Neural Stem Cell Tumorigenicity and Biodistribution Assessment for Phase I Clinical Trial in Parkinson's Disease

Ibon Garitaonandia, Rodolfo Gonzalez, Trudy Christiansen-Weber, Tatiana Abramihina, Maxim Poustovoitov, Alexander Noskov, Glenn Sherman, Andrey Semechkin, Evan Snyder, Russell Kern, Ibon Garitaonandia, Rodolfo Gonzalez, Trudy Christiansen-Weber, Tatiana Abramihina, Maxim Poustovoitov, Alexander Noskov, Glenn Sherman, Andrey Semechkin, Evan Snyder, Russell Kern

Abstract

Human pluripotent stem cells (PSC) have the potential to revolutionize regenerative medicine. However undifferentiated PSC can form tumors and strict quality control measures and safety studies must be conducted before clinical translation. Here we describe preclinical tumorigenicity and biodistribution safety studies that were required by the US Food and Drug Administration (FDA) and Australian Therapeutic Goods Administration (TGA) prior to conducting a Phase I clinical trial evaluating the safety and tolerability of human parthenogenetic stem cell derived neural stem cells ISC-hpNSC for treating Parkinson's disease (ClinicalTrials.gov Identifier NCT02452723). To mitigate the risk of having residual PSC in the final ISC-hpNSC population, we conducted sensitive in vitro assays using flow cytometry and qRT-PCR analyses and in vivo assays to determine acute toxicity, tumorigenicity and biodistribution. The results from these safety studies show the lack of residual undifferentiated PSC, negligible tumorigenic potential by ISC-hpNSC and provide additional assurance to their clinical application.

Conflict of interest statement

I.G., R.G., M.P., T.A., T.C.W., A.N., G.S., A.S. and R.K. are employees and stock holders of International Stem Cell Corporation.

Figures

Figure 1. Characterization of ISC-hpNSC manufactured under…
Figure 1. Characterization of ISC-hpNSC manufactured under cGMP.
(a) Derivation and cGMP manufacturing scheme of ISC-hpNSC. (b) Immunocytochemical analysis of ISC-hpNSC for neural markers Nestin (NES), Musashi (MSI1) and SOX2. (c) Quantitation by flow cytometry analysis of neural markers NES, MSI1, and SOX2 and pluripotency markers OCT-4 and SSEA-4. Percentage of positive cells (blue) is calculated based on isotype control stained cells (red). (d) G-banding karyotyping analysis shows that the cells have a normal female 46 XX karyotype.
Figure 2. Flow cytometry analysis after magnetic…
Figure 2. Flow cytometry analysis after magnetic separation of ISC-hpNSC and hpSC populations.
Flow cytometry analysis of OCT-4 expression of the different cell populations in this experiment: 100% ISC-hpNSC, 100% hpSC, 1% hpSC in ISC-hpNSC, 0.1% hpSC in ISC-hpNSC, 0.01% hpSC in ISC-hpNSC, and 0.001% hpSC in ISC-hpNSC population. The pure 100% ISC-hpNSC was the only cell population without OCT-4 expression signal.
Figure 3. qRT-PCR analysis of ISC-hpNSC.
Figure 3. qRT-PCR analysis of ISC-hpNSC.
Log curves for quantitation of the qRT-PCR analysis of ISC-hpNSC with dotted squares indicating the groupings of each sample run in triplicate. ISC-hpNSC and human fibroblasts group with the same level of POU5F1 expression of 10 hpSC. The negative control (NC) samples are below the 1 hpSC (100 hpSC) cell reaction. Linearity of curves Ct was between 17–35 cycles.
Figure 4. Acute toxicity study of ISC-hpNSC.
Figure 4. Acute toxicity study of ISC-hpNSC.
(a) Engraftment of ISC-hpNSC in the striatum of athymic nude rats. On the left is the coronal section with the dotted rectangle showing the location where the ISC-hpNSC graft was found. The black arrow represents the direction of the injection site. ST: striatum; CC: corpus callosum. On the right is a higher magnification micrograph showing the ISC-hpNSC graft stained for anti-STEM121 (red), anti-tyrosine hydroxylase (green), and DAPI (blue). (b) IBA-1+ staining comparison between rats receiving ISC-hpNSC or vehicle control. On the left is the ISC-hpNSC graft and on the right is the injection site in the vehicle control animal stained for anti-STEM121 (red), anti-IBA-1 (green) and DAPI. The number of IBA-1+ microglia cells surrounding the ISC-hpNSC graft is comparable to the number found at the injection site in the vehicle control animals. (c) TUNEL staining and quantification of apoptotic cells in animals injected with vehicle control and escalating doses of ISC-hpNSC. White arrows point to the apoptotic cells. Data was expressed as average ± SEM, one-factor ANOVA with Dunnett test comparing number of apoptotic cells of ISC-hpNSC doses against vehicle control: n = 3; α = 0.05; ***P < 0.001; *P < 0.05.
Figure 5. Tumorigenicity and biodistribution study of…
Figure 5. Tumorigenicity and biodistribution study of ISC-hpNSC.
(a) Number of animals with teratomas, ataxia, stereotypy, tremors, head tilt, vocalization, and leaning in the hpSC, ISC-hpNSC, and vehicle control groups. (b) Average body weight of the hpSC, ISC-hpNSC, and vehicle control groups with no statistically significant differences between the groups. (c) Representative image of a teratoma found in the brain of a rat injected with undifferentiated hpSC with disorderly growth with red blood cells (red area) and numerous clear spaces. The black dotted square represents the area where the higher magnification image on the right was taken, showing the presence of cartilage (bottom left corner), mucous-producing cells (top left) and nervous tissue, representing disorderly endodermal and ectodermal differentiation as assessed by an experienced board certified pathologist.

