Cell Reprogramming Leaps Ahead: First Transplant of Primate Induced Pluripotent Cell-Derived Neurons into Donor Brain

"Reprogramming" of adult differentiated cells into pluripotent stem cells is an exciting method in biology that holds enormous promise for rejuvenation biotechnology. Now, for the first time, Dr. Su-Chun Zhang and coworkers at the University of Wisconsin-Madison have successfully generated neurons from reprogrammed nonhuman primate cells, transplanted them back into the same animal's brain, and seen them successfully and cleanly integrate into the local tissue.
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The motion disorders that are most characteristic of Parkinson’s disease (PD) are principally caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc). The first major use of cell therapy as a rejuvenation biotechnology was the use of fetal/embryonic mixed mesencephalic tissue grafts to replace some of the functional properties of these lost cells, by engrafting them at a location closer to their striatal target sites for local DA delivery.(1) While there is some evidence that the rapid and specific loss of these neurons in PD may involve distinct pathological processes,(2) any such mechanisms emerge as additive insults to the universal process of slower and more linear progressive loss of DA neurons during the course of “normal” degenerative aging. Orthotopic replacement of these neurons to fully rebuild DA neuronal circuitry will be a key component of restoring and maintaining motor function and other aspects of brain functioning with aging.

In the early mixed cell graft studies, many patients enjoyed striking temporary improvements in the major motion disorder symptoms. But the magnitude of clinical response has been highly variable, gains have proven impermanent, and ≈15% of transplanted patients have developed substantial dyskinesias during the “off” phase of levodopa treatment.(1) There have been additional problems arising from the tissue source for the transplants: the supply of tissue is necessarily severely limited, and allogenic tissue grafts immunogenic, leading to immune rejection in some cases and necessitating immunosuppression in a majority of transplant recipients.(1)

Many of these limitations could be overcome with the availability of a better cell source for transplant: pure DA neurons from pluripotent stem cells, such as embryonic stem cells (ESC), or preferably patient-identical pluripotent cell types such as induced pluripotent stem cells (iPS) or somatic cell nuclear transfer (SCNT — “therapeutic cloning”). iPS- or SCNT-derived cells in particular would be immunologically “self,” and therefore free of the risk of rejection (with the possible exception of mitochondrial immunogenicity in SCNT cells). As well, a pure DA neuron source would likely eliminate the graft-induced dyskinesias that emerged in the trials, which appear to have been the result of “contamination” of the tissue grafts with the serotonergic neurons that are present in the mixed cell population used in these early trials (3, and see a previous CSO Team blog post on improved cell differentiation for dopaminergic brain repair). And the nearly-unlimited replicative capacity of pluripotent cells, combined with an efficient and stable means of differentiation, holds the promise of generating sufficient numbers of DA neurons to meet the clinical need for brain repair presented by frank PD; and by “normal” age-related SNc DA brain aging.

Now, for the first time, Dr. Su-Chun Zhang and coworkers at the University of Wisconsin-Madison have successfully generated neurons from reprogrammed nonhuman primate cells, transplanted them back into the originating animal’s brain, and seen them successfully and cleanly integrate into the local tissue.(4) And in the process, they have substantially removed additional concerns that have recently been raised with iPS technology.

The authors first derived iPS from fibroblasts taken from skin samples from three adult rhesus monkeys, and transfected them using the original four-factor Yamanaka cocktail of reprogramming genes (OCT4, SOX2, KLF4, and c-MYC). The resulting iPS exhibited the characteristic morphology of primate ESC, were positive for expression of SOX2 and NANOG, and formed teratomas upon injection into immunodeficient mice. These cells were then transfected with green fluorescent protein (GFP) labeling, and moved through a neural rosette intermediate to derive neurons and glia, being allowed to differentiate for 42 days before transplantation (based on protocols previously established in rodent transplantation studies). DA neurons were identified based on expression of tyrosine hydroxylase and on regionally-specific DA neuronal markers; other cell types were identified on similar grounds.(4)

