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Proof-of-principle study of the first-ever autologous iPSC-derived cell transplant in non-human primates

Shinya Yamanaka was awarded the Noble Prize for Medicine last year for his work on cellular reprogramming and creating induced pluripotent stem cells (iPSC).  Shinya Yamanaka found four transcription factors (Oct-3/4, Sox2, c-Myc, and Klf4) that determine pluripotency and he was able to reprogram differentiated adult cells into pluripotent cells that can then be re-differentiated into fully specialized tissue.  His findings raised great expectations, especially in the field of cellular therapy and regenerative medicine.  However, the path to the therapeutic use of iPSC is long and not without complications.  It was believed that iPSCs would avoid any immunogenic response because these cells can be developed from a patient’s own somatic cells. However, Zhao et al. challenged this notion when they discovered that iPSCs derived from C57BL/6 (B6) mice by the standard retroviral approach formed teratomas once transplanted into syngenic host mice and induced a rapid T cell dependent immune response1.

Recently, a group of researchers led by Dr. Su-Chun Zhang at the Waisman Center on the University of Wisconsin-Madison campus have shown that utilizing iPSCs as an autologous cell therapy is feasible and without any immunological reaction or rejection.  Dr. Zhang was the first to derive neural cells from embryonic stem cells (ESCs), as well as from iPSCs, and now has shown the first proof-of-principle that autologous iPSC-derived cells can engraft and survive in the primate brain.

In a new study published in Cell Reports, Zhang’s group reports on the successful generation of iPSCs from fibroblasts obtained from 8-10 year old rhesus monkeys (Macaca mulatta) using retroviruses containing the four Yamanaka factors, subsequent neuronal differentiation and cell transplantation back into the donor monkeys2.  The rhesus iPSCs were differentiated into neuroepithelia with the characteristic neural tube-like rosettes and expression of neuroectoderm transcription factors Pax6 and Sox1.  The neuronal rosettes were then expanded and further differentiated into neurons so that by the time for cell transplantation (day 42), 37% of the cells were bIII-tubulin+ neurons, 16% were S100b+ immature astrocytes, and 47% were Nestin+ progenitors.

primate ipsc cell pic
To examine the feasibility of transplanting autologous iPSC-derived neural progenitors in Parkinson’s disease, Zhang created parkinsonism in the rhesus monkeys by unilateral intracarotid artery injection of a neurotoxin 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP).  A year to 18 months after the MPTP infusion, all the monkeys developed a stable hemiparkinsonian condition distinguished by the characteristic tremors, bradykinesia, imbalance and impairment in motor skills.  At day 30 of cell culture the iPSC-derived neural progenitors were labeled with a GFP (green fluorescent protein) lentivirus and cells were transplanted at day 42 into the striatum and substantia nigra of the same monkey from which the iPSC were derived.

The monkey did not receive any immune suppression and engraftment of the GFP-labeled iPSC-derived neural progenitors was assessed at 6 months post-transplantation using stereological analysis on serial coronal sections.  Distinct grafts were present in the injected regions where 63% of the cells were microtubule-associated protein 2 (MAP2)+ neurons, 22% were glial fibrillary acidic protein (GFAP)+ astrocytes, and 10% were myelin basic protein (MBP)+ oligodendrocytes.  The GFP+ neurons showed long fibers extending into the surrounding host tissue and some neurons were present outside of the graft region expressing markers of mature neuronal differentiation.  Additionally, there was an absence of markers for pluripotent stem cells (OCT4, NANOG, SOX17, and Brachyury) and no positive staining for Ki67 (labels mitotic cells) indicating that the grafted progenitors had terminally differentiated.

Zhao et al. showed that the teratomas formed from transplanted iPSCs had an immunological rejection by syngenic mice1.  However, Zhang demonstrates that the autologous iPSC-derived neural cell transplants in the primate brain do not undergo immunological rejections suggested by the lack of CD3 and CD8 (lymphocyte markers) staining.  There was minimal response of endogenous glia (astrocytes and microglia) since staining for human leukocyte antigen D-related (HLA-DR; microglia and macrophage marker) was seen throughout the brain including the grafted regions.

Despite the positive engraftment and differentiation of the iPSC-derived neural progenitors, they did not see any behavioral improvement in the parkinsonian monkeys.  A potential explanation is that the GFP+ neurons were mostly g-Aminobutyric acid (GABA+) and few were positive for tyrosine hydroxylase (TH+).  TH catalyzes the formation of L-DOPA, the rate-limiting step in the biosynthesis of dopamine.  Deficiency in TH has been implicated in giving rise to parkinsonian characteristics3.  Also, the number of transplanted cells may not have been enough to replace the dopamine-making cells in the primate brain.

However, this study provides hope for cell therapy using autologous iPSC-derived cells.  The iPSC-derived neural progenitors survived and differentiated into mature neurons, astrocytes, and oligodendrocytes in the primate brain with no evidence of immune rejection or teratoma formation.  The transplanted cells structurally integrated into the host brain and with characteristic features of neurons indicating by extending long processes and features of oligodendrocytes indicated by staining for myelin basic protein, suggestive of myelination.  This proof-of-principle study of the first-ever transplant of iPSC-derived cells back into the same non-human primate presents hope for personalized regenerative medicine and the neurodegenerative patient population.

Further reading:

1. Zhao, Tongbiao; Zhang, Zhen-Ning; Rong, Zhili; and Xu, Yang.  Immunogenicity of induced pluripotent stem cells.  Nature. 474, 212–215, 9 June 2011

2. Emborg ME, Liu Y, Xi J, Chang X, Yin Y, Lu J, Joers V, Swanson C, Holden JE, and Zhang Su.  Induced pluripotent stem cell-derived neural stem cells survive and mature in the nonhuman primate brain.  Cell Reports 3, 1-5, March 28, 2013.

3. Goodwill KE, Sabatier C, Marks C, Raag R, Fitzpatrick PF, Stevens RC. Crystal Structure of tyrosine hydroxylase at 2.3a and its implications for inherited neurodegenerative diseases. Nature Structural Biology 4 (7): 578–585, 1997

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ATM INHIBITOR SENSITIZES GLIOBLASTOMA TO IONIZING RADIATION THERAPY

A brain tumor is an abnormal growth of tissue in the brain. Brain tumors may develop from neural elements within the brain (primary brain tumor), or they may represent spread of distant cancers (secondary brain tumor). According to the National Cancer Institute, in 2013 the estimated new cases and deaths from brain tumor in the United States would be 23,130 and 14,080 respectively. There are many types of primary brain tumors. Primary brain tumors are named according to the type of cells or the part of the brain in which they originate. For example, most primary brain tumors begin in glial cells. This type of tumor is called a glioma. Among adults, the most common types are:

  • Meningioma: This tumor arises in the meninges. It’s usually benign (grade I) and grows slowly.
  • Oligodendroglioma: This tumor arises from cells that make the fatty substance that covers and protects nerves. It usually occurs in the cerebrum. It’s most common in middle-aged adults.
  • Astrocytoma: This tumor arises from star-shaped glial cells called astrocytes.

Among these, glioblastoma multiforme (GBM) is the most common and deadliest of malignant primary brain tumors in adults. Classified as a Grade IV (most serious) astrocytoma, GBM develops from the lineage of star-shaped glial cells, called astrocytes that support nerve cells. Even though GBM is one of the most aggressive forms of human cancers, the incident of this disease is fairly less.  Every year, in the United States approximately 7 out of 100,000 people are diagnosed with high-grade glioblastoma. Uncontrolled cellular proliferation, diffuse infiltration, and resistance to apoptosis are considered as hallmarks of GBM. The current standard of treatment in glioblastoma involves surgery followed by radiation therapy, and chemotherapy with the DNA alkylating agent temozolomide (TMZ).  The efficacy of this current treatment strategy is limited as the median survival is just over one year because of development of resistance. Therefore, more effective treatment plans need to be developed and explored. In a recent study published in the peer review journal Clinical Cancer Research (2013 Apr 25) Thorpe and colleagues reported that combined treatment of ataxia telangiectasia mutated (ATM) inhibitor and radiation significantly increased survival of mice bearing glioblastoma with p53 mutation compared to treatment with ATM inhibitor or radiation alone.

The nuclear protein kinase ataxia-telangiectasia mutated (ATM) plays a crucial role in DNA-damage response by transducing double-strand breaks. This kinase phosphorylates several effectors that play key roles in cell cycle progression and DNA repair pathways.

gLIOBLASYTOMA -resized-600 (1)

Ionizing radiation is the most effective therapy for glioblastoma. Ionizing radiation works by damaging the DNA of exposed tissue leading to cellular death. However, tumor cells evade death and contribute to radioresistance through preferential activation of the DNA damage checkpoint response and an increase in DNA repair capacity. Therefore, combined treatment of ionizing radiation with inhibitors of DNA damage checkpoint may overcome the problem of resistance in glioblastoma. Since ATM is associated with DNA damage repair there are several small molecule ATM inhibitors that disrupt ATM function and enhances sensitivity of tumor cells to radiation as well as chemotherapeutic agents. ATM inhibitor KU-60019 is a potent inhibitor effectively blocking activation of key ATM targets and radiosensitizing human glioma cells in vitro. In their study Thorpe et al. (2013) showed that mice bearing glioblastoma survived longer periods of time (145 days) when treated with KU-60019 and radiation than mice treated with radiation alone (58 days) or KU-60019 alone (66 days). In addition, this study also noted that mice bearing glioma with mutant p53 are more sensitive to KU-60019 dependent radiosensitization than mice bearing glioma with p53 wild-type. Altogether this study suggests that ATM kinase inhibition may be an effective strategy as adjuvant therapy for patients with mutant p53 brain cancers.

References:

1.Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB, Dewhirst MW, Bigner DD, Rich JN: Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 2006, 444(7120):756-760.

2.Biddlestone-Thorpe L, Sajjad M, Rosenberg E, Beckta JM, Valerie NC, Tokarz M, Adams BR, Wagner AF, Khalil A, Gilfor D et al: ATM kinase inhibition preferentially sensitizes p53 mutant glioma to ionizing radiation. Clin Cancer Res 2013.

3. http://www.cancer.gov/cancertopics/wyntk/brain/page2