Scaffold-free brain organoids differentiated from patient-derived IPSCs model familial Alzheimer's Disease by

Raja et al, 2016

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Alzheimer's Disease (AD)

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^{Tender Rose Dementia Care Specialists, Mar 2010: https://www.youtube.com/watch?v=Eq_Er-tqPsA}

AD Molecular Characteristics

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Amyloid Beta accumulation (Aβ40 and Aβ42 for example) into plaques (Ballatore et al, 2007; Goate , 2006)
Tauopathy: Aberrant phosphorylation leading to aggregation of hyperphosphorylated tau protein (pTau) into neurofibrillary tangles. (Ballatore et al, 2007; Buée et al, 2000; Feany et al. 1996; Lee et al, 2001).
Endosome abnormalities: enlarged endosomes (Cataldo et al, 2000; Cataldo et al, 2001) and defects in endosome trafficking (Iovino et al, 2015; Wren et al, 2015)

Animal Models of AD

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Many different invertebrate and vertebrate species are used for modelling: drosophila (Dissel et al, 2017), zebrafish (Bhattarai et al, 2012), and rodents (Elder et al, 2010; Do Carmo, Cuello, 2013) where mouse models are the most prevalent

Advantages:

Mouse models successfully recapitulate many molecular mechanisms involved in AD listed above (Elder et al, 2010; Webster et al, 2014; Guo, Zhou, 2017)
Use of genetic (ex: mutations in genes controlling amyloid beta deposition) and non-genetic (ex: transverse aortic constriction to induce hypertension) models can be used to model both fAD and sAD (Elder et al, 2010; Carnevale et al, 2012)
Evaluation of behaviour with cognitive and memory tasks with findings that are translatable to human AD (Webster et al, 2014).

Disadvantages:

Genetic models often require multiple mutations or copies of mutations to create a phenotype that resembles human AD (Elder et al, 2010; Webster et al, 2014). The observable molecular and behavioural phenotype measured in these models therefore does not rely on the same molecular etiologies as human AD.
Many drug therapies developed from mouse models fail in clinical trials, indicating a lack of efficiency in representing human AD phenotypes. (DeMattos et al, 2001; Cedernaes et al, 2014; Raja et al, 2016)

Research Question

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Hypothesis: Scaffold-free brain organoids grown from fAD patient iPSCs can recapitulate the molecular mechanisms of AD phenotype and will be responsive to AD treatments.

