Maria Sundberg

Pharmacological Rescue of Mitochondrial Deficits in iPSC-Derived Neural Cells from Patients with Familial Parkinson’s Disease

Neural cells derived from induced pluripotent stem cells from patients with genetic forms of Parkinson’s disease provide insights into disease pathogenesis. Understanding Mitochondrial Deficits in Parkinson’s Disease Parkinson’s disease (PD) is a common, progressive neurodegenerative disease characterized by loss of dopaminergic neurons in the nigrostriatal pathway of the brain, resulting in motor and cognitive deficits. Rodent and primate models only partially predict disease mechanisms. In a new study, Cooper et al. set out to make a human cellular model of PD. First, the authors obtained fibroblasts from members of families with genetically defined forms of PD and generated induced pluripotent stem cells (iPSCs) from the fibroblasts. They then induced differentiation of these PD patient–derived iPSCs into neural cells including dopaminergic neurons to study how the genetic mutations influenced the responses of neural cells to various cellular stressors. Mitochondrial dysfunction has already been implicated in the pathogenesis of PD, so the authors decided to treat their iPSC-derived neural cells from patients with rare familial forms of PD with chemical stressors and toxins known to disrupt mitochondrial function. The researchers observed a gradual increase in sensitivity to cellular stress as the cell type analyzed became functionally closer to the vulnerable cell types in the PD brain; that is, fibroblasts taken directly from PD patients were less sensitive to the chemical stressors than iPSC-derived neural cells. Several drugs helped iPSC-derived neural cells to resist the damaging effects of the cellular stressors. These studies with human neural cells from iPSCs from patients with familial PD highlight opportunities to characterize disease pathways and to screen for new therapeutic agents. Parkinson’s disease (PD) is a common neurodegenerative disorder caused by genetic and environmental factors that results in degeneration of the nigrostriatal dopaminergic pathway in the brain. We analyzed neural cells generated from induced pluripotent stem cells (iPSCs) derived from PD patients and presymptomatic individuals carrying mutations in the PINK1 (PTEN-induced putative kinase 1) and LRRK2 (leucine-rich repeat kinase 2) genes, and compared them to those of healthy control subjects. We measured several aspects of mitochondrial responses in the iPSC-derived neural cells including production of reactive oxygen species, mitochondrial respiration, proton leakage, and intraneuronal movement of mitochondria. Cellular vulnerability associated with mitochondrial dysfunction in iPSC-derived neural cells from familial PD patients and at-risk individuals could be rescued with coenzyme Q10, rapamycin, or the LRRK2 kinase inhibitor GW5074. Analysis of mitochondrial responses in iPSC-derived neural cells from PD patients carrying different mutations provides insight into convergence of cellular disease mechanisms between different familial forms of PD and highlights the importance of oxidative stress and mitochondrial dysfunction in this neurodegenerative disease.

Successful Function of Autologous iPSC-Derived Dopamine Neurons following Transplantation in a Non-Human Primate Model of Parkinson’s Disease

Autologous transplantation of patient-specific induced pluripotent stem cell (iPSC)-derived neurons is a potential clinical approach for treatment of neurological disease. Preclinical demonstration of long-term efficacy, feasibility, and safety of iPSC-derived dopamine neurons in non-human primate models will be an important step in clinical development of cell therapy. Here, we analyzed cynomolgus monkey (CM) iPSC-derived midbrain dopamine neurons for up to 2 years following autologous transplantation in a Parkinsons disease (PD) model. In one animal, with the most successful protocol, we found that unilateral engraftment of CM-iPSCs could provide a gradual onset of functional motor improvement contralateral to the side of dopamine neuron transplantation, and increased motor activity, without a need for immunosuppression. Postmortem analyses demonstrated robust survival of midbrain-like dopaminergic neurons and extensive outgrowth into the transplanted putamen. Our proof of concept findings support further development of autologous iPSC-derived cell transplantation for treatment of PD.

Improved cell therapy protocols for Parkinson's disease based on differentiation efficiency and safety of hESC-, hiPSC-, and non-human primate iPSC-derived dopaminergic neurons

The main motor symptoms of Parkinsons disease are due to the loss of dopaminergic (DA) neurons in the ventral midbrain (VM). For the future treatment of Parkinsons disease with cell transplantation it is important to develop efficient differentiation methods for production of human iPSCs and hESCs‐derived midbrain‐type DA neurons. Here we describe an efficient differentiation and sorting strategy for DA neurons from both human ES/iPS cells and non‐human primate iPSCs. The use of non‐human primate iPSCs for neuronal differentiation and autologous transplantation is important for preclinical evaluation of safety and efficacy of stem cell‐derived DA neurons. The aim of this study was to improve the safety of human‐ and non‐human primate iPSC (PiPSC)‐derived DA neurons. According to our results, NCAM+/CD29low sorting enriched VM DA neurons from pluripotent stem cell‐derived neural cell populations. NCAM+/CD29low DA neurons were positive for FOXA2/TH and EN1/TH and this cell population had increased expression levels of FOXA2, LMX1A, TH, GIRK2, PITX3, EN1, NURR1 mRNA compared to unsorted neural cell populations. PiPSC‐derived NCAM+/CD29low DA neurons were able to restore motor function of 6‐hydroxydopamine (6‐OHDA) lesioned rats 16 weeks after transplantation. The transplanted sorted cells also integrated in the rodent brain tissue, with robust TH+/hNCAM+ neuritic innervation of the host striatum. One year after autologous transplantation, the primate iPSC‐derived neural cells survived in the striatum of one primate without any immunosuppression. These neural cell grafts contained FOXA2/TH‐positive neurons in the graft site. This is an important proof of concept for the feasibility and safety of iPSC‐derived cell transplantation therapies in the future. STEM Cells 2013;31:1548–1562

Glucocorticoid hormones decrease proliferation of embryonic neural stem cells through ubiquitin-mediated degradation of cyclin D1.

Corticosteroids can influence brain function, and glucocorticoid hormone receptors (GRs) are present in brain tissue. We observed that GR and also mineralocorticoid receptor (MR) are expressed by embryonic rat neural stem cells (NSCs). NSCs in developing ventricular epithelium were positive for GR. Stimulation of cultured NSCs with the specific receptor ligands dexamethasone and corticosterone reduced cell proliferation, shown by 5′-bromo-2-deoxy-uridine labeling. The effect of the hormones was dose dependent and inhibited by the GR blocker mifepristone but not by spironolactone, blocking MR. Dexamethasone inhibited the cell cycle by decreasing the levels of cyclin D1 in NSCs. The hormone-induced decline was inhibited by MG132 (benzyloxycarbonyl-leucyl-leucyl-leucinal), showing an involvement of the ubiquitin proteasome system, In keeping with this, dexamethasone increased the ubiquitination of cyclin D1. In embryonic brain, dexamethasone inhibited cell proliferation of NSCs. This demonstrates that embryonic NSCs are critically influenced by glucocorticoids, which can have long-term effects in the brain.

CD marker expression profiles of human embryonic stem cells and their neural derivatives, determined using flow-cytometric analysis, reveal a novel CD marker for exclusion of pluripotent stem cells.

Human embryonic stem cells (hESCs) are pluripotent cells that can differentiate into neural cell lineages. These neural populations are usually heterogeneous and can contain undifferentiated pluripotent cells that are capable of producing teratomas in cell grafts. The characterization of surface protein profiles of hESCs and their neural derivatives is important to determine the specific markers that can be used to exclude undifferentiated cells from neural populations. In this study, we analyzed the cluster of differentiation (CD) marker expression profiles of seven undifferentiated hESC lines using flow-cytometric analysis and compared their profiles to those of neural derivatives. Stem cell and progenitor marker CD133 and epithelial adhesion molecule marker CD326 were more highly expressed in undifferentiated hESCs, whereas neural marker CD56 (NCAM) and neural precursor marker (chemokine receptor) CD184 were more highly expressed in hESC-derived neural cells. CD326 expression levels were consistently higher in all nondifferentiated hESC lines than in neural cell derivatives. In addition, CD326-positive hESCs produced teratomas in SCID mouse testes, whereas CD362-negative neural populations did not. Thus, CD326 may be useful as a novel marker of undifferentiated hESCs to exclude undifferentiated hESCs from differentiated neural cell populations prior to transplantation.

Production and isolation of NG2+ oligodendrocyte precursors from human embryonic stem cells in defined serum-free medium

Human embryonic stem cells (hESCs) are a promising source of oligodendrocyte precursor cells (OPCs) and oligodendrocytes. These cells can be used to repair myelin in central nervous system deficits such as multiple sclerosis or traumas such as spinal cord injury. Here, we introduce a novel differentiation method for the production of OPCs from hESCs. OPCs were differentiated as spheres in defined serum-free medium supplemented with recombinant human growth factors. A broad gene expression analysis revealed that this OPC population expressed Olig1/2, Sox10, PDGFR, Nkx2.2, Nkx6.2, oligodendrocyte-myelin glycoprotein, myelin basic protein (MBP), and proteolipid protein (PLP). According to quantitative RT-PCR analyses addition of ciliary neurotrophic factor (CNTF) upregulated the Olig2 mRNA levels in the OPC population. According to the flow cytometry analyses the OPC population was >90% NG2-positive, >80% PDGFR-positive, and >60% CD44-positive, and further matured into O4- (45%) and GalC- (80%) positive oligodendrocyte populations when cultured on top of human extracellular matrix proteins, which were used instead of Matrigel. In addition, OPCs matured into myelin-forming cells when cocultured with neuronal cells. The multilayered myelin sheet formation around axons was detected with transmission electron microscopy in cocultures. Further, the OPC populations could be purified with sorting of NG2(+) cells. These NG2(+) cells reformed spheres that remained stable during prolonged culturing (7weeks), and matured into GalC-positive oligodendrocytes. Importantly, these NG2(+) spheres were free of pluripotent Tra1-81, Oct-4, and CD326-positive hESCs. Thus, this method is suitable for the efficient production of OPCs and in the future for therapeutic graft production.

Markers of pluripotency and differentiation in human neural precursor cells derived from embryonic stem cells and CNS tissue.

Cell transplantation therapies for central nervous system (CNS) deficits such as spinal cord injury (SCI) have been shown to be effective in several animal models. One cell type that has been transplanted is neural precursor cells (NPCs), for which there are several possible sources. We have studied NPCs derived from human embryonic stem cells (hESCs) and human fetal CNS tissue (hfNPCs), cultured as neurospheres, and the expression of pluripotency and neural genes during neural induction and in vitro differentiation. mRNA for the pluripotency markers Nanog, Oct-4, Gdf3, and DNMT3b were downregulated during neural differentiation of hESCs. mRNA for these markers was found in nonpluripotent hfNPC at higher levels compared to hESC-NPCs. However, Oct-4 protein was found in hESC-NPCs after 8 weeks of culture, but not in hfNPCs. Similarly, SSEA-4 and CD326 were only found in hESC-NPCs. NPCs from both sources differentiated as expected to cells with typical features of neurons and astrocytes. The expressions of neuronal markers in hESC-NPCs were affected by the composition of cell culture medium, while this did not affect hfNPCs. Transplantation of hESC-NPC or hfNPC neurospheres into immunodeficient mouse testis or subcutaneous tissue did not result in tumor formation. In contrast, typical teratomas appeared in all animals after transplantation of hESC-NPCs to injured or noninjured spinal cords of immunodeficient rats. Our data show that transplantation to the subcutaneous tissue or the testes of immunodeficient mice is not a reliable method for evaluation of the tumor risk of remaining pluripotent cells in grafts.

Crystal structure of the second PDZ domain of SAP97 in complex with a GluR-A C-terminal peptide

