Tag Archives: iPSC

Creative Development, Project

Revised Ethics – Blood cells for iPSCs

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Due to COVID supply issues, we are still having issues sourcing key reagents etc. for the project. As an alternative iPSC protocol, we are now planning on using blood cells. The main reason is that it is a regular and active protocol in the broader lab area with clear in-house expertise. This also works better conceptually for me than harvesting cells from a skin biopsy – after all blood is strongly associated with notions of kinship . It is also nice to move into the footsteps of my dear colleague Dr Trish Adams who used iPSC technology to turn blood cells into heart cells for the project Machina Carnis.

This does entail a further ethics amendment, but since we have prior approval for skin biopsy harvest, I do not foresee any major issues. I hope to submit this before holidays – ready for the new year!

Laboratory, Project

Final Sign Off

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We are minor amendments away from final IBC approval to move ahead with iPSC and Cell Immortalisation processes.

I previously compiled a list of possible options based on available kits and associated literature. Brad and Jo-Maree recommended companies with Australian distributors due to delays in International shipping due to COVID. With this in mind, we identified the ThermoFisher
Epi5 Episomal iPSC Reprogramming Kit for reprogramming the fibroid (Tumour Baby) cells into a stem cell like state. The online product listing also has a comprehensive manual which provides clear instruction regarding the required materials and reagents and protocol. Epi5 Protocol

Overview of key steps in the reprogramming process from Epi5 manual.

We can now move forward with ordering the kit and other required/associated elements.

For cell immortalisation Brad also recommended we use a company with an Australian outlet.  Fischer Scientific may be the best option as they have a range of Alstem Immortalization  Products. We have identified the SV40 T Antigen and hTERT Cell Immortalization Kits as the most applicable for our cells.

Both kits have good product documentation and manuals available via the Alstem Bio website.

My preference is to ue the SV40 T Antigen. The protocol looks deceptively simple:

  1. Plate the target cells in one well of 6-well plate at density of 1-2 x 105 cells/well.
  2. The next day, take one vial of the concentrated recombinant lentivirus from -80 °C freezer and thaw it on ice.
  3. Infect the target cells in a 6-well plate with 4-20 μl/well viral supernatant in the presence of 4 μl TransPlus reagent (ALSTEM, cat#V050). Note: TransPlus reagent is a polycation that neutralizes charge interactions to increase binding between the pseudoviral capsid and the cellular membrane.
  4.  The next day, aspirate medium containing viral supernatant and add the appropriate complete growth medium to the cells and incubate at 37 °C.
  5. After 72 hours incubation, subculture the cells into 2 x 100 mm dishes and add the appropriate amount of puromycin for stable cell-line generation.
  6. 10-15 days after selection, pick clones for expansion and screen for positive ones. Note: Since the virus-titer will decrease significantly, we recommend that adding 25% v/v virus protection medium (ALSTEM, cat# VF050) into the thawed supernatant before frozen again for future use.

See: https://www.alstembio.com/web/protocol/SV40_T_Antigen_Cell_Immortalization_Kit_Protocol.pdf 

I hope it works out as simply as this sounds…

Creative Development, Laboratory, Project

LAB SCHEDULE

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UPDATED LAB SCHEDULE

Now that the project has the formal go-ahead, I am moving into lab mode and have determined some of the key milestones for the next months.

1: Training & Prep: 1 – 2 weeks

Training with HBVP cells include:

  • Thawing and culturing cells, making media, working in a biosafety cabinet and maintaining sterility, light microscopy
  • Learn to use the autoclave and prepare petri dishes and glass vessels for culture
  • Coat petri dishes and glass vessels with poly-l-lysine for cell adhesion, test with HBVP cells
  • Order media, reagents and kits
  • Submit IBC approval forms

2: Cell culture of fibroid cells – 4 – 8 weeks

  • Thawing and culture – grow up and freeze stocks of cells, light microscopy
  • Ask Dietmar to send 3D scaffolds
  • Grow and fix cells in petri dishes and glass vessels
  • Fluorescent microscopy of cells
  • Scanning Electron Microscopy (SEM) of cultured cells
  • Transmission Electron Microscopy (TEM) of cultured cells
  • Timelapse microscopy
  • 3D cell seeding HBVPs and Fibroid cells – see differences in cell response.
  • Wait for IBC approval

PROJECT: 3 months

3: Cell Immortalisation +

  • Immortalisation of primary fibroid cells via established commercial kit (Applied Biological Materials (ABM) or Alstem cell immortalisation kits)
  • Cell genetic profiling
  • Cell culture of immortalised fibroid cells (optimisation of culture methods for 2D and 3D environments, cellular response and proliferation testing)
  • Grow and fix cells in petri dishes and glass vessels
  • Timelapse microscopy

4: iPSC production

  • Reprogramming of primary cells to generate induced pluripotent stem cells (iPSCs) via established commercial kit (e.g. Epi5™ Episomal iPSC Reprogramming Kit available via Thermo Fisher)
  • Development of Gastruloids, Organoids or Neurospheres (self-organised 3D cell masses)
  • Cell culture of reprogrammed or immortalised fibroid cells (optimisation of culture methods for 2D and 3D environments, cellular response and proliferation testing)
  • Timelapse microscopy
  • If iPSC successful – create neurons and heart cells
Project

Ethics GRANTED! …but one more approval to go…

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We have approval to move forward with the key aims of the project. This is great news as it means I can start working with own cells. I am still a bit precious as there are limited vials, so I will do a couple of weeks of training on HBVPs before I move on to my own cells.

While we can get started on the fibroid cell culture, Brad realised that cell immortalisation will require further Institutional Biosafety Committee (IBC) approval. This is because the process will require the uses of lentiviral vectors. As such, it is considered Notifiable Low Risk Dealing (NLRD) and the committee will need to ensure that we have the appropriate facilities and training in place to move forward.  iPSC cell reprogramming is exempt, but we still need to let the IBC know what we are doing.

The application is due tomorrow, so we had a meeting this morning to go over the protocols and identify the particular kits we are going to use. There are a range of biomedical research supply companies, but the important thing is to make sure that we use an organisation that has an Australian supplier.  hTERT and SV40 T Antigen kits are the best options for our project as they are suitable for a range of  cell types including fibroblasts.  Fischer Scientific have Alstem Immortalisation Kits available, but ABM may also be a good option. They also have a good overview of Cell Immortalisation Protocols for anyone interested in the process.

For iPSC reprogramming, we are going to use the Epi5™ Episomal iPSC Reprogramming Kit by Thermo Fischer. Another group has used this product previously – so we can get tips on how to get the best results.  Lovely Jo-Maree is looking into the best purchasing options. With lead time for purchase and delivery, the products will likely arrive around the same time as final approval.

Project

iPSC Protocols

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We are still waiting for formal ethical clearance to undertake work using my fibroid (fibroblast) cells. In the interim, Brad sent through a Nature protocol detailing options for producing iPSCs from human keratinocytes derived from plucked hair follicles or skin biopsies. I have isolated keratinocytes from hair follicles before at QUT when I was part of the Tissue Repair and Regeneration Group for  the HSE (Human Skin Equivalent/Experience Project).  It was quite a mission as keratinocytes require a feeder layer of fibroblast cells. These needed to be irradiated to ensure that they did not outgrow the keratinocytes 🙁

Since we are only doing a skin biopsy if the fibroid cells are not viable, I am parking this option and scouting for protocols that are fibroblast specific. With that in mind, the company Sigma-Aldrich has specified an iPSC reprogramming protocol – Reprogramming of Human Fibroblasts using Non-Integrating Self-Replicating RNA Vectors – designed for fibroblasts and with a 30-day creation estimate. Of course, you always have to double or triple timeframes when you are undertaking a protocol for the first time. Of course, I will need to discuss this option with Brad and seek a pricing and availability estimate.

Fisher Scientific also has a reprogramming kit – the CytoTune-iPS Sendai Reprogramming kit. However, the latest version and full kit carries a hefty price tag at over $18, 500. They also offer a potentially more affordable option via ‘Episomal Vectors’ or Epi5™ Episomal iPSC Reprogramming Kit. These could be an option but Brad is of course the best advisor.

There are also a number of journal articles detailing iPSC reprogramming including: Reprogramming fibroblasts into induced pluripotent stem cells with Bmi1 [Nature], Human Pluripotent Stