Notes on Direct Conversion of Human Fibroblasts into Neural Progenitors

Article Overview

Direct Conversion of Human Fibroblasts into Neural Progenitors Using Transcription Factors Enriched in Human ESC-Derived Neural Progenitors

This article, published in Stem Cell Reports, explores a novel method for generating neural progenitors directly from human fibroblasts, aiming to overcome limitations of iPSC technology, particularly regarding timing and safety concerns.

Timeline for NSC Treatment

Original iPSC-based Pipeline (Total: $112$ days or approximately $3.7$ months)

Fibroblast Isolation and Expansion: $14$ days
Fibroblast Quality Assessment: $7$ days
- Karyotype analysis.
- Sterility testing (e.g., bacteria, fungi, mycoplasma).
iPSC Reprogramming: $14$ days
iPSC Expansion & Banking: $21$ days (for $15$ million cells).
iPSC Gene Expression Analysis, Flow Cytometry: $7$ days
NSC Differentiation: $14$ days
NSC Expansion & Banking: $21$ days (for $15$ million cells: $10$ million for treatment/ $5$ million for testing).
NSC Gene Expression Analysis, Flow Cytometry: $7$ days
NSC Quality Assessment: $7$ days
- Karyotype analysis.
- Sterility testing (e.g., bacteria, fungi, mycoplasma).

Implications for Stroke Treatment

Stroke Statistics:
- Second leading cause of death globally.
- Number one cause of long-term disability in the U.S.
- Someone has a stroke every $40$ seconds.
- Someone dies of stroke every $4$ minutes.
Golden Window for Stroke Treatment/Recovery: Roughly $3$ months.
Mismatch: The original iPSC-based NSC treatment timeline of $3.7$ months exceeds the $3$ -month golden window for stroke treatment, indicating this approach is too slow for acute stroke intervention.

Diseases Where Timing is Less Critical (Beyond 3-Month Window)

Huntington's Disease (HD)
Alzheimer's Disease (AD)
These conditions are suitable for modeling because they are genetic, allowing for the creation of disease-specific cell lines for studying disease mechanisms and screening for treatments.

Direct Conversion (iN technology) - A Faster Alternative

The transcript suggests a potential saving of $42$ days, reducing the total timeline to $70$ days through direct NSC reprogramming, which could fall within the critical treatment window for some conditions.

Key Elements From the Introduction

Induced Pluripotent Stem Cells (iPSCs) Technology

Advantages:
- Leverages pluripotency reprogramming technology.
- Enables the generation of disease-specific iPSCs from patients' somatic cells.
- Allows in vitro differentiation into various disease-relevant cell types.
- Useful for disease modeling and drug development (e.g., HD iPSC Consortium, 2012).
- Offers a virtually unlimited supply of specialized cells for transplantation in patients with organ failure or degenerative diseases.
Concerns:
- Tumorigenic potential.
- Spontaneous differentiation of iPSCs.

Induced Neurons (iN) Technology - Direct Conversion

Advantages:
- Provides a fast and simple method for generating specific neuronal subtypes.
- May avoid problems associated with human iPSCs (hiPSCs), such as uncontrolled cell differentiation and tumor formation.
- Multiple research groups have successfully demonstrated direct reprogramming of fibroblasts into NSCs in both mouse and human.

Focus on Embryonic Neural Progenitors (ENPs)

Most studies have concentrated on differentiating NSCs/NPs into central nervous system (CNS) cell types, often neglecting peripheral nervous system (PNS) cell types.
The authors aim to generate embryonic neural progenitors (ENPs) instead of neural stem cells (NSCs) or neural progenitors (NPs), because ENPs possess a greater level of plasticity.

Cell Types and Functions in the Nervous System

Central Nervous System (CNS) Cell Types:
- Neurons: Conduct electrical impulses; enable cognition, sensation, motor control.
- Astrocytes: Regulate the blood-brain barrier, ion balance, and neurotransmitter recycling.
- Oligodendrocytes: Form myelin sheaths around axons in the CNS.
- Microglia: Act as resident immune cells of the CNS (analogous to macrophages).
Peripheral Nervous System (PNS) Cell Types:
- Neurons: Sensory and motor neurons connect the CNS to limbs and organs.
- Schwann Cells: Myelinate axons in the PNS (analogous to oligodendrocytes).
- Satellite Glial Cells: Support and insulate neuron cell bodies in ganglia.
- Macrophage-like Immune Cells: Provide immune surveillance in the PNS (non-microglial).