References

    1. Thomson J. A. et al.. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).
    1. Takahashi K. et al.. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872, doi: 10.1016/j.cell.2007.11.019 (2007).
    1. Revazova E. S. et al.. Patient-specific stem cell lines derived from human parthenogenetic blastocysts. Cloning and stem cells 9, 432–449, doi: 10.1089/clo.2007.0033 (2007).
    1. Tachibana M. et al.. Human embryonic stem cells derived by somatic cell nuclear transfer. Cell 153, 1228–1238, doi: 10.1016/j.cell.2013.05.006 (2013).
    1. Ratcliffe E., Glen K. E., Naing M. W. & Williams D. J. Current status and perspectives on stem cell-based therapies undergoing clinical trials for regenerative medicine: case studies. British medical bulletin 108, 73–94, doi: 10.1093/bmb/ldt034 (2013).
    1. Carpenter M. K., Frey-Vasconcells J. & Rao M. S. Developing safe therapies from human pluripotent stem cells. Nature biotechnology 27, 606–613, doi: 10.1038/nbt0709-606 (2009).
    1. Frey-Vasconcells J., Whittlesey K. J., Baum E. & Feigal E. G. Translation of stem cell research: points to consider in designing preclinical animal studies. Stem cells translational medicine 1, 353–358, doi: 10.5966/sctm.2012-0018 (2012).
    1. Kuroda T. et al.. Highly sensitive in vitro methods for detection of residual undifferentiated cells in retinal pigment epithelial cells derived from human iPS cells. PloS one 7, e37342, doi: 10.1371/journal.pone.0037342 (2012).
    1. Kanemura H. et al.. Tumorigenicity studies of induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) for the treatment of age-related macular degeneration. PloS one 9, e85336, doi: 10.1371/journal.pone.0085336 (2014).
    1. Kawamata S., Kanemura H., Sakai N., Takahashi M. & Go M. J. Design of a Tumorigenicity Test for Induced Pluripotent Stem Cell (iPSC)-Derived Cell Products. Journal of clinical medicine 4, 159–171, doi: 10.3390/jcm4010159 (2015).
    1. Bailey A. M. Balancing tissue and tumor formation in regenerative medicine. Science translational medicine 4, 147fs128, doi: 10.1126/scitranslmed.3003685 (2012).
    1. Hentze H. et al.. Teratoma formation by human embryonic stem cells: evaluation of essential parameters for future safety studies. Stem cell research 2, 198–210, doi: 10.1016/j.scr.2009.02.002 (2009).
    1. Lee A. S. et al.. Effects of cell number on teratoma formation by human embryonic stem cells. Cell cycle 8, 2608–2612 (2009).
    1. Daley G. Q. et al.. Setting Global Standards for Stem Cell Research and Clinical Translation: The 2016 ISSCR Guidelines. Stem cell reports, doi: 10.1016/j.stemcr.2016.05.001 (2016).
    1. Kimmelman J. et al.. New ISSCR guidelines: clinical translation of stem cell research. Lancet, doi: 10.1016/S0140-6736(16)30390-7 (2016).
    1. Trounson A. & McDonald C. Stem Cell Therapies in Clinical Trials: Progress and Challenges. Cell stem cell 17, 11–22, doi: 10.1016/j.stem.2015.06.007 (2015).
    1. Alper J. Geron gets green light for human trial of ES cell-derived product. Nature biotechnology 27, 213–214, doi: 10.1038/nbt0309-213a (2009).
    1. Schwartz S. D. et al.. Human embryonic stem cell-derived retinal pigment epithelium in patients with age-related macular degeneration and Stargardt’s macular dystrophy: follow-up of two open-label phase 1/2 studies. Lancet 385, 509–516, doi: 10.1016/S0140-6736(14)61376-3 (2015).
    1. Agulnick A. D. et al.. Insulin-Producing Endocrine Cells Differentiated In Vitro From Human Embryonic Stem Cells Function in Macroencapsulation Devices In Vivo. Stem cells translational medicine 4, 1214–1222, doi: 10.5966/sctm.2015-0079 (2015).
    1. Gonzalez R. et al.. Proof of concept studies exploring the safety and functional activity of human parthenogenetic-derived neural stem cells for the treatment of Parkinson’s disease. Cell transplantation 24, 681–690, doi: 10.3727/096368915X687769 (2015).
    1. Peterson S. E. & Loring J. F. Genomic instability in pluripotent stem cells: implications for clinical applications. The Journal of biological chemistry 289, 4578–4584, doi: 10.1074/jbc.R113.516419 (2014).
    1. Kimbrel E. A. & Lanza R. Hope for regenerative treatments: toward safe transplantation of human pluripotent stem-cell-based therapies. Regenerative medicine 10, 99–102, doi: 10.2217/rme.14.89 (2015).
    1. Piltti K. M., Salazar D. L., Uchida N., Cummings B. J. & Anderson A. J. Safety of human neural stem cell transplantation in chronic spinal cord injury. Stem cells translational medicine 2, 961–974, doi: 10.5966/sctm.2013-0064 (2013).
    1. Schwartz S. D. et al.. Embryonic stem cell trials for macular degeneration: a preliminary report. Lancet 379, 713–720, doi: 10.1016/S0140-6736(12)60028-2 (2012).
    1. Trounson A. & DeWitt N. D. Pluripotent stem cells progressing to the clinic. Nature reviews. Molecular cell biology 17, 194–200, doi: 10.1038/nrm.2016.10 (2016).
    1. Lee A. S., Tang C., Rao M. S., Weissman I. L. & Wu J. C. Tumorigenicity as a clinical hurdle for pluripotent stem cell therapies. Nature medicine 19, 998–1004, doi: 10.1038/nm.3267 (2013).
    1. . A Study to Evaluate the Safety of Neural Stem Cells in Patients With Parkinson’s Disease, (2016).
    1. Gonzalez R. et al.. Neural Stem Cells Derived from Human Parthenogenetic Stem Cells Engraft and Promote Recovery in a Nonhuman Primate Model of Parkinson’s Disease. Cell transplantation, doi: 10.3727/096368916X691682 (2016).
    1. Revazova E. S. et al.. HLA homozygous stem cell lines derived from human parthenogenetic blastocysts. Cloning and stem cells 10, 11–24, doi: 10.1089/clo.2007.0063 (2008).
    1. Lo B. & Parham L. Ethical issues in stem cell research. Endocrine reviews 30, 204–213, doi: 10.1210/er.2008-0031 (2009).
    1. Mansnerus J. Patentability of Parthenogenic Stem Cells: International Stem Cell Corporation v. Comptroller General of Patents. European journal of health law 22, 267–286 (2015).
    1. Daughtry B. & Mitalipov S. Concise review: parthenote stem cells for regenerative medicine: genetic, epigenetic, and developmental features. Stem cells translational medicine 3, 290–298, doi: 10.5966/sctm.2013-0127 (2014).
    1. Johannesson B. et al.. Comparable frequencies of coding mutations and loss of imprinting in human pluripotent cells derived by nuclear transfer and defined factors. Cell stem cell 15, 634–642, doi: 10.1016/j.stem.2014.10.002 (2014).
    1. Cuellar C. A. et al.. Propagation of sinusoidal electrical waves along the spinal cord during a fictive motor task. The Journal of neuroscience: the official journal of the Society for Neuroscience 29, 798–810, doi: 10.1523/JNEUROSCI.3408-08.2009 (2009).