These cells were then tested in a “hemiparkinsonian” nonhuman primate model, generated via unihemispheric lesioning with the DA neuronal-selective neurotoxin MPTP, resulting in loss of motor control on the hand contralateral to the hemisphere of the lesions, as well as more global PD-like symptoms. Twelve to eighteen months later, each monkey then received a transplant of mature neurons and still-dividing neural progenitors derived from its own iPS cells, grafted into the SNc and striatum.(4)

Six months after transplantation, serial coronal sections of the iPSC-derived graft sites were examined in the three monkeys. These exhibited appropriate regionally-specific neuronal organization. The investigators state that “the grafts were not readily discernible in any of the monkeys,” yet GFP immunostaining or the use of fluorometric microscope revealed striatal and nigral grafts in all three monkeys with between 11,556 and 34,321 transplanted cells, concentrated on the graft sites. Additionally, the authors speculate that yet more transplanted cells may also have been present, but not detected due to underexpression of the GFP label; this would have been particularly likely in fully-differentiated, postmitotic cell types such as neurons.(4)

An institutional press release quotes two investigators:

“When you look at the brain, you cannot tell that it is a graft,” says senior author Su-Chun Zhang … “Structurally the host brain looks like a normal brain; the graft can only be seen under the fluorescent microscope.”

Marina Emborg, … lead co-author of the study, says, “This is the first time I saw, in a nonhuman primate, that the transplanted cells were so well integrated, with such a minimal reaction. And after six months, to see no scar, that was the best part.”

None of the cells present in the grafts labeled as mitotically-active, nor did any cells express the pluripotency genes OCT4, NANOG, or SOX17, nor Brachyury (a marker of mesodermal commitment), providing reassurance against the concerns about the possibly tumorigenic properties of (unstably-differentiated) transplanted cells derived from pluripotent stem cells. Most neurons displaying the GFP label were confined to the graft site, and innervated the surrounding tissue for up to 1.5 mm.(4) And unlike in two previous studies of transplantation of neurons derived from human ESC into nonhuman primate brain,(5,6) the transplant sites exhibited no lymphocyte infiltration or other signs of rejection(4) — a concern that the authors themselves had raised in a previous study of iPS-derived cells in mice.(7) A moderate increase in GFAP+ staining, concentrated at graft sites and potentially a worrisome finding, was instead at least partially attributable to astrocytes whose GFP labeling marked them as derived from grafted cells rather than from activation of endogenous astrocytes.(4) The investigators also found a mild increase in human leukocyte antigen D-related staining, which they attributed to nonspecific trauma rather than infiltration of the graft by immune cells drawn in as a result of a rejection process.(4)

Unfortunately, “The vast majority of the GFP+ neurons were GABA+ and few were positive for TH. This may explain why we did not observe obvious behavioral recovery and PET changes.”(4) So this study is good proof of safety, but not proof-of-principle for DA neuronal repair using iPS-derived DA neurons in nonhuman primates. Fortunately, in independent work in nonhuman primates, Drs. Donald Redmond, Jr. and John R. Sladek, Jr. have made significant progress in overcoming the key hurdles to human DA circuit repair, including addressing some of the weaknesses in the present study.(4) These investigators have developed methods to encourage orthotopic transplants to grow outward toward their striatal targets and rebuild the DA circuit (incompletely, so far),(8,9) and for stable and high-conversion derivation of DA neurons from human ESC that are viable and to a degree functional in nonhuman primate brain.(9) Meanwhile, Japanese researchers have shown that with suitable growth factors, maturing neurons derived from a human ESC-derived cell line can not only stably and consistently differentiate into a population enriched with DA neurons, but can attenuate the Parkinsonian symptoms of MPTP-treated cynomolgus monkeys for at least 12 months post-transplant.(10)

As these methods are perfected and disseminated through the rejuvenation biotechnology research community, the day when we can use a person’s own cells to return her fine motor control to youthful smoothness and precision is increasingly in reach — waiting to be grasped with unshaking hands.

