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Cell Technologies and Engineering Core

Salk-CIRM Shared Resources Laboratory

Salk Institute for Biological Studies - Cell Technologies and Engineering Core - Salk-CIRM Shared Resources Laboratory

Salk-CIRM Shared Resources Laboratory

The Cell Technologies and Engineering Core Facility (CTEC) at the Salk Institute supports research on human fibroblasts and stem cells, providing dedicated lab space, equipment, validated reagents, and extensively characterized cell lines. It generates patient-derived fibroblasts from healthy donors and Alzheimer’s patients, which are either studied directly, converted to induced pluripotent stem cells (iPSCs), or transdifferentiated into brain cell types for aging and neurodegeneration studies. iPSCs reset aging hallmarks, while directly reprogrammed cells retain donor aging signatures, making them ideal for studying the aging brain. The core ensures rigor and reproducibility through standardized protocols, reagent packages, and rigorous quality control. Beginning Summer 2026, CTEC Core will offer hands-on training in stem cell techniques, genome editing, and transdifferentiation, partnering with CIRM programs and California research institutions to expand access and impact.

SOPs

Training Course References

Basic Stem Cell Techniques [View References]

  • Takahashi, K. et al. Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors. Cell 131, 861–872 (2007).
  • Mali, P. et al. Butyrate greatly enhances derivation of human induced pluripotent stem cells by promoting epigenetic remodeling and the expression of pluripotency-associated genes. Stem Cells 28, 713–720 (2010).
  • Watanabe, K. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol 25, 681–686 (2007).
  • Codner, G. F. et al. Aneuploidy screening of embryonic stem cell clones by metaphase karyotyping and droplet digital polymerase chain reaction. BMC Cell Biol 17, (2016).

Genome Editing [View References]

  • Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A., et al. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823. 10.1126/science.1231143.
  • Lin, S., Staahl, B.T., Alla, R.K., and Doudna, J.A. (2014). Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife 3, e04766. 10.7554/eLife.04766.
  • Hockemeyer, D., and Jaenisch, R. (2016). Induced pluripotent stem cells meet genome editing. Cell Stem Cell 18, 573–586. 10.1016/j.stem.2016.04.013.
  • Kime, C., Mandegar, M.A., Srivastava, D., Yamanaka, S., Conklin, B.R., and Rand, T.A. (2016).
  • Efficient CRISPR/Cas9-Based Genome Engineering in Human Pluripotent Stem Cells. Curr. Protoc. Hum. Genet. 88, 21.4.1-21.4.23. 10.1002/0471142905.hg2104s88.

Direct Reprogramming (Fibro-iN) [View References]

  • Mertens J, Paquola ACM, Ku M, Hatch E, Böhnke L, Ladjevardi S, McGrath S, Campbell B, Lee H, Herdy JR, Gonçalves JT, Toda T, Kim Y, Winkler J, Yao J, Hetzer MW, Gage FH. Directly Reprogrammed Human Neurons Retain Aging-Associated Transcriptomic Signatures and Reveal Age-Related Nucleocytoplasmic Defects. Cell Stem Cell. 2015 Dec 3;17(6):705-718. doi: 10.1016/j.stem.2015.09.001. Epub 2015 Oct 8. PMID: 26456686; PMCID: PMC5929130.
  • Zheng X, Boyer L, Jin M, Mertens J, Kim Y, Ma L, Ma L, Hamm M, Gage FH, Hunter T. Metabolic reprogramming during neuronal differentiation from aerobic glycolysis to neuronal oxidative phosphorylation. Elife. 2016 Jun 10;5:e13374. doi: 10.7554/eLife.13374. PMID: 27282387; PMCID: PMC4963198.
  • Mertens J, Reid D, Lau S, Kim Y, Gage FH. Aging in a Dish: iPSC-Derived and Directly Induced Neurons for Studying Brain Aging and Age-Related Neurodegenerative Diseases. Annu Rev Genet. 2018 Nov 23;52:271-293. doi: 10.1146/annurev-genet-120417-031534. Epub 2018 Sep 12. PMID: 30208291; PMCID: PMC6415910.
  • Mertens J, Herdy JR, Traxler L, Schafer ST, Schlachetzki JCM, Böhnke L, Reid DA, Lee H, Zangwill D, Fernandes DP, Agarwal RK, Lucciola R, Zhou-Yang L, Karbacher L, Edenhofer F, Stern S, Horvath S, Paquola ACM, Glass CK, Yuan SH, Ku M, Szücs A, Goldstein LSB, Galasko D, Gage FH. Age-dependent instability of mature neuronal fate in induced neurons from Alzheimer’s patients. Cell Stem Cell. 2021 Sep 2;28(9):1533-1548.e6. doi: 10.1016/j.stem.2021.04.004. Epub 2021 Apr 27. PMID: 33910058; PMCID: PMC8423435.