    1. Gonzalez R. et al.. Deriving dopaminergic neurons for clinical use. A practical approach. Scientific Reports 3, 1–5, doi: 10.1038/srep01463 (2013).
    1. Robinson S. et al.. A European pharmaceutical company initiative challenging the regulatory requirement for acute toxicity studies in pharmaceutical drug development. Regulatory toxicology and pharmacology: RTP 50, 345–352, doi: 10.1016/j.yrtph.2007.11.009 (2008).
    1. Buckley L. A. & Dorato M. A. High dose selection in general toxicity studies for drug development: A pharmaceutical industry perspective. Regulatory toxicology and pharmacology: RTP 54, 301–307, doi: 10.1016/j.yrtph.2009.05.015 (2009).
    1. Andersson C., Hamer R. M., Lawler C. P., Mailman R. B. & Lieberman J. A. Striatal volume changes in the rat following long-term administration of typical and atypical antipsychotic drugs. Neuropsychopharmacology: official publication of the American College of Neuropsychopharmacology 27, 143–151, doi: 10.1016/S0893-133X(02)00287-7 (2002).
    1. Shimizu S. In The Laboratory Mouse (eds Hedrich H. J. & Bullock G.) Ch. 32, 527–541 (Elsevier, 2004).
    1. Hardman C. D. et al.. Comparison of the basal ganglia in rats, marmosets, macaques, baboons, and humans: volume and neuronal number for the output, internal relay, and striatal modulating nuclei. The Journal of comparative neurology 445, 238–255 (2002).
    1. Yin D. et al.. Striatal volume differences between non-human and human primates. Journal of neuroscience methods 176, 200–205, doi: 10.1016/j.jneumeth.2008.08.027 (2009).
    1. Krabbe K. et al.. Increased intracranial volume in Parkinson’s disease. Journal of the neurological sciences 239, 45–52, doi: 10.1016/j.jns.2005.07.013 (2005).
    1. Chen L. et al.. Human neural precursor cells promote neurologic recovery in a viral model of multiple sclerosis. Stem cell reports 2, 825–837, doi: 10.1016/j.stemcr.2014.04.005 (2014).
    1. Redmond D. E. Jr. et al.. Behavioral improvement in a primate Parkinson’s model is associated with multiple homeostatic effects of human neural stem cells. Proceedings of the National Academy of Sciences of the United States of America 104, 12175–12180, doi: 10.1073/pnas.0704091104 (2007).
    1. Prockop D. J. Defining the probability that a cell therapy will produce a malignancy. Molecular therapy: the journal of the American Society of Gene Therapy 18, 1249–1250, doi: 10.1038/mt.2010.99 (2010).
    1. Sierra A. et al.. Microglia shape adult hippocampal neurogenesis through apoptosis-coupled phagocytosis. Cell stem cell 7, 483–495, doi: 10.1016/j.stem.2010.08.014 (2010).
    1. Garitaonandia I. et al.. Increased risk of genetic and epigenetic instability in human embryonic stem cells associated with specific culture conditions. PloS one 10, e0118307, doi: 10.1371/journal.pone.0118307 (2015).
    1. Laurent L. C. et al.. Dynamic changes in the copy number of pluripotency and cell proliferation genes in human ESCs and iPSCs during reprogramming and time in culture. Cell stem cell 8, 106–118, doi: 10.1016/j.stem.2010.12.003 (2011).
    1. Peterson S. E., Garitaonandia I. & Loring J. F. The tumorigenic potential of pluripotent stem cells: What can we do to minimize it? BioEssays: news and reviews in molecular, cellular and developmental biology 38 Suppl 1, S86–S95, doi: 10.1002/bies.201670915 (2016).

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