This study is an impressive technological attempt to interrogate the neuro-immune axis within a three-dimensional tri-culture disease model system. Their elegant mechanistic experiments implicate TLR4 activation and microglial-derived IFN-γ and TNF as important factors contributing to neuronal loss in this three-dimensional model culture of Alzheimer’s disease. This study is a significant advance because it is one of the first neurodegenerative disease models to use human-derived cells in three-dimensional culture that included both neural ectoderm and myeloid lineages, and to conclusively demonstrate that myeloid cells can initiate the inflammatory cascade and neuronal loss. As systems like these improve, they'll provide powerful avenues for interrogating pathogenic mechanisms underlying human neurodegenerative diseases and eventually for screening therapeutic candidates.
However, as the authors aptly point out, overexpression of APP mutations that cause familial AD in immortalized human neural progenitor cells to trigger activation of immortalized microglia is a significant caveat/limitation of this study. Therefore, extrapolation of the conclusions in this paper to three-dimensional models involving primary cells needs to be explored and confirmed empirically.
Specifically, primary microglia are highly heterogeneous and represent a vastly different cell population than SV40-immortalized microglia. Furthermore, to consider these cells activated requires a suite of data including functional properties as well as characterization with multiple surface protein and transcription factor markers (not just CD68 immunocytochemistry).
Within the spatiotemporal context of this study, the large amount (>20 percent) of neuronal death reported is surprising and raises the interesting possibility that the infiltrating microglia formed and/or replaced a cellular niche within the three-dimensional tri-culture rather than distributing homogeneously throughout the culture, although this was not investigated. Moreover, while the News and Views article points out that “the microglia are plated days or weeks after the neurons and astrocytes, which mimics the waves of microglial infiltration into the developing brain and allows investigation of neuroinflammation in a more mature culture of functional neurons supported by astrocytes,” it is now well accepted that microglia progenitors migrate from the yolk sac to the brain prior (~E8.5) to brain circuit formation and are intimately involved in shaping and sculpting neuronal development.
The use of iPSCs in some of the experiments is a good attempt to extend the findings beyond immortalized cell lines, although these cells also overexpress mutated APP. One significant caveat with human iPSCs is their functional transcriptional age. Specifically, the authors mention that their three-dimensional tri-culture model exhibits late-stage AD markers. Given that iPSC-derived neurons are reset to model fetal brain transcriptomes, as opposed to the aged transcriptome of the donor, adult protein isoforms, including some forms of tau, are not expressed or expressed at very low levels. Therefore, caution must be used when interpreting the significance of late-stage AD markers in this model with APP overexpression. Aging the cells or using direct differentiation approaches may provide a better model system in future iterations.
In short, the three-dimensional tri-culture presented in this study is a significant advance toward efforts in the neuroscience community to more accurately model the human brain and AD in a dish. Future studies may integrate three-dimensional co-cultures of neurons, microglia, and astrocytes that are all derived from the same iPSC lines generated from individuals with various mutations that cause or increase the risk of AD.
In the future the refinement and extension of this type of in vitro three-dimensional model system will allow scientists to examine the role of other physiological components (i.e., vascular system, peripheral immune system) in AD and other neurodegenerative diseases. This will be important given the growing body of evidence that peripheral immune cells modify or contribute to both inflammation and neurodegeneration in many of these diseases.
The authors have devised an ingenious upgrade to their three-dimensional human brain culture system. Not only do they now capture critical neuroinflammatory/neuroimmune AD components, but they are also modeling brain migration of microglia. This new and improved three-dimensional culture holds great promise for understanding the contribution of innate immunity to AD evolution and for preclinically evaluating experimental therapeutics that target it.
This paper represents a significant advancement in the generation of human cell models of Alzheimer's disease. Building upon previous work demonstrating that three-dimensional neuronal cultures overexpressing APP with mutations linked to familial AD (fAD) develop Aβ and tau pathologies more rapidly than two-dimensional cultures (Choi et al., 2014), Park et al. extend their three-dimensional model to include the introduction of microglial cells into the co-culture via the elegant use of microfluidic chambers, which allow the activation and migration of microglia to be investigated. This is one of the first studies to investigate the AD inflammatory response in a human cell culture system, and is a shift away from studying single cell types in isolation and toward more complex co-culture models. This system permits the investigation of non-cell-autonomous disease mechanisms, the importance of which is highlighted through the high representation of microglial genes as risk factors for AD.
The work in this paper uses immortalized neuronal precursor cells overexpressing APP with fAD mutations, iPSC engineered to overexpress mutant APP, and an immortalized microglial cell line.
