Week 1 HW: Principles and Practices

Programming living neural systems: design of iPSC-derived neuronal organoids for personalized neurodegenerative disease research.

Neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and amyotrophic lateral sclerosis (ALS) remain poorly understood and pose a significant challenge to modern medicine due to their high public health burden and negative impact on patients’ quality of life. There have been significant advances in experimental neuroscience, but current experimental models fail to accurately reproduce the specific neural development or progressive nature of these disorders.

This project explores the design of programmable neural organoids derived from induced pluripotent stem cells (iPSCs), intended to function as an experimental platform for the personalized study of neurodegenerative diseases. Taking advantage of the pluripotent nature of iPSCs, it is possible to culture neural tissues in three-dimensional environments that promote self-organization and intercellular interactions that are most relevant from a physiological point of view. It should be noted that iPSCs can be generated from individual patients, allowing the resulting organoids to retain the specific genetic and molecular characteristics of each patient.

In this context, when we talk about programmable organoids, we are referring to the controlled modulation of cell fate, synchronization, and microenvironment during their development. Thanks to the precise application of developmental signals, temporal differentiation protocols, and engineered three-dimensional matrices, the system can be oriented toward specific neuronal subtypes and disease-relevant architectures. Furthermore, additional layers of control, such as the inclusion of glial cell populations or specific genetic modifications, allow the organoid to function as a customizable biological system.

The goal of this project is not to create a clinically applicable model, but rather to explore how living neural systems can be designed, programmed, and iteratively modified to better understand disease mechanisms. Looking ahead, this platform could lay the groundwork for other applications in disease modeling, drug screening, and personalized medicine, and help answer broader questions about how complex living systems can be designed and controlled.

Governance Goals

Research using organoids offers unique opportunities to study human neurodegenerative diseases, but their functional complexity and biological origin also raise important ethical and governance challenges. This project is grounded in the core bioethical principles of autonomy, non-maleficence, beneficence, and justice. Among these, I consider the principles of non-maleficence and autonomy to be the fundamental pillars guiding the ethical framework for this project

GOAL 1

The principle of non-maleficence is central due to the nature of the project. Ensuring this principle guarantees that the research is carried out within clear ethical limits, protecting both the participants and the integrity of the biological system being manipulated. To this end:

1.1. Protection of the biological integrity of the organoid: it must be ensured that the organoids do not develop unforeseen functions.

1.2. Minimization of the ethical and functional risks associated with neuronal complexity: control of size, connectivity, culture time, and monitoring of neuronal activity.

GOAL 2

The principle of autonomy ensures respect for the moral relationship with key participants, in this case, the iPSC donor.

2.1 Informed consent: Donors explicitly agree to the use of their cells for research purposes.

2.2 Protection of donor identity and privacy: This involves safeguarding the donor’s identity by anonymizing genetic data. It must be ensured that the organoids are used solely for research purposes.

Governance Actions

Action 1: Restricted use of neuronal organoids to certified laboratories

Purpose: To prevent the misuse of neuronal organoids and ensure their ethical and responsible use.

Design: The generation and modification of neuronal organoids would be permitted only in laboratories that hold recognized certifications in biosafety and bioethics, and that operate under institutional oversight and regulatory supervision.

Assumptions: Certified laboratories comply with strict biosafety, ethical, and procedural protocols, and have trained personnel capable of handling advanced biological systems responsibly.

Risks: This restriction may limit access to organoid research in regions with fewer resources or limited access to certified infrastructure, potentially reinforcing global research inequalities.

Benefits: Enhanced safety, traceability, and institutional accountability in the use of neuronal organoids, reducing the risk of misuse or unethical applications.

Action 2: Explicit Prohibition of Implantation in Humans

Purpose: To prevent medical risks and ethical dilemmas associated with the implantation of neuronally programmed tissue developed through synthetic biology approaches.

Design: Implementation of institutional regulations that restrict the use of neuronal organoids exclusively to in vitro applications, such as scientific research, disease modeling, and evaluation of therapeutic strategies, explicitly prohibiting their implantation in human subjects.

Assumptions: It is assumed that this technology is not yet prepared for direct clinical application and that its primary value lies in disease modeling and monitoring rather than in the replacement of human neural tissue.

