which is commonly used to grow murine iPSCs and ESCs. STAP stem cells also retained the pluripotent features seen in STAP cells, including, the expression of Oct4, Nanog, and Klf4, the ability to form teratomas, and the ability to produce viable, fertile mice after blastocyst injection.
Volume 15 Issue 6
©2014 Landes Bioscience. Do not distribute.
murine cells are generated by subjecting differentiated cells to harsh physical and chemical stimuli. This method, which they refer to as stimulus-triggered acquisition of pluripotency (STAP), could potentially overcome critical drawbacks of previous iPSC techniques and increase the feasibility of applying cellular reprogramming to the clinic. Obokata hypothesized that somatic cells could be stressed into pluripotency after noticing that cultured cells exhibited a small, ESC-like morphology when passed through a capillary tube.8 This observation prompted Obokata et al. to query whether different types of physical and chemical stimuli could reprogram somatic cells to a pluripotent state. To do this, they monitored Oct4 expression, a marker of pluripotency, in stressed somatic cells from week-old Oct4-GFP reporter mice. Using CD45 + splenic leukocytes, Obokata et al. found that, in the right culture conditions, a 30 min exposure to a pH of 5.7 transformed CD45 + cells to GFP+ CD45− cells with the highest efficiency of all stimuli tested. Most CD45 + cells died after exposure to low pH, yet after one week in culture, ~30% of surviving cells were GFP+ CD45− (representing approximately 10% of the starting CD45 + population). CD45 + cells left untreated showed no endogenous GFP after a week in culture. Interestingly, the authors note that identical treatment of adult spleen leukocytes resulted in lower numbers of surviving cells, but do not report if mature CD45 + or other differentiated cells could effectively be reprogrammed via the STAP method. To confirm the Oct4-GFP+ cells were reprogrammed leukocytes, the authors had to rule out the possibility of a contaminating population of stem cells that were able to expand following exposure to low pH. First, they showed that this possibility was unlikely, as the splenic cells did not proliferate during the treatment protocol. Obokata et al. also demonstrated that a portion of the GFP+ CD45− cells had genomic rearrangements in the T-cell receptor β locus, suggesting they arose from a differentiated CD45 + T-lymphocyte. Finally, individual CD45 + cells were followed by video microscopy and were observed to shrink in size, lose CD45 staining, and gain GFP expression
www.landesbioscience.com
could theoretically be expanded and differentiated in vitro into T-lymphocytes or myeloid cells engineered to attack a patient’s tumor. Factor-induced iPSCs have already been used to generate chimeric antigen receptor (CAR) transduced T cells capable of producing anti-cancer activity in mouse xenografts of B-cell lymphomas.9 Another approach would be to use STAP technology to expand autologous tumor-infiltrating lymphocytes (TILs). TILs are immune cells that infiltrate solid tumors and are associated with a better prognosis. Collection and expansion of TILs for adoptive T-cell therapy has been used to treat patients with melanoma, but expanded TILs are difficult to maintain in culture.10 STAP could be used to reprogram TILs to stem cells that would retain their T-cell receptor rearrangements. These STAP stem cells could then be used to generate large numbers of autologous anti-tumor T cells for adoptive therapy. Time will tell whether STAP technology can accelerate cellular reprogramming to a practical level for cancer treatments. Regardless, this report by Obokata et al. has potentially radical implications for all applications of pluripotent stem cell technology and will likely be heavily scrutinized in the years to come. Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Acknowledgments
References 1.
Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126:66376; PMID:16904174; http://dx.doi.org/10.1016/j. cell.2006.07.024 2. Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells. Nature 2007; 448:313-7; PMID:17554338; http:// dx.doi.org/10.1038/nature05934 3. Wernig M, Meissner A, Foreman R, Brambrink T, Ku M, Hochedlinger K, Bernstein BE, Jaenisch R. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 2007; 448:31824; PMID:17554336; http://dx.doi.org/10.1038/ nature05944 4. Yu J, Vodyanik MA, Smuga-Otto K, AntosiewiczBourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 2007; 318:1917-20; PMID:18029452; http://dx.doi. org/10.1126/science.1151526 5. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007; 131:861-72; PMID:18035408; http://dx.doi.org/10.1016/j. cell.2007.11.019 6. Rajasingh J. Progress in molecular biology and translational science. Academic Press; c2012. Chapter 3, 51-82. 7. Obokata H, Wakayama T, Sasai Y, Kojima K, Vacanti MP, Niwa H, Yamato M, Vacanti CA. Stimulustriggered fate conversion of somatic cells into pluripotency. Nature 2014; 505:641-7; PMID:24476887; http://dx.doi.org/10.1038/nature12968 8. Cyranoski D. Acid bath offers easy path to stem cells. Nature 2014; 505:596; PMID:24476866; http:// dx.doi.org/10.1038/505596a 9. Themeli M, Kloss CC, Ciriello G, Fedorov VD, Perna F, Gonen M, Sadelain M. Generation of tumortargeted human T lymphocytes from induced pluripotent stem cells for cancer therapy. Nat Biotechnol 2013; 31:928-33; PMID:23934177; http://dx.doi. org/10.1038/nbt.2678 10. Wu R, Forget MA, Chacon J, Bernatchez C, Haymaker C, Chen JQ, Hwu P, Radvanyi LG. Adoptive T-cell therapy using autologous tumorinfiltrating lymphocytes for metastatic melanoma: current status and future outlook. Cancer J 2012; 18:160-75; PMID:22453018; http://dx.doi. org/10.1097/PPO.0b013e31824d4465
This publication was supported by the National Institutes of Health Intramural Research Program, Center of Cancer Research, National Cancer Institute. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.
Cancer Biology & Therapy 677
©2014 Landes Bioscience. Do not distribute.
Interestingly, Obokata et al. note that, unlike STAP cells, these proliferating cells resembled murine ESCs in morphology, chromatin ultrastructure, and absence of X-chromosome inactivation. They also found that STAP stem cells express Essrβ, an ESC marker not seen in normal STAP cells. These latter findings suggest that culturing STAP cells in ACTH transforms them to a state more closely resembling ESCs. In this study, Obokata et al. show a potentially groundbreaking somatic cell reprogramming technique with the ability to overcome common drawbacks of factorbased iPSC technologies. The next major tests facing STAP technology are replicating these findings in other laboratories and its application to adult murine cells and human cells. If human STAP stem cells can be generated with the reported ease and efficiency cited in this study, this would dramatically increase the availability of patient-derived pluripotent cells for use in research, disease treatment, and regenerative medicine. One of the notable advantages of STAP stem cells is that they eliminate the risk of spontaneous oncogenesis and genomic aberrations associated with factor-based reprogramming techniques. In addition, STAP reprogramming appears to have a 10-fold higher efficiency than factor-based iPSC protocols. Low reprogramming efficiency is a serious drawback to iPSC techniques, with the most efficient methods yielding just 1% iPSCs from the starting somatic population.6 Replicating the features of murine STAP cells in humans could have major implications for cancer treatment as well. Many have pointed out the potential application of iPSCs to generate large numbers of autologous anti-tumor immune cells. STAP stem cells from any tissue source