Astrocytes [View References]

  • Santos R, Vadodaria KC, Jaeger BN, Mei A, Lefcochilos-Fogelquist S, Mendes APD, Erikson G, Shokhirev M, Randolph-Moore L, Fredlender C, Dave S, Oefner R, Fitzpatrick C, Pena M, Barron JJ, Ku M, Denli AM, Kerman BE, Charnay P, Kelsoe JR, Marchetto MC, Gage FH. Differentiation of Inflammation-Responsive Astrocytes from Glial Progenitors Generated from Human Induced Pluripotent Stem Cells. Stem Cell Reports. 2017 Jun 6;8(6):1757-1769. doi: 10.1016/j.stemcr.2017.05.011. PMID: 28591655; PMCID: PMC5470172.
  • Santos R, Mei A, Marchetto MC. Generation of inflammation-responsive astrocytes from glial progenitors derived from human pluripotent stem cells. STAR Protoc. 2022 Mar 17;3(2):101261. doi: 10.1016/j.xpro.2022.101261. PMID: 35313707; PMCID: PMC8933838.
  • Patani R, Hardingham GE, Liddelow SA. Functional roles of reactive astrocytes in neuroinflammation and neurodegeneration. Nat Rev Neurol. 2023 Jul;19(7):395-409. doi: 10.1038/s41582-023-00822-1. Epub 2023 Jun 12. PMID: 37308616.
  • Derevyanko A, Tao T, Allen NJ. Common alterations to astrocytes across neurodegenerative disorders. Curr Opin Neurobiol. 2025 Feb;90:102970. doi: 10.1016/j.conb.2025.102970. Epub 2025 Jan 28. PMID: 39879721.

Microglia [View References]

  • Abud EM, Ramirez RN, Martinez ES, Healy LM, Nguyen CHH, 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. doi: 10.1016/j.neuron.2017.03.042. PMID: 28426964; PMCID: PMC5482419.
  • Munro DAD, Bestard-Cuche N, McQuaid C, Chagnot A, Shabestari SK, Chadarevian JP, Maheshwari U, Szymkowiak S, Morris K, Mohammad M, Corsinotti A, Bradford B, Mabbott N, Lennen RJ, Jansen MA, Pridans C, McColl BW, Keller A, Blurton-Jones M, Montagne A, Williams A, Priller J. Microglia protect against age-associated brain pathologies. Neuron. 2024 Aug 21;112(16):2732-2748.e8. doi: 10.1016/j.neuron.2024.05.018. Epub 2024 Jun 18. PMID: 38897208.

Salk-CIRM SRL Training Course

The Salk Institute has designed a detailed course with two optional subcourses that will introduce researchers to foundational stem cell concepts and techniques and then delve into the more specialized topics of genome editing and the creation of stem cell-based neuronal models for studying aging and disorders of the brain.

Outline of the Curriculum

Basic Stem Cell Techniques Course
Techniques that will be taught include:

  • Basic hPSC culture techniques including feeder free culture as well as culture on feeders
  • Cryopreservation and thawing of hPSCs
  • Basic characterization of pluripotency by immunocytochemistry and alkaline phosphatase
  • Differentiation via embryoid body formation

Transdifferentiation of Somatic Cells to Neurons (led by Salk with contributions from UCSD)
Topics will include:

  • History of direct conversion/transdifferentiation technology
  • Deriving fibroblasts from skin biopsies that will be used for transdifferentiation
  • How to assess the quality of fibroblasts prior to transdifferentiation
  • The rationale for choosing different transcription factors for neuronal conversion
  • Making and stable fibroblast lines for transdifferentiation
  • Using cell sorting to select PSA-NCAM-expressing neurons after transdifferentiation
  • Phenotyping neuronal cells (e.g., via immunochemistry and live cell imaging)
  • Using transdiferentiation technology to study human aging and neurodegenerative conditions
  • Latest technological advances to improve neuronal conversion

Genome Editing of Stem Cells and Primary Fibroblasts
Discussion topics include:

  • The rationale and choice of iPSCs for genome editing
  • Genome editing technologies and formats, when each is most appropriate
  • The latest technological advances, CRISPR spin-off technologies
  • Designing and generating DNA donor molecules for homologous recombination
  • Special considerations when single-cell cloning iPSCs
  • Editing primary fibroblasts prior to neural conversion

Hands-on activities will include:

  • Delivery of CRISPR materials into cells in different formats, by different methods
  • Validation by target site PCR and Sanger sequencing
  • Colony picking to isolate clones
  • Generation of a ssDNA donor molecule for knockin of a transgene
  • Special considerations when single-cell cloning iPSCs
  • Editing primary fibroblasts prior to neural conversion

Course Instructors

  • Basic Stem Cell Techniques will be designed and co-taught by Drs. Maria Carolina Marchetto and Jennifer Page.
  • Genome Editing of Stem Cells and Primary Fibroblasts will be taught by Dr. Jennifer Page.
  • Transdifferentiation of Somatic Cells to Neurons will be designed and taught by Drs. Gage, Allen, and Maria Carolina Marchetto.

Course syllabus (subject to change)

Day of the Week Basic Stem Cell Course Activity Gene Editing Course Activity Transdifferentiation Course Activity
Monday Morning Theory:
  • Lecture on history of Reprogramming and Pluripotent Stem Cell culture techniques
Afternoon Practical:
  • Prepare plates and thaw stem cells
Tuesday Morning Practical:
  • Observe Stem cell morphology after thawing
  • Learn to recognize stem cell morphology
Afternoon Practical:
  • Passing stem cells
Wednesday Morning Practical:
  • Cryopreservation
Afternoon
  • Free
 Afternoon Theory:
  • History of Genome Editing; Current status of Technology
Practical:
  • Assembly of CRISPR RNPs and delivery
Thursday Morning Practical:
  • Differentiation via embryoid body formation
  • Basic characterization of pluripotency by immunocytochemistry and alkaline phosphatase
 Afternoon Theory:
  • Design of donor vectors
Practical:
  • Generation of ssDNA donor
Friday Morning Practical:
  • Observe embryo bodies
  • Analyze slides on fluorescent microscope
Afternoon Free		 Afternoon Theory:
  • Analysis tools
Practical:
  • Picking colonies to isolate clones; Using analysis tools to analyze previous day’s sequencing (data provided by instuctors)
Saturday / Sunday
Monday Afternoon Theory:
  • Assessing genomic integrity beyond desired edit
Practical:
  • Target site PCR, Sanger sequencing for validation
 Morning Theory:
  • Lecture on History of direct conversion/transdifferentiation technology
  • Transdiferentiation technology to study aging and neurodegenerative conditions
Practical:
  • Thaw fibroblasts for neural conversion
Afternoon Free
Tuesday Morning Theory:
  • Fibroblast morphology and the quality of fibroblasts prior to transdifferentiation
Afternoon Theory:
  • Choosing different transcription factors for neuronal conversion
Wednesday Morning Practical:
  • Making stable fibroblast lines
Afternoon Practical:
  • Fluorescent Activated Cell sorting to select PSA-NCAM-expressing neurons
Thursday Morning Practical:
  • Immunostaining on neuronal cells (Fix and block)
Afternoon Practical:
  • Incubate with primary antibodies overnight
Friday Morning Practical:
  • Incubate with secondary antibodies and mount slides
Afternoon Practical:
  • Analyze slides on fluorescent microscope
Theory:
  • Discuss latest technological advances to improve neuronal conversion

Salk-CIRM SRL offerings