It will be interesting to see if the phenotypes described here are recapitulated using iPSC-derived neurons/microglia with genotypes relevant to both familial and sporadic AD. These have the important advantage of endogenous levels of gene expression, avoiding the overexpression of genes with multiple mutations in order to drive a cellular phenotype. Of particular note, several papers have shown that three-dimensional cerebral organoids derived from fAD iPSC develop Aβ deposits and increased tau phosphorylation, supporting the premise that patient-derived iPSC capture disease relevant phenotypes in vitro (Raja et al., 2016, and Lin et al., 2018).
Up until recently, a barrier in the development of such a model has been a lack of protocols for the differentiation of iPSC into microglia, however this is no longer the case and iPSC-microglia can now be robustly generated (Abud et al., 2017; Haenseler et al., 2017).
Taken together with the availability of multiple transcriptomic data sets from microglia in aging and disease states (e.g., Olah et al., 2018; Keren-Shaul et al., 2017), the scene is set for the development of iPSC tri-cultures and deep interrogation of how closely the in vitro models capture signatures of aging and disease.
References:
Choi SH, Kim YH, Hebisch M, Sliwinski C, Lee S, D'Avanzo C, Chen H, Hooli B, Asselin C, Muffat J, Klee JB, Zhang C, Wainger BJ, Peitz M, Kovacs DM, Woolf CJ, Wagner SL, Tanzi RE, Kim DY.
A three-dimensional human neural cell culture model of Alzheimer's disease.
Nature. 2014 Nov 13;515(7526):274-8. Epub 2014 Oct 12
PubMed.
Raja WK, Mungenast AE, Lin YT, Ko T, Abdurrob F, Seo J, Tsai LH.
Self-Organizing 3D Human Neural Tissue Derived from Induced Pluripotent Stem Cells Recapitulate Alzheimer's Disease Phenotypes.
PLoS One. 2016;11(9):e0161969. Epub 2016 Sep 13
PubMed.
Lin YT, Seo J, Gao F, Feldman HM, Wen HL, Penney J, Cam HP, Gjoneska E, Raja WK, Cheng J, Rueda R, Kritskiy O, Abdurrob F, Peng Z, Milo B, Yu CJ, Elmsaouri S, Dey D, Ko T, Yankner BA, Tsai LH.
APOE4 Causes Widespread Molecular and Cellular Alterations Associated with Alzheimer's Disease Phenotypes in Human iPSC-Derived Brain Cell Types.
Neuron. 2018 Jun 27;98(6):1141-1154.e7. Epub 2018 May 31
PubMed.
Abud EM, Ramirez RN, Martinez ES, Healy LM, Nguyen CH, Newman SA, Yeromin AV, Scarfone VM, Marsh SE, Fimbres C, Caraway CA, Fote GM, Madany AM, Agrawal A, Kayed R, Gylys KH, Cahalan MD, Cummings BJ, Antel JP, Mortazavi A, Carson MJ, Poon WW, Blurton-Jones M.
iPSC-Derived Human Microglia-like Cells to Study Neurological Diseases.
Neuron. 2017 Apr 19;94(2):278-293.e9.
PubMed.
Haenseler W, Sansom SN, Buchrieser J, Newey SE, Moore CS, Nicholls FJ, Chintawar S, Schnell C, Antel JP, Allen ND, Cader MZ, Wade-Martins R, James WS, Cowley SA.
A Highly Efficient Human Pluripotent Stem Cell Microglia Model Displays a Neuronal-Co-culture-Specific Expression Profile and Inflammatory Response.
Stem Cell Reports. 2017 Jun 6;8(6):1727-1742.
PubMed.
Olah M, Patrick E, Villani AC, Xu J, White CC, Ryan KJ, Piehowski P, Kapasi A, Nejad P, Cimpean M, Connor S, Yung CJ, Frangieh M, McHenry A, Elyaman W, Petyuk V, Schneider JA, Bennett DA, De Jager PL, Bradshaw EM.
A transcriptomic atlas of aged human microglia.
Nat Commun. 2018 Feb 7;9(1):539.
PubMed.
Keren-Shaul H, Spinrad A, Weiner A, Matcovitch-Natan O, Dvir-Szternfeld R, Ulland TK, David E, Baruch K, Lara-Astaiso D, Toth B, Itzkovitz S, Colonna M, Schwartz M, Amit I.
A Unique Microglia Type Associated with Restricting Development of Alzheimer's Disease.
Cell. 2017 Jun 15;169(7):1276-1290.e17. Epub 2017 Jun 8
PubMed.
Caution is warranted here. We must not jump to the conclusion, based on this data, that the "inflammatory" or microglial response is the cause of destruction. It accompanies destruction, to be sure, but there are reasons to wonder whether the microglial reaction is a response to the injury, rather than the cause.