Results

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Future Directions

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This paper has successfully recapitulated AD phenotypes in organoids, which importantly are derived from human patient cells and not animals. Hence, it could be a promising model to retry therapies that failed translation from mouse models to human clinical trials (Doody et al, 2012; Doody et al, 2014; Green et al, 2009; Salloway et al, 2014). Mouse models have supported the therapeutic benefit of treatment with monoclonal antibodies specific for Aβ to resolve the AD phenotype (DeMattos et al, 2001). When this research was translated into clinical trials, many forms of anti-Aβ drugs were attempted that did not show therapeutic benefit. One such drug, called bapineuzumab, showed a significant decrease in CSF levels of pTau, but no difference in levels of Aβ and no improvement in cognitive symptoms (Blennow et al., 2012). It would be of interest to apply bapineuzumab to these organoids to assess its efficacy of resolving AD phenotypes.
- If bapineuzumab is successful in resolving the Aβ and the pTau phenotypes, this would be significant in demonstrating this model's responsiveness to treatments. It would further indicate that there are differences between the organoid model and human fAD that prevent these models from responding in the same fashion as human AD would.
- If bapineuzumab is successful in resolving only the pTau phenotype, this would be an indication that the fAD organoids hold high representational power of human AD. As anti-Aβ would be inducing a decrease in pTau, it would also support the hypothesis that the hyperphosphrylation of tau protein is causally connected to increased Aβ deposition.
- If bapineuzumab does not resolve the Aβ or pTau phenotypes, this could indicate a lack of responsiveness of the organoid, reflecting poorly on its representational abilities, indicating that the organoid is less sensitive than human AD.
Due to its closer resemblance to humans, this is a model that can serve as an additional step in the translation process to test the potential of proposed therapies before these therapies move on to the more costly and time-consuming clinical trials.
Because organoids can be generated by cells from specific patients, there is also potential to develop patient specific therapies using this model. Patients can donate fibroblasts to make organoids, and for each of patient's organoids we can try a set of different drugs to see which drug works best for that patient's organoid, and consequently decide which is the drug best given to the actual patient.
When it comes to sAD, genetic factors only partially explain the risk of AD. One widely accepted notion is that the onset of AD is most likely the consequence of complicated interactions of multiple genetic and non-genetic factors (Gatz, 2006; Yang et al, 2016). Several non-genetic risk factors have been proposed, including aging, cerebro-cardiovascular diseases, metabolic disorders, traumatic brain injury, sleep disorders, chronic hypoxia, environmental toxins and high cholesterol (Yang et al, 2016; Grant et al, 2002; Styczyńska et al, 2008; Dosunmu et al, 2007). One model has even looked at sAD iPSC cells grown in culture clustered with fetal brain messenger RNA samples, and showed AD phenotypes to appear in these sAD patient-derived iPSC cells (Israel et al. 2012). However, the mechanisms underlying them still remain largely unknown, due to the lack of non-postmortem and AD patient-specific research models (Yang et al, 2016). Although the organoids produced in this paper are based on fAD mutations, they have the possibility of being used to model sAD as well. For example, researchers could take cells from sAD patients, derive iPSCs, and grow multiple organoids under some environmental conditions that may induce AD, so AD will arise in these organoids sporadically, similarly to sAD patients. Specifically, this means that for a specific sAD patient whose disease arose in a way that cannot be fully accounted for by genetic mutation, we can take these cells and grow multiple organoids, some in neutral conditions as control, and others subjected to some environment such as high cholesterol. This will allow us to better observe how the proposed environmental factors can influence disease progression in sAD patient organoids, if they are an influence at all. In addition to studying sAD disease progression, we may even be able to take one step further and use this organoid model to help design sAD patient-specific therapy.

Figure 2B: Amyloid Beta

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In terms of Aβ aggregates, Organoids derived from the APP^Dp2-3 (blue bar) and the PSEN1^A264E (red bar) lines exhibited increased numbers of Aβ aggregates verses the controls. Organoids from the PSEN1^M146I (green bar) patient cell line exhibited a trend towards higher amyloid levels, but this was not significant. Values between the two control lines did not significantly differ.

Figure 3: fAD organoids respond to treatment, resolving the AD phenotype

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fAD organoids are treated with β-secretase and γ-secretase inhibitors which inhibit the formation of the complex that cleaves amyloid precursor protein into Aβ. The immunohistochemical stains below show that this treatment reduces Aβ and pTau deposition, two strong molecular indicators of the AD phenotype

These results indicate responsiveness of the model to treatments, meaning that this could be a possible model for screening prospective clinical therapies. There is some debate as to the timeline of emergence of human AD molecular mechanisms, but the time-dependent change in Aβ and pTau in these organoids in response to treatment also mimic human AD progression (Jack, 2014) with the appearance of Aβ plaques preceding neurofibrillary tangle formation.

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^{Can Brain ORGANOIDS help to unlock human brain cells. (2016, March 7). http://technologyinfoo.com/724/can-brain-organoids-help-to-unlock-human-brain- cells/}

Background and Introduction

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Induced Pluripotent Stem Cell Models of AD

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Induced pluripotent stem cells (iPSCs) cultured from fAD patients can be differentiated into neurons to model human AD in vitro in 2D cultures

Advantages:

Successfully produces the hallmarks of AD pathology (Yahata, 2011; Sproul, 2014; Hossini, 2015; Lullo, Kriegstein, 2017)
Because of their closeness to human AD, once they become more well established, these models may have the capacity to produce findings pertaining to the molecular phenotype of AD that hold more translational power than previous models.

Disadvantages

Due to the nature of 2D models, the phenotypes of aberrant extracellular protein aggregation are lost.
As for 3D models, previous methods of organoid production require a scaffold, are labor-intensive, and require exogenous over-expression of disease-relevant mutations or factors in order to express AD phenotype (Zhang et al, 2014).
While not yet well characterized, papers such as this one by Raja et al (2016) aim to develop high fidelity, well-characterized iPSC-derived 3D organoid models of fAD.

Major Findings

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The authors conducted three sets of experiments. The experiments in Figure 1 demonstrate that the organoids were able to recapitulate hallmark symptoms of AD. Figure 2 demonstrates that the hallmark AD phenotypes generated by the organoid could be generated from multiple cell lines with different AD-inducing mutations. Finally, Figure 3 demonstrates that the fAD organoids were responsive to common AD treatments

Conclusion & Discussion

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Advantages of the fAD organoids proposed in this article

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These organoids were able to develop and respond to AD therapies within 90 days. Previously, scientists only insights into human AD pathology were from behavioural assessments, imaging techniques, and postmortem analysis. This will allow scientists to access live, human-derived tissue that strongly represents the human AD phenotype.
The recapitulation of the phenotype is evidenced by the induction of Aβ and pTau deposition, as well as endosomal abnormalities. The response to therapy is also evidenced by a reduction in Aβ and pTau deposition.
fAD derived organoids may be more representative of human AD pathology than traditional animal models. While many mouse models of fAD rely on genetic manipulation to produce an AD-like phenotype, fAD organoids are directly derived from human AD patients. The artificial mutagenesis required to create these animal models may create a pathology that does not replicate the course of human disease, and is therefore unsuitable for developing human therapies. By directly recreating human AD pathology, fAD organoids may provide researchers with better tools to develop AD therapies.

Disadvantages of fAD Organoids proposed in this article

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While organoids may be better able to replicated the molecular etiology of AD than animals models, organoids cannot show behavioural responses. Murine AD models can be subjected to behavioural tests, allowing researchers to identify specific behavioural deficits. Researchers testing a drug in a murine AD model can examine both behavioural and molecular rescue of phenotype. While drugs may affect the molecular pathology or improve biomarkers, this does not guarantee that they will have any functional impact (Salloway et al 2014). As functional recovery cannot be measured with fAD organoids, their scope as a model system is limited. Furthermore, AD is a heterogenous pathology affecting multiple cognitive domains. Without behaviour, researchers cannot identify how molecular pathologies and treatments relate to specific cognitive deficits (Ex attentional vs memory deficits).
While this model can replicate several strains of human fAD, it does not represent sAD. Familial cases of AD are much rarer than sAD. As such, it is critical the better models of sAD be developed. The scope of fAD organoids is somewhat limited as they only represent a minority of AD cases.
As these organoids are constructed without a scaffold there is significant tissue necrosis in the centre. To combat the potential influence of the necrotic tissue the authors only examined the peripheral 250um of the organoids. However, this is a rather arbitrary decision. The figure below (supplemental figure 3) shows cleaved cascade 3 staining (white) for apoptosis, and Hoechst staining (blue) for nuclear material. It is obvious that even in the peripheral 250um there is tissue necrosis. While this may have no effect on the organoids AD relevance, the proinflammatory environment may influence the disease progression, especially given the growing epidemiological findings indicating that immune activation plays an important role in AD pathogenesis (Carnevale et al, 2012).

References

d_oliver1 — 2017-12-04 16:36:00 UTC

All figures adapted from Raja et al. 2016, unless otherwise cited

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Possible questions for the field

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While iPSC induction erases epigenetic factors, direct reprogramming has been shown to maintain epigenetic memory (Hou, Lu, 2016). With more research being conducted on the epigenetics of AD, one question in the field of AD organoinds going forward could be whether it would be feasible and practical to differentiate a brain organoid through direct reprogramming as opposed to IPSC induction. By direct reporgramming brain organoids from sAD patient cells, this may provide a possible model for studying the epigenetic factors contributing to sAD.

AD Symptoms

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(AD) is an age-related, progressive neurodegenerative disorder associated with cognitive decline, severe memory impairments, anxiety, circadian rhythm disturbances, activity disturbances, and aggression. It is associated with brain atrophy, neuronal losses, dense extracellular deposits of amyloid plaques and neurofibrillary tangles (Webster et al, 2014).