Synaptic targeting of GluR‐A subunit‐containing glutamate receptors involves an interaction with synapse‐associated protein 97 (SAP97). The C‐terminus of GluR‐A, which contains a class I PDZ ligand motif (‐x‐Ser/Thr‐x‐φ‐COOH where φ is an aliphatic amino acid) associates preferentially with the second PDZ domain of SAP97 (SAP97PDZ2). To understand the structural basis of this interaction, we have determined the crystal structures of wild‐type and a SAP97PDZ2 variant in complex with an 18‐mer C‐terminal peptide (residues 890–907) of GluR‐A and of two variant PDZ2 domains in unliganded state at 1.8–2.44 Å resolutions. SAP97PDZ2 folds to a compact globular domain comprising six β‐strands and two α‐helices, a typical architecture for PDZ domains. In the structure of the peptide complex, only the last four C‐terminal residues of the GluR‐A are visible, and align as an antiparallel β‐strand in the binding groove of SAP97PDZ2. The free carboxylate group and the aliphatic side chain of the C‐terminal leucine (Leu907), and the hydroxyl group of Thr905 of the GluR‐A peptide are engaged in essential class I PDZ interactions. Comparison between the free and complexed structures reveals conformational changes which take place upon peptide binding. The βΑ−βΒ loop moves away from the C‐terminal end of αB leading to a slight opening of the binding groove, which may better accommodate the peptide ligand. The two conformational states are stabilized by alternative hydrogen bond and coulombic interactions of Lys324 in βΑ−βΒ loop with Asp396 or Thr394 in βΒ. Results of in vitro binding and immunoprecipitation experiments using a PDZ motif‐destroying L907A mutation as well as the insertion of an extra alanine residue between the C‐terminal Leu907 and the stop codon are also consistent with a ‘classical’ type I PDZ interaction between SAP97 and GluR‐A C‐terminus.

A xeno-free culturing protocol for pluripotent stem cell-derived oligodendrocyte precursor cell production

AIM To show that human embryonic stem cells (hESCs) can be efficiently differentiated into oligodendrocyte precursor cells (OPCs) in a xeno-free medium with a specific medium supplement and specific human recombinant growth factors. MATERIALS & METHODS The xeno-free OPC-differentiation medium for pluripotent stem cells was developed by using StemPro® neural stem cell xeno-free medium supplement together with human recombinant growth factors SHH, PDGF-AA, IGF-1, EGF, basic FGF and CNTF, in addition to RA, T3, human laminin and ascorbic acid. We analyzed the differentiated hESC-derived OPCs and oligodendrocytes with quantitative real-time (RT)-PCR, RT-PCR, flow cytometry and immunocytochemistry, and we performed NG2-positive selection for OPC cultures with fluorescence-activated cell sorting. RESULTS Based on quantitative RT-PCR analysis, OPCs after 9 weeks of differentiation in xeno-free medium expressed OLIG2, SOX10 and NKX2.2 at elevated levels compared with control conditions. According to the flow cytometric analysis, the cells expressed A2B5 (>70%) and NG2 (40-60%) at 5 weeks time point whereas maturing oligodendrocytes expressed O4 (60-80%) at 11 weeks time point. In addition, hESC-derived OPC populations were purified based on NG2-positive selection using fluorescence-activated cell sorting. NG2-positive OPC populations survived and differentiated further into O4 expressing oligodendrocytes in xeno-free medium, and the sorted cell populations were free from pluripotent Tra1-81 and Oct-4 -positive cells. CONCLUSIONS This study confirms that the xeno-free culturing method can support the differentiation and purification of hESC-derived OPC populations and provides an initial step toward safe cell graft production for the future clinical applications.

Advances in stem-cell–generated transplantation therapy for Parkinson's disease

Introduction: Human pluripotent stem cells have the potential to differentiate into different cell lineages of the human body, including dopaminergic (DA) neurons. Previous studies have shown that stem-cell–derived DA neurons can improve the motor deficits of Parkinsons disease (PD) animal models. That is why current research interests focus on the development of stem-cell–derived neural cells for transplantation therapies for PD patients. Areas covered: This review article emphasizes the safety and efficacy requirements of human pluripotent stem-cell–derived neural cells and usage of reliable preclinical animal models prior to clinical trials. The current advances and hurdles related to cell production, differentiation and transplantation are also summarized. Expert opinion: Before entering the clinic, transplantable cell populations must be differentiated and characterized according to good manufacturing practice (GMP) regulations both in vitro and in vivo. Taking into account the rapid development of the stem-cell field and technological improvements in cell preparations and GMP facilities, we think that pluripotent stem-cell–derived DA neurons will offer a relevant cell therapy option for treatment of PD in the near future.