Cell-Based Therapies

What type of stem cells are used as the starting material to grow blood stem cells in the lab?

Pluripotent hESCs

What is the differentiation pathway from hESCs to haematopoietic progenitors?

Mesoderm → Haemangioblast → Haemogenic endothelium → Haematopoietic progenitors.

What role do morphogens and cytokines play in generating HSCs?

They guide the differentiation of hESCs into haemogenic endothelium using factors like BMP4, VEGF, bFGF, CHIR, IGF1, IL6, IL11, SB, and SCF.

What is the significance of transcription factors in making lab-grown blood cells?

A library of 26 TFs was tested; 7 were found to be sufficient to induce functional HSCs from hESC-derived haemogenic endothelium.

How can iPSCs be used in Parkinson’s disease?

iPSCs can be differentiated into midbrain dopaminergic (mDA) neurons to replace those lost in PD by replicating developmental signaling and contact cues.

What was the outcome of transplanting iPSC-derived neurones into a simian PD model?

Transplanted cells matured, improved function (movement), survived long-term, and produced dopamine.

What marker can you use to identify floor plate-derived progenitors during the differentiation of pluripotent stem cells into midbrain dopaminergic (mDA) neurones?

Corin

What was the first approved gene therapy stem cell treatment?

Strimvelis for SCID (adenosine deaminase deficiency) approved in Europe.

How was the first patient cured of HIV using HSCs?

Timothy Ray Brown received an allogeneic HSC transplant from a donor with the CCR5 Δ32 mutation during leukemia treatment, resulting in HIV-resistant immune cells.

What is the CCR5 Δ32 mutation, and how does it relate to HIV resistance?

CCR5 Δ32 is a 32-base pair deletion that produces a non-functional CCR5 receptor, preventing HIV from entering T helper cells.

Why couldn’t HIV infect the transplanted immune system in Timothy Ray Brown?

The donor cells lacked functional CCR5 receptors, so HIV could not enter the newly generated immune cells.

Explain the stem-cell treatment for ADA-SCID.

It uses autologous CD34+ HSCs that have been edited to express the normal ADA gene.

How could CRISPR-Cas9 be used to treat HIV?

Delete CCR5 in a patient’s own HSCs (autologous therapy).

How are HSCs used in ADA-SCID treatment?

Patient HSCs are extracted, genetically modified to express functional ADA and expanded in culture, and reinfused to repopulate the immune system with corrected cells.

Do SCID patients require full myeloblative irradiation?

No. Their own (mutant) HSCs may remain, but they are deficient in ADA, so their progeny don’t survive well and the corrected ones gradually dominate the immune system.

What is a major risk associated with retroviral vector-based gene therapy?

Insertional mutagenesis where random viral insertion near oncogenes may lead to leukaemia.

Why is Strimvelis particularly valuable despite the risks?

Over 75% of ADA-SCID patients lack an HLA-matched donor so allogenic transplant would not work.

What genetic mutation causes sickle cell disease?

A single nucleotide polymorphism (A → T) in the β-globin gene, leading to abnormal haemoglobin (sickle shape not biconcave) which causes blockages in blood vessels.

How is sickle cell disease treated with engineered HSCs?

Autologous HSCs are transfected with an antisickling β-globin gene (analogue that just synthesises the B-globin) using a lentiviral vector and reinfused to repopulate the blood with functional red cells.

What was the advantage of using lentiviruses over retroviruses?

Lentiviruses tend to integrate into actively transcribed genes, but not preferentially near proto-oncogenes, unlike retroviruses.

What did the 2022 NEJM sickle-cell gene therapy study on 35 patients show?

The majority of hemoglobin in treated patients was the engineered type, showing strong efficacy and no severe vaso-occlusive events across individuals.

Which two gene editing therapies are approved for treating sickle cell disease?

Casgevy (CRISPR-Cas9) and Lyfgenia (Lentiviral).

How do cytotoxic T cells recognize and kill cancer cells?

Cytotoxic T cells are activated by antigen-presenting cells (APCs) and kill target cells expressing specific antigens by releasing perforin and granzyme, which induce apoptosis.

How can the efficacy of cytotoxic T cell-mediated killing of cancer cells be improved?

Cell engineering.

What is the role of APCs in cancer immunotherapy?

APCs present tumour-specific antigens to prime T cells, enhancing immune recognition and destruction of cancer cells.

What are artificial antigen-presenting cells (aAPCs)?

Engineered cells or microparticles decorated with antigens that mimic natural APCs to activate T cells without needing patient-derived cells (off the shelf).

How can dendritic cells be used as cancer vaccines?

A tumour biopsy is taken and total RNA is extracted. RNA is reverse-transcribed to cDNA, then amplified to make tumour-specific RNA. Mononuclear cells are collected from the patient via leukapheresis and cultured to isolate monocytes. Monocytes are differentiated into immature dendritic cells (DCs). These DCs are loaded with tumour RNA and matured. The RNA-loaded mature DCs are formulated into a DC vaccine and reinfused into the patient to trigger a tumour-specific immune response.

What are CAR T cells?

T cells that are genetically engineered to express a chimeric antigen receptor (CAR), allowing them to recognize and kill cancer cells that express specific antigens.