Degrees of Plasticity

Totipotent Stem Cells: Can differentiate into all cell types, including embryonic and extraembryonic tissues (e.g., two-cell stage, four-cell stage, eight-cell stage).
Pluripotent Embryonic Stem Cells (ESCs): Can differentiate into all cell types of the three germ layers (endoderm, mesoderm, ectoderm) but not extraembryonic tissue (e.g., inner cell mass of a blastocyst).
- Induced Pluripotent Stem Cells (iPSCs): Artificially reprogrammed somatic cells with pluripotent capabilities.
Multipotent Stem Cells: Can differentiate into multiple cell types within a specific lineage or tissue (e.g., hematopoietic stem cells).
- Differentiation Pathways:
  - Endoderm Line: Gives rise to organs like the lung and pancreas.
  - Mesoderm Line: Gives rise to tissues like heart muscle and red blood cells.
  - Ectoderm Line: Gives rise to tissues like skin and neurons.

Epigenetic Modifications and Reprogramming

Differentiation is an Epigenetic Change

Epigenetic Modifications: Heritable phenotype changes that do not involve alterations in the DNA sequence but affect gene activity and expression.
Most Common Types:
- DNA Methylation: Addition of a methyl group to DNA, typically at CpG sites, often leading to gene silencing.
  - DNMT3 (DNA Methyltransferase 3): A family of enzymes that catalyze the transfer of a methyl group to DNA.
- Histone Blocking (Histone Modifications): Chemical alterations to histone proteins that can affect chromatin structure and gene accessibility.
  - Histone Acetylation: Transfer of an acetyl functional group from acetyl coenzyme A to histones.
    - Catalyzed by Histone Acetyltransferases (HATs).
    - Removes positive charges on histones, decreasing their interaction with negatively charged DNA.
    - Leads to a more relaxed chromatin structure, associated with greater gene transcription.
  - Histone Deacetylation: Removal of an acetyl group from histones.
    - Catalyzed by Histone Deacetylases (HDACs).
    - Results in condensed chromatin, typically repressing gene transcription.

Reprogramming is the Reverse of Differentiation

This concept, the ability to revert differentiated cells to a pluripotent state, was notably recognized by the $2012$ Nobel Prize in Physiology or Medicine.

Major Objectives/Findings Claimed by the Authors

Defined two transcription factor (TF) combinations that can efficiently convert human fibroblasts (FBs) into multipotent induced embryonic neural progenitors (iENPs).
Demonstrated that different TF combinations induce iENP populations with varying proliferative features and regional differentiation preferences.
Showed that neurons derived from AD- and HD-iENPs recapitulated major disease pathological features in vitro.

Figure 1 Analysis: Initial Identification of hESC-ENP Enriched TFs and iENP Generation

(a) Heatmap Analysis: Compares global gene expression profiles of hESC-ENPs (NP1 from ArrayExpress database, Zhang et al., 2010; NP2 from H9-SOX1:EGFP sorted ND day $18$ -NP) and Fibroblasts (FB1, -2, and -3).
(b) Selected TFs: Identifies transcription factors with higher expression in hESC-ENPs compared to fibroblasts.
(B) Experimental Strategy: Schematic depicting the direct conversion of FBs into iENPs using $25$ identified TFs.
- PAX6:EGFP and SOX1:EGFP Reporters: These are genetic constructs where the promoter of a neural-specific gene (PAX6 or SOX1) drives the expression of Enhanced Green Fluorescent Protein (EGFP).
  - Importance: They serve as indicators of neural progenitor identity, allowing researchers to visualize and quantify cells that have successfully reprogrammed into neural progenitors.
  - Gene Promoter: A DNA sequence that regulates gene transcription by acting as a binding site for proteins that initiate and control RNA synthesis from a DNA template.
(C) Lentivirus Infection and Cell Sorting:
- FBs are infected with lentiviruses encoding the hESC-ENP TFs (25$TF) and the neural reporter (PAX6:EGFP or SOX1:EGFP).
- Cells infected with UbC:EGFP serve as controls.
- Fluorescence-Activated Cell Sorting (FACS) is used to isolate EGFP-positive cells (i.e., those expressing the neural reporter), and their subsequent growth is monitored.
(D) ICC Analysis: Immunocytochemistry analysis of iENP-$25$F clusters (resembling neural progenitor colonies/spheres) using antibodies against indicated antigens (e.g., NESTIN, SOX1, OTX2, ZO1, PAX6, SOX2, ID1).
(E) RT-PCR Analysis of Gene Expression in iENP-$25$F:
- RT-PCR (Reverse Transcription Polymerase Chain Reaction): A molecular biology technique used to detect and quantify RNA by converting it into complementary DNA (cDNA) and then amplifying specific DNA sequences.
  - Process: RNA is first reverse-transcribed into cDNA, and then the cDNA is amplified using PCR (Polymerase Chain Reaction).
  - PCR: Exponentially amplifies specific DNA sequences. The primer at the PCR step is designed to be specific for particular gene targets.
- Gel Electrophoresis: A laboratory technique used to separate DNA, RNA, or protein molecules based on their size and electrical charge. Smaller molecules move faster through the gel's pores.
  - The system for primer design typically provides the expected DNA fragment size.
- Ethidium Bromide (EtBr): A fluorescent dye that intercalates between DNA base pairs, making DNA glow under UV light for visualization after gel electrophoresis.
- Controls in RT-PCR:
  - P (Positive Control): A sample known to contain the target RNA/DNA, ensuring the assay is working correctly.
  - N (Negative Control): A sample lacking the target, to check for contamination or non-specific amplification.
  - NC (No Template Control): Contains all reaction components except the RNA/cDNA template, essential for detecting primer-dimer formation or reagent contamination.

Figure 2 Analysis: Optimizing TF Combinations for PAX6:EGFP iENPs

(A) Experimental Strategy: Schematic depicting the stepwise reduction of TFs from 25 $to$ 6 $for iENP induction, using the PAX6:EGFP neural reporter.</li><li>(B and C) Stepwise TF Selection: Potent iENP factors are selected through single TF dropouts from the original$ 25 $-TF set (B) and the$ 15 $-TF set (C). Results are expressed as the relative percentage of PAX6:EGFP+ cells after each TF removal, indicating the importance of each factor.</li><li>(D) Comparison of Induction Efficiency: Compares the efficiency of PAX6:EGFP+ cell induction from FBs using$ 25 $-,$ 15 $-, and$ 6 $-TF combinations (FACS-based quantification).<ul><li>The$ 6-TF combination (iENP-$6$F) yielded 10.54 % ext{ } {+}}{ } 0.47 % of PAX6:EGFP+ cells, demonstrating efficient induction with fewer factors.

(E) Global Gene Expression Heatmap: Microarray analysis comparing gene expression profiles of FB, hESC-ENP, iENP-$6$F, and iENP-$15$F.

(F) ICC Staining: Immunocytochemistry staining of iENP-$6$F using antibodies against indicated NP markers (e.g., ZO1, NESTIN, NCAD, PAX6, SOX1, SOX2).

(G) RT-PCR of Endogenous and Exogenous TFs: Analysis of mRNA from iENP-$6$F to confirm expression of the 6 TFs (both from the introduced plasmid and endogenous activation).

(H) RT-PCR of Neural Genes: Analysis of mRNA from iENP-$6$F to confirm expression of indicated neural genes (e.g., NESTIN, DACH1, LHX2, OTX2, PAX6, SOX1, SOX2, ACTB as a loading control).

Figure 3 Analysis: Optimizing TF Combinations for SOX1:EGFP iENPs

(Similar to Figure 2, but for SOX1:EGFP Reporter): The authors perform a similar stepwise reduction of TFs but optimize for SOX1:EGFP expression.
- They identify a 7-TF combination (iENP-$7$F) that yields 11.22 % ext{ } {+}}{ } 0.44 %$$ of SOX1:EGFP+ cells.

Figure 4 Analysis: Characterization and Differentiation Potential of iENP-$6$F

(A-E) Differentiation and Marker Expression:
- ICC staining of differentiated iENP-$6$F using antibodies against:
  - The glial marker GFAP (A).
  - The oligodendrocyte marker GALC (B).
  - Neuronal markers like MAP2, NEUN, NFH, TUJ1 (C, D).
  - The synapse marker SYN (E).
(F) Quantification of Differentiated Cells: Compares the percentage of TUJ1+ (neurons), GFAP+ (astrocytes), and GALC+ (oligodendrocytes) cells in differentiated hESC-NPs, iENP-$6$F, and iENP-$15$F.
(G-M) CNS and PNS Neuronal Subtypes: ICC staining of differentiated iENP-$6$F using antibodies against specific markers for various CNS and PNS neuronal antigens:
- GABAergic Neuron (CNS)
- TBR1 Cortical Neuron (CNS)
- TH Dopaminergic Neuron (CNS)
- HB9 / ISL1 / BRN3A Motor Neuron (CNS/PNS)
- PRPH PNS Neuron
(N) Lineage-Specific Differentiation:
- (a) Schematic: Experimental procedures outlining distinct cocktail of lineage-specific cues (e.g., N2B27+SHH+Noggin+DKK1+XAV979+LDN93189+SB431542 for TBR1+ cortical neurons) used to induce specific neuronal subtypes from iENP-$6$F.
- (b) ICC Characterization: ICC analysis of differentiated iENP-$6$F under these subtype-specific conditions using relevant CNS and PNS neuronal antibodies.
- (c) Quantification: Percentage of indicated neuronal subtypes induced, comparing conditions without inducers (GF-) vs. with inducers (GF+).

Neuron Action Potential

Definition: A brief electrical impulse, or