References

1. Lindvall O, Björklund A. Cell therapy in Parkinson’s disease. NeuroRx. 2004 Oct;1(4):382-93. Review. PubMed PMID: 15717042; PubMed Central PMCID: PMC534947.

2. Levy G. The relationship of Parkinson disease with aging. Arch Neurol. 2007 Sep;64(9):1242-6. Review. PubMed PMID: 17846263.

3. Politis M, Wu K, Loane C, Quinn NP, Brooks DJ, Rehncrona S, Bjorklund A, Lindvall O, Piccini P. Serotonergic neurons mediate dyskinesia side effects in Parkinson’s patients with neural transplants. Sci Transl Med. 2010 Jun 30;2(38):38ra46. PubMed PMID: 20592420.

4. Emborg ME, Liu Y, Xi J, Zhang X, Yin Y, Lu J, Joers V, Swanson C, Holden JE, Zhang SC. Induced pluripotent stem cell-derived neural cells survive and mature in the nonhuman primate brain. Cell Rep. 2013 Mar 28;3(3):646-50. doi: 10.1016/j.celrep.2013.02.016. Epub 2013 Mar 14. PubMed PMID: 23499447.

5. Emborg ME, Zhang Z, Joers V, Brunner K, Bondarenko V, Ohshima S, Zhang SC. Intracerebral Transplantation of Differentiated Human Embryonic Stem Cells to Hemiparkinsonian Monkeys. Cell Transplant. 2012 Jun 15. doi: 10.3727/096368912X47144. [Epub ahead of print] PubMed PMID: 22710103.

6. Kriks S, Shim JW, Piao J, Ganat YM, Wakeman DR, Xie Z, Carrillo-Reid L, Auyeung G, Antonacci C, Buch A, Yang L, Beal MF, Surmeier DJ, Kordower JH, Tabar V, Studer L. Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease. Nature. 2011 Nov 6;480(7378):547-51. doi: 10.1038/nature10648. PubMed PMID: 22056989; PubMed Central PMCID: PMC3245796.

7. Zhao T, Zhang ZN, Rong Z, Xu Y. Immunogenicity of induced pluripotent stem cells. Nature. 2011 May 13;474(7350):212-5. doi: 10.1038/nature10135. PubMed PMID: 21572395.

8. Redmond DE Jr, Weiss S, Elsworth JD, Roth RH, Wakeman DR, Bjugstad KB, Collier TJ, Blanchard BC, Teng YD, Synder EY, Sladek JR Jr. Cellular repair in the parkinsonian nonhuman primate brain. Rejuvenation Res. 2010 Apr-Jun;13(2-3):188-94. Review. PubMed PMID: 20370501; PubMed Central PMCID: PMC2946058.

9. Daadi MM, Grueter BA, Malenka RC, Redmond DE Jr, Steinberg GK. Dopaminergic neurons from midbrain-specified human embryonic stem cell-derived neural stem cells engrafted in a monkey model of Parkinson’s disease. PLoS One. 2012;7(7):e41120. doi: 10.1371/journal.pone.0041120. Epub 2012 Jul 17. PubMed PMID: 22815935; PubMed Central PMCID: PMC3398927.

10. Doi D, Morizane A, Kikuchi T, Onoe H, Hayashi T, Kawasaki T, Motono M, Sasai Y, Saiki H, Gomi M, Yoshikawa T, Hayashi H, Shinoyama M, Refaat MM, Suemori H, Miyamoto S, Takahashi J. Prolonged maturation culture favors a reduction in the tumorigenicity and the dopaminergic function of human ESC-derived neural cells in a primate model of Parkinson’s disease. Stem Cells. 2012 May;30(5):935-45. doi: 10.1002/stem.1060. PubMed PMID: 22328536.

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