Risks: This restriction may slow or limit future research aimed at long-term clinical applications once the technology reaches higher levels of safety and maturity.

Benefits: Prevention of potential harm to patients, establishment of clear ethical boundaries, and avoidance of ethical and legal conflicts related to the misuse of programmed neuronal organoids.

Action 3: Informed Consent and Donor Anonymization

Purpose: To respect donor autonomy by ensuring that biological material is used in an ethical, transparent, and responsible manner.

Design: Obtaining explicit informed consent from donors, anonymizing genetic and personal data, maintaining a clear separation between donor identity and derived organoids, and restricting the use of biological material exclusively to research purposes.

Assumptions: Donors fully understand the nature and scope of the research, communication is clear and accessible, and donor identity is adequately protected throughout the research process.

Risks: Incomplete or poorly communicated information to donors, data breaches, and potential legal or ethical conflicts resulting from misuse of sensitive information.

Benefits: Ethical and legal use of biological material, increased trust in scientific research, and compliance with established bioethical and regulatory standards.

Action 4: Functional delimitation of the organoid

Purpose: To ensure that neuronal organoids do not develop unintended functions, in accordance with the principle of non-maleficence.

Design: To establish clear limits on organoid size, connectivity, and culture duration, and to implement continuous monitoring of neuronal activity.

Assumptions: It is assumed that the defined parameters and continuous monitoring are sufficient to prevent the emergence of undesirable functions.

Risk: Loss of control over organoid complexity, emergence of unexpected neuronal activity, ethical concerns, and potential loss of trust in the project.

Benefits: Stable experimental models, ethically validated research, and reliable data necessary for the study of neurodegenerative diseases.

Action 5: Documentation and traceability

Purpose: To ensure reproducibility and scientific accountability by enabling a clear and transparent record of all modifications throughout the project.

Design: To document the origin of iPSCs, experimental protocols, procedural changes, and results, while maintaining an updated and accessible project history.

Assumptions: It is assumed that rigorous documentation of the process enables reproducibility, supports risk evaluation, and facilitates adjustments when necessary.

Risk: Incomplete protocols, recording errors, or lack of traceability, leading to inconsistent data and consequently reduced validity or loss of trust within the scientific community.

Benefits: Reproducible and reliable experiments that provide a solid foundation for continued progress in the project.

Governance Actions Scoring

Governance actions considered: Action 1: Restricted use of neuronal organoids to certified laboratories

Action 2: Explicit prohibition of implantation in humans

Action 3: Informed consent and donor anonymization

Action 4: Functional delimitation of the organoid

Action 5: Documentation and traceability

Scoring system:

1 = strongest contribution to the policy objective

2 = moderate contribution

3 = weak or indirect contribution

Priority actions based on the scoring

Based on the assigned scores, priority actions are those that show a strong contribution (score = 1) across multiple key objectives, particularly biosecurity, laboratory safety, and incident prevention, while remaining feasible and not unduly restricting research.

Action 1: Restricted use of neuronal organoids to certified laboratories This action consistently receives low scores (1–2) in objectives related to incident prevention, laboratory safety, and environmental protection. This indicates that restricting research to certified laboratories is highly effective in mitigating risks at the source, ensuring that neuronal organoids are handled only in facilities with appropriate infrastructure, expertise, and oversight. Although certification requirements may introduce additional costs, their overall contribution to safety and governance justifies this action as a top priority.

Action 5: Documentation and traceability Documentation and traceability score highly in objectives related to both incident prevention and response. This action strengthens accountability by allowing the origin, handling, and downstream use of neuronal organoids to be monitored over time. While it may impose some administrative burden, its critical role in oversight, transparency, and post-incident investigation makes it a central governance priority.

Action 2: Explicit prohibition of implantation in humans This action stands out for its high feasibility, low implementation cost, and strong preventive effect. By clearly prohibiting human implantation, it directly addresses major ethical and biosecurity concerns and establishes a clear regulatory boundary. Although its contribution to incident response is limited, its clarity and risk-reduction potential make it a foundational priority action.

Complementary actions] Action 3: Informed consent and donor anonymization This action primarily supports ethical standards and public trust rather than direct risk mitigation. It is therefore considered essential but complementary, reinforcing responsible research practices.