Comments
Emory University
Emory University
Emory University
Emory University
Emory University School of Medicine
The University of Florida College of Medicine
This study is an impressive technological attempt to interrogate the neuro-immune axis within a three-dimensional tri-culture disease model system. Their elegant mechanistic experiments implicate TLR4 activation and microglial-derived IFN-γ and TNF as important factors contributing to neuronal loss in this three-dimensional model culture of Alzheimer’s disease. This study is a significant advance because it is one of the first neurodegenerative disease models to use human-derived cells in three-dimensional culture that included both neural ectoderm and myeloid lineages, and to conclusively demonstrate that myeloid cells can initiate the inflammatory cascade and neuronal loss. As systems like these improve, they'll provide powerful avenues for interrogating pathogenic mechanisms underlying human neurodegenerative diseases and eventually for screening therapeutic candidates.
However, as the authors aptly point out, overexpression of APP mutations that cause familial AD in immortalized human neural progenitor cells to trigger activation of immortalized microglia is a significant caveat/limitation of this study. Therefore, extrapolation of the conclusions in this paper to three-dimensional models involving primary cells needs to be explored and confirmed empirically.
Specifically, primary microglia are highly heterogeneous and represent a vastly different cell population than SV40-immortalized microglia. Furthermore, to consider these cells activated requires a suite of data including functional properties as well as characterization with multiple surface protein and transcription factor markers (not just CD68 immunocytochemistry).
Within the spatiotemporal context of this study, the large amount (>20 percent) of neuronal death reported is surprising and raises the interesting possibility that the infiltrating microglia formed and/or replaced a cellular niche within the three-dimensional tri-culture rather than distributing homogeneously throughout the culture, although this was not investigated. Moreover, while the News and Views article points out that “the microglia are plated days or weeks after the neurons and astrocytes, which mimics the waves of microglial infiltration into the developing brain and allows investigation of neuroinflammation in a more mature culture of functional neurons supported by astrocytes,” it is now well accepted that microglia progenitors migrate from the yolk sac to the brain prior (~E8.5) to brain circuit formation and are intimately involved in shaping and sculpting neuronal development.
The use of iPSCs in some of the experiments is a good attempt to extend the findings beyond immortalized cell lines, although these cells also overexpress mutated APP. One significant caveat with human iPSCs is their functional transcriptional age. Specifically, the authors mention that their three-dimensional tri-culture model exhibits late-stage AD markers. Given that iPSC-derived neurons are reset to model fetal brain transcriptomes, as opposed to the aged transcriptome of the donor, adult protein isoforms, including some forms of tau, are not expressed or expressed at very low levels. Therefore, caution must be used when interpreting the significance of late-stage AD markers in this model with APP overexpression. Aging the cells or using direct differentiation approaches may provide a better model system in future iterations.
In short, the three-dimensional tri-culture presented in this study is a significant advance toward efforts in the neuroscience community to more accurately model the human brain and AD in a dish. Future studies may integrate three-dimensional co-cultures of neurons, microglia, and astrocytes that are all derived from the same iPSC lines generated from individuals with various mutations that cause or increase the risk of AD.
In the future the refinement and extension of this type of in vitro three-dimensional model system will allow scientists to examine the role of other physiological components (i.e., vascular system, peripheral immune system) in AD and other neurodegenerative diseases. This will be important given the growing body of evidence that peripheral immune cells modify or contribute to both inflammation and neurodegeneration in many of these diseases.
View all comments by Malu G. TanseyUniversity of Southern California
The authors have devised an ingenious upgrade to their three-dimensional human brain culture system. Not only do they now capture critical neuroinflammatory/neuroimmune AD components, but they are also modeling brain migration of microglia. This new and improved three-dimensional culture holds great promise for understanding the contribution of innate immunity to AD evolution and for preclinically evaluating experimental therapeutics that target it.
View all comments by Terrence TownInstitute of Neurology, University College London
This paper represents a significant advancement in the generation of human cell models of Alzheimer's disease. Building upon previous work demonstrating that three-dimensional neuronal cultures overexpressing APP with mutations linked to familial AD (fAD) develop Aβ and tau pathologies more rapidly than two-dimensional cultures (Choi et al., 2014), Park et al. extend their three-dimensional model to include the introduction of microglial cells into the co-culture via the elegant use of microfluidic chambers, which allow the activation and migration of microglia to be investigated. This is one of the first studies to investigate the AD inflammatory response in a human cell culture system, and is a shift away from studying single cell types in isolation and toward more complex co-culture models. This system permits the investigation of non-cell-autonomous disease mechanisms, the importance of which is highlighted through the high representation of microglial genes as risk factors for AD.
The work in this paper uses immortalized neuronal precursor cells overexpressing APP with fAD mutations, iPSC engineered to overexpress mutant APP, and an immortalized microglial cell line.