AD Prevalence

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It is currently the 6th leading cause of death in the United States. With medical advances increasing life expectancies and the growing population of seniors, the incidence of AD in the US is said to increase from 5.3 million patients to 13.8 million by 2050 (Alzheimer's Association, 2015). This reflects an urgent need for reserach to be conducted with models that can yield relevant results that can be translated to a clinical setting.

AD Causes

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The full etiology of AD is unknown. It can be broadly divided into sAD (sporadic) and fAD (familial). fAD has been attributed to a series of mutations in the amyloid pathway. Epidemiological studies have considered risk factors for AD that may result in sAD.

Figure 1: Amyloid Beta

d_oliver1 — 2017-12-07 03:24:48 UTC

Immunohistochemisty using an Aβ specific antibody on 60 and 90 day old cultures shows a time dependant increase in Aβ deposition. Organoids created from fAD cells showed significantly higher levels of Aβ deposition compared to controls. Staining for MAP2, a neuronal protein, suggests both intracellular and extracellular plaques. This suggests that fAD organoids mayrecapitulate the degenerative Aβ pathology central to alzheimer's disease.

Figure 1: Hyperphosphorylated Tau

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Using an antibody specific for pTau, the authors show increased pTau deposition in fAD organoids compared to controls. It is important to note that this increase is only significant at the 90 day time point, suggesting that Tau pathology may be antecedent to Aβ pathology. This finding is replicated using a second antibody specific to pTau, although this data is not displayed here. This suggests that fAD organoids may recapitulate AD Tau pathology.

Figure 1: Endosomal Abnormalities

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Immunohistochemical staining for EEA1, an early end-somalia marker, shows a significant increase in the number of large endosomes in fAD organoids compared to controls. The authors also measured functional endocytosis by allowing cells to take up labelled transferrin. They noted that cells from fAD organoids had significantly larger endosomes compared to controls. This suggests that fAD organoids may model the endosomal abnormalities associated with AD.

Creation of Organoids

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Organoids were produced using donated fibroblasts from patients with fAD resulting from the APP^Dp1-1 mutation, and healthy age matched controls. Donated cells were induced to pluripotency, which was validated by staining cells for TRA-1-60 and TRA-1-81. Pluripotent cells were then plated to form embryoid bodies (EB) in a growth medium promoting neural lineage differentiation. After 18–20 days the EBs were separated and allowed to mature for a further 15–20 days in a growth medium with factors promoting neural epithelial formation. Following this, cells were moved to a growth medium containing 2% matrigel for the remainder of the experiment (supplemental figure 2).

Validation of Organoids

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Immunohistochemical staining for Sox2, a marker of neural progenitor cells, and MAP2, a marker of mature neurons, shows that organoids develop stratified layers (supplemental figure 2). Further, organoids display characteristic neural rosettes. Based on cleaved caspase expression, all data was collected from the outer 250um of tissue so as to exclude any necrotic tissue in the centre of the organoid.

Significant Conclusions

d_oliver1 — 2017-12-07 14:41:58 UTC

The authors were able to create scaffold-free organoids from multiple lines of fAD iPSCs that both recapitulated the major symptoms of AD pathology, and responded to common AD treatments. The evidence presented supports the authors hypothesis, and suggests that fAD organoids may be a useful model system. The ease of organoid growth could allow for high throughput screening of novel AD therapies.

The authors were able to demonstrate that fAD organoids show the major molecular symptoms of AD (Figure 1). Without this, these organoids would show little promise as a model system.
Organoids grown using tissue from multiple different fAD patients shows that these organoids are a useful model for studying multiple forms of fAD (Figure 2). The potential for performing experiments on cells with different fAD causing mutations will increase the robustness of any experimental findings.
By culturing organoids in growth media containing common AD therapeutics, the authors were able to reduce the severity of AD symptomology (Figure 3). This demonstrates the usefulness of fAD organoids as a model system for drug development.

Experimental Data Issues

d_oliver1 — 2017-12-07 15:20:13 UTC

Despite the stated ease of growing fAD organoids, the authors include very small samples sizes in many experiments. While the authors do manage to achieve statistical significance, there is extremely high variability in some groups. For example, the vehicle treated groups in figure 3 show very high variability, with some vehicle treated organoids showing no pathology. In conjunction with the small sample size, this raises the possibility that the observed results are merely an artifact of a small highly variable sample.
While the paper was broadly well constructed, there were a number of significant errors in figure design. For example, Figure 1 B in the original paper is missing an arrow head which should indicate intracellular amyloid beta. These omissions are compounded by inconsistencies in font size and colour coding throughout. While this does not directly impact on the validity of the author's results, it reduces the readability and credibility of the paper.
The authors examined endosomal abnormalities as a characteristic phenotype of AD in their effort to recapitulate AD phenotypes in the initial organoids derived from the APP^Dp1-1 line. But when they showed the results of recapitulating AD pathology in organoids derived from additional lines, there was no mentioning of any efforts to analyze whether there was presence of endosomal abnormalities in this section. This is an inconsistency they should address.

Figure 3: Resolution of pTau phenotype

saige_power — 2017-12-07 17:02:27 UTC

Immunohistochemical staining for Serine 396, a phosphotylated tau residue, shows significant decrease in pTau deposition in the treated organoids than the vehicle-treated controls. This signifcant difference appeared 60 days after treatment began, indicating that there is a temporal difference in resolution of the pTau phenotype and the Aβ phenotype.

Figure 3: Resolution of Aβ phenotype

saige_power — 2017-12-07 17:54:00 UTC

Immunohistochemical staining for D54D2, indicative of Aβ deposition, indicates that at 30 days, there are significantly fewer 3-6um plaques in the treated organoids than in the vehicle-treated controls. At 60 days, there are significantly fewer 1-3um, 3-6um, and 6um< plaques in the treated organoids than in the control group. There are no significant differences in plaque size between the treatment and control groups

Figure 2: Organoids from multiple AD lines recapitulate AD pathology

ce_shang — 2017-12-07 18:56:23 UTC

To confirm that observed pathologies were not limited to the APP^Dp1-1 line , but rather generalizable to fAD, the researchers created organoids from 3 other lines of patient-derived iPSC with different mutations. They saw similar significant phenotypes in 2 of the 3 lines after 90d of culture. Specifically, the 3 lines are an additional APP duplication line APP^Dp2-3, and two PSEN1 fAD mutant lines PSEN1^M146I, PSEN1^A264E. Below is the immunohistochemistry of these 3 lines compared to 2 lines of control. Visually, staining of D45D2 and MAP2 suggests higher levels of Aβ aggregation, and staining of Ser396 suggests higher levels of pTau, compared to the controls.

Figure 2C: Tauopathy

ce_shang — 2017-12-07 19:15:15 UTC

Examining the presence of pTau immunoreactivity, again the APP^Dp2-3 and the PSEN1^A264E organoids (blue and red) exhibited increased pTau (Ser396) immunoreactivity. However, the PSEN1^M146I organoids (green) did not differ from control. Each data point represent one organoid, and values between the two control lines did not significantly differ.

fAD Mutations

ce_shang — 2017-12-07 20:19:53 UTC

Much of the current understanding comes from studying mutation of fAD patients
For example, mutations involving the Aβ processing enzymes presenilin 1 and 2 (PSEN1, PSEN2), which are part of the γ-secretase complex that cuts APP into Aβ (Lamere et al, 1996; Gerrish et al, 2012)
Or, mutations/duplications of the amyloid precursor protein (APP) gene itself (Rovelet-Lecrux et al, 2006; Kosaka et al, 1996).

saige_power — 2017-12-07 22:32:55 UTC

^{The mouse: the scientist’s best friend. (n.d.) https://www.medicalnewstoday.com/articles/277169.php}

Experiment Flow Chart

ce_shang — 2017-12-08 05:01:19 UTC

By Ce Shang, David Oliver, and Saige Power, demonstratingthe experimental protocol by Raja et al, 2016