Action 4: Functional delimitation of the organoid Functional delimitation helps prevent unintended developments and supports responsible innovation without significantly impeding research. Its impact is more indirect, making it a supportive rather than a primary governance measure.

Conclusion Overall, Actions 1, 5, and 2 emerge as the priority governance measures, as they provide the strongest balance between risk reduction, feasibility, and effective oversight, while the remaining actions play an important supporting role within the governance framework.

Ethical Concerns and Appropriate Governance Actions

One of the main ethical concerns emerging from the study of neuronal organoids is the moral status of increasingly complex biological models, particularly as they acquire more advanced neural organization and functional capabilities. As neuronal organoids become more sophisticated, questions arise regarding the possibility of sentience, consciousness, or the capacity to experience harm, even if only in a rudimentary form. A related ethical concern involves the blurring of boundaries between research models and human subjects, especially in scenarios involving potential implantation or integration with living systems. This challenges existing ethical frameworks, which were not designed for entities that are biologically human-derived but lack full organismal status. Additionally, issues of donor autonomy, informed consent, and long-term use of biological material raise concerns about respect for persons, ownership of biological data, and public trust in biomedical research.

Governance measures to address these concerns

To address these ethical challenges, several governance measures are particularly relevant: Explicit prohibition of implantation in humans helps establish clear moral and regulatory boundaries, preventing ethically unacceptable applications and reducing the risk of crossing into morally ambiguous territory. Functional delimitation of neuronal organoids ensures that research remains within defined and ethically acceptable limits, avoiding the development of functionalities that could raise concerns about sentience or moral status. Restricted use of neuronal organoids to certified laboratories provides institutional oversight and ensures that ethically sensitive research is conducted only in environments with appropriate expertise, review mechanisms, and accountability structures. Informed consent and donor anonymization protect donor autonomy and dignity, ensuring transparency regarding the use, storage, and future applications of donated biological material. Documentation and traceability support ethical accountability over time, enabling monitoring, review, and intervention if ethical or safety concerns emerge as research evolves.

Conclusion

Overall, the central ethical concern lies in managing the uncertain moral status and future potential of neuronal organoids. A combination of preventive governance measures, clear regulatory boundaries, and ongoing oversight is necessary to address these concerns responsibly, while still allowing scientific innovation to proceed.

Statement on the use of AI tools AI tools were used exclusively to assist with translation and language refinement from the author’s original Spanish text. The content, analysis, and conclusions are entirely the author’s own.

Week 2 HW: Lecture Prep

Homework Questions from Professor Jacobson

Nature’s machinery for copying DNA is called polymerase. What is the error rate of polymerase? How does this compare to the length of the human genome. How does biology deal with that discrepancy?

Polymerase is an enzyme whose function is to produce a chain of nucleic acids by copying another chain that serves as a template. It is basically the enzyme that copies DNA during replication. The error made by DNA polymerase consists of incorporating an incorrect nucleotide, that is, placing a base that is not complementary to that of the template (for example, placing A where C should go).

The error rate of DNA polymerase is approximately 1 error per 10⁵ nucleotides incorporated when acting alone. However, thanks to its proofreading activity and cellular DNA repair mechanisms, the effective error rate during replication is reduced to approximately 1 error per 10⁹–10¹⁰ nucleotides.

The error rate of DNA polymerase, even with correction mechanisms, must be compared to the large length of the human genome, which is approximately 3 × 10⁹ base pairs. If the polymerase did not have correction systems, thousands of errors would occur for each replication of the genome. However, thanks to proofreading and DNA repair mechanisms, the final number of errors is reduced to about one mutation per genome per cell division.

Biology addresses the discrepancy between the error rate of DNA polymerase and the large length of the human genome through various control and correction mechanisms. These include the high specificity of the polymerase for the correct nucleotides, its error correction (proofreading) activity, post-replication error repair systems such as mismatch repair, and the elimination of severely damaged cells through apoptosis. Together, these mechanisms allow a very low mutation rate to be maintained despite the large size of the genome.

How many different ways are there to code (DNA nucleotide code) for an average human protein? In practice what are some of the reasons that all of these different codes don’t work to code for the protein of interest?

An average human protein, with a length of approximately 400 amino acids, can theoretically be encoded by an extremely large number of different DNA sequences. This is due to the degeneracy of the genetic code, meaning that multiple codons can specify the same amino acid. On average, each amino acid is encoded by about three different codons, which implies that a protein of this size could be encoded by on the order of 3¨400 distinct nucleotide sequences, an astronomically large number.

However, in practice, not all of these sequences are functional for encoding the protein of interest. This is because biological systems impose several constraints on gene expression. Factors such as codon usage bias and tRNA availability influence the efficiency and accuracy of translation. In addition, mRNA secondary structures can interfere with translation initiation or elongation. Certain nucleotide sequences may also unintentionally introduce regulatory signals, such as alternative splicing sites or premature polyadenylation signals, which can disrupt proper mRNA processing. Furthermore, GC content, mRNA stability, and the presence of repetitive or toxic sequences further limit the set of sequences that are biologically viable.

Taken together, although many theoretical nucleotide sequences can encode the same human protein, only a small fraction are effective in a real cellular context.

Homework Questions from Dr. LeProust

What’s the most commonly used method for oligo synthesis currently?

The most commonly used method for oligonucleotide synthesis today is solid-phase chemical synthesis using phosphoramidite chemistry.

In this method, the oligonucleotide is constructed base by base on a solid support. Each synthesis cycle includes the deprotection of the terminal nucleotide, the incorporation of an activated nucleotide (phosphoramidite), the oxidation or sulfidation of the bond formed, and blocking steps to prevent unwanted reactions. This process allows for highly automated, rapid, and efficient synthesis.

Why is it difficult to make oligos longer than 200nt via direct synthesis?

It is difficult to manufacture oligonucleotides longer than 200 nucleotides by direct chemical synthesis because the efficiency of each nucleotide incorporation cycle is not 100%. In phosphoramidite synthesis, each step has a small probability of error or coupling failure. As the length of the oligonucleotide increases, these errors accumulate, causing a drastic decrease in the yield of the complete product and an increase in truncated or incorrect sequences.

In addition, long oligonucleotides are more difficult to purify because the size differences between the correct sequence and incomplete sequences are small. Factors such as the formation of secondary structures during synthesis and chemical limitations associated with the stability of reagents and the solid support also play a role. For these reasons, direct synthesis of long oligonucleotides is inefficient and unreliable beyond approximately 200 nucleotides.

Why can’t you make a 2000bp gene via direct oligo synthesis?

A gene of approximately 2000 base pairs cannot be created by direct oligonucleotide synthesis because chemical DNA synthesis is less than 100% efficient in each nucleotide incorporation cycle. In phosphoramidite synthesis, each step has a small probability of failure, and these errors accumulate exponentially as the sequence length increases. As a result, the yield of the complete product decreases dramatically, and truncated or erroneous sequences predominate.

Furthermore, purification of such long fragments is extremely difficult, as the differences between the correct sequence and incomplete sequences are minimal. Added to this are chemical and physical limitations, such as reagent degradation, secondary structure formation, and the instability of long DNA during solid-phase synthesis. For these reasons, direct synthesis is not feasible for long genes, and in practice, genes of this size are obtained by assembling multiple shorter oligonucleotides using molecular biology techniques.

Homework Question from George Church

Given slides #2 & 4 (AA:NA and NA:NA codes)] What code would you suggest for AA:AA interactions?

For AA:AA (amino acid–amino acid) interactions, there is no discrete, symbolic code equivalent to the genetic code. Instead, the most appropriate type of “code” is one based on the physicochemical properties of amino acids, such as electrical charge, polarity, hydrophobicity, and residue size. These properties determine the non-covalent interactions between amino acids, including hydrophobic interactions, electrostatic interactions, hydrogen bonds, and disulfide bonds, which govern protein folding and stability.

Unlike NA:NA or NA:AA codes, where there is a defined correspondence between symbols, AA:AA interactions emerge from the three-dimensional context of the protein and the chemical characteristics of the residues involved. Therefore, a code based on physicochemical properties allows for a more realistic description and prediction of amino acid–amino acid interactions within a protein.

Source: Alberts, B. et al. Molecular Biology of the Cell. Garland Science. Chapters on protein structure and non-covalent interactions .

Week 1 Lab: Pipetting

Individual Final Project

Group Final Project