It will be interesting to see if the phenotypes described here are recapitulated using iPSC-derived neurons/microglia with genotypes relevant to both familial and sporadic AD. These have the important advantage of endogenous levels of gene expression, avoiding the overexpression of genes with multiple mutations in order to drive a cellular phenotype. Of particular note, several papers have shown that three-dimensional cerebral organoids derived from fAD iPSC develop Aβ deposits and increased tau phosphorylation, supporting the premise that patient-derived iPSC capture disease relevant phenotypes in vitro (Raja et al., 2016, and Lin et al., 2018).
Up until recently, a barrier in the development of such a model has been a lack of protocols for the differentiation of iPSC into microglia, however this is no longer the case and iPSC-microglia can now be robustly generated (Abud et al., 2017; Haenseler et al., 2017).
Taken together with the availability of multiple transcriptomic data sets from microglia in aging and disease states (e.g., Olah et al., 2018; Keren-Shaul et al., 2017), the scene is set for the development of iPSC tri-cultures and deep interrogation of how closely the in vitro models capture signatures of aging and disease.
References:
Choi SH, Kim YH, Hebisch M, Sliwinski C, Lee S, D'Avanzo C, Chen H, Hooli B, Asselin C, Muffat J, Klee JB, Zhang C, Wainger BJ, Peitz M, Kovacs DM, Woolf CJ, Wagner SL, Tanzi RE, Kim DY. A three-dimensional human neural cell culture model of Alzheimer's disease. Nature. 2014 Nov 13;515(7526):274-8. Epub 2014 Oct 12 PubMed.
Raja WK, Mungenast AE, Lin YT, Ko T, Abdurrob F, Seo J, Tsai LH. Self-Organizing 3D Human Neural Tissue Derived from Induced Pluripotent Stem Cells Recapitulate Alzheimer's Disease Phenotypes. PLoS One. 2016;11(9):e0161969. Epub 2016 Sep 13 PubMed.
Lin YT, Seo J, Gao F, Feldman HM, Wen HL, Penney J, Cam HP, Gjoneska E, Raja WK, Cheng J, Rueda R, Kritskiy O, Abdurrob F, Peng Z, Milo B, Yu CJ, Elmsaouri S, Dey D, Ko T, Yankner BA, Tsai LH. APOE4 Causes Widespread Molecular and Cellular Alterations Associated with Alzheimer's Disease Phenotypes in Human iPSC-Derived Brain Cell Types. Neuron. 2018 Jun 27;98(6):1141-1154.e7. Epub 2018 May 31 PubMed.
Abud EM, Ramirez RN, Martinez ES, Healy LM, Nguyen CH, Newman SA, Yeromin AV, Scarfone VM, Marsh SE, Fimbres C, Caraway CA, Fote GM, Madany AM, Agrawal A, Kayed R, Gylys KH, Cahalan MD, Cummings BJ, Antel JP, Mortazavi A, Carson MJ, Poon WW, Blurton-Jones M. iPSC-Derived Human Microglia-like Cells to Study Neurological Diseases. Neuron. 2017 Apr 19;94(2):278-293.e9. PubMed.
Haenseler W, Sansom SN, Buchrieser J, Newey SE, Moore CS, Nicholls FJ, Chintawar S, Schnell C, Antel JP, Allen ND, Cader MZ, Wade-Martins R, James WS, Cowley SA. A Highly Efficient Human Pluripotent Stem Cell Microglia Model Displays a Neuronal-Co-culture-Specific Expression Profile and Inflammatory Response. Stem Cell Reports. 2017 Jun 6;8(6):1727-1742. PubMed.
Olah M, Patrick E, Villani AC, Xu J, White CC, Ryan KJ, Piehowski P, Kapasi A, Nejad P, Cimpean M, Connor S, Yung CJ, Frangieh M, McHenry A, Elyaman W, Petyuk V, Schneider JA, Bennett DA, De Jager PL, Bradshaw EM. A transcriptomic atlas of aged human microglia. Nat Commun. 2018 Feb 7;9(1):539. PubMed.
Keren-Shaul H, Spinrad A, Weiner A, Matcovitch-Natan O, Dvir-Szternfeld R, Ulland TK, David E, Baruch K, Lara-Astaiso D, Toth B, Itzkovitz S, Colonna M, Schwartz M, Amit I. A Unique Microglia Type Associated with Restricting Development of Alzheimer's Disease. Cell. 2017 Jun 15;169(7):1276-1290.e17. Epub 2017 Jun 8 PubMed.
View all comments by Selina WrayMcGill University Faculty of Medicine
Caution is warranted here. We must not jump to the conclusion, based on this data, that the "inflammatory" or microglial response is the cause of destruction. It accompanies destruction, to be sure, but there are reasons to wonder whether the microglial reaction is a response to the injury, rather than the cause.
View all comments by John BreitnerMake a Comment
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