How do CAR T cells work?

Upon recognizing their target antigen, CAR T cells proliferate and kill tumour cells using perforin and granzyme, similar to normal T cell responses.

What are CAR T cells primarily used to treat?

Primarily used for leukemias and lymphomas (blood cancers), though there is active development for solid tumours.

Why does CAR T therapy not work in solid tumours?

The TME blocks T-cell activation and IL-2 signalling.

What modifications are being explored to adapt CAR T cells for solid tumours?

Adding extra transgenes, such as enzymes to break down the extracellular matrix (ECM) for better tumour penetration or synNotch receptors to activate IL-2 secretion.

Name three FDA-approved CAR T therapies and their targets.

Kymriah – CD19, for acute lymphoblastic leukemia. Yescarta – CD19, for large B-cell lymphoma, Abecma – BCMA, for multiple myeloma

How do CAR T cells activate without MHC presentation?

CARs contain a stimulatory domain (e.g., CD3ζ, CD28) that mimics T cell receptor (TCR) signalling, allowing direct activation when the CAR binds an antigen on the tumour cell, bypassing the need for MHC-mediated antigen presentation.

How can iPSCs be used as a cancer vaccine?

iPSCs express a variety of tumour-associated antigens because they are primitive cells, many of which are shared with cancer cells, making them suitable for training the immune system.

How do iPSCs function as a cancer vaccine in preclinical models?

In mouse models, vaccination with irradiated iPSCs + CpG adjuvant led to tumour regression after tumour inoculation.

Why are the iPSCs used in cancer vaccines irradiated?

Irradiation ensures the iPSCs are non-proliferative and cannot form teratomas.

What is the purpose of cell encapsulation in therapy?

It shields implanted cells (usually allogeneic) from the host immune system, reducing the risk of rejection/HVGD while allowing therapeutic protein diffusion and mass transfer through a porous membrane.

What are common targets for encapsulated cell therapies?

Eye diseases, neurodegenerative diseases, diabetes, and cancers.

Why are cells better than direct protein delivery for some therapies?

Cells can continuously produce proteins in situ, potentially offering a sustained and cost-effective therapeutic strategy.

What is ENCELTO used to treat?

Idiopathic macular telangiectasia type 2, a condition that causes photoreceptor cell loss in the macula, leading to progressive blindness.

What type of cells are encapsulated in ENCELTO?

Allogeneic retinal pigment epithelial (RPE) cells engineered to express recombinant human ciliary neurotrophic factor (rhCNTF).

How does ENCELTO help prevent vision loss?

The encapsulated cells release rhCNTF, which promotes photoreceptor survival, preserving retinal structure and function.

Where is ENCELTO implanted?

It is sutured in place and floats at the back of the eye, near the retina.

What is Viacyte?

A dense cell network of insulin-secreting cells encapsulated in a semi-permeable barrier that respond to glucose and restore blood sugar control in diabetic patients.

What type of cells are used in Viacyte's diabetes therapy, and how are they protected?

Allogeneic stem cell-derived pancreatic beta-like cells.

What are the differences between PEC-Encap, PEC-Direct, and PEC-QT?

PEC-Encap: Shielded from immune system (no immunosuppression needed). PEC-Direct: Visible to immune system, requires immunosuppression. PEC-QT: Cells engineered to be immune-evasive, no immunosuppression required.

Why is HLA matching important in cell therapy and transplantation?

It determines whether the immune system accepts cells as self or non-self

How are HLA genes inherited?

HLAs are co-dominantly expressed and inherited as haplotypes (one set from each parent).

Why is it difficult to find perfectly matched HLA donors?

There are a huge number of possible allele combinations at each HLA locus (e.g., 125 B alleles, 68 A alleles).

What happens if transplanted cells are not HLA matched?

The immune system can recognize them as foreign and trigger rejection.

What is required to prevent rejection of allogeneic transplants?

Immunosuppressive therapy

What is the HLA system?

A set of genes located on chromosome 6 that encode proteins essential for immune recognition. It is the human version of MHC.

What is class I HLA?

Found on almost all nucleated cells that present endogenous antigens.

What is class II HLA?

Found on APCs and present exogenous antigens.

Are human pluripotent stem cells (hPSCs) immune privileged?

No, they express HLA class I.

What affects the immune visibility of hPSCs?

Their differentiation state and cell lineage. Retained in cardiomyocytes but reduced in hepatocytes.

What are hypoimmunogenic iPSCs?

Genetically engineered iPSCs that evade immune detection by disrupting HLA class I and II genes and/or express CD47 (tumour antigen involved in avoiding phagocytosis).

What inspired hypoimmunogenic iPSCs?

Studying the interface between mother and foetus revealed syncytiotrophopblasts.

Why is hypoimmunogenicity important for stem cell therapies?

It enables the potential for off-the-shelf banks of iPSCs which could be scaled for multiple applications.

How can bacteria be used as therapeutic agents in cancer?

Obligate anaerobes can be engineered to colonise hypoxic tumour regions (resistant to radio/chemotherapy and release cytotoxic agents, followed by clearance with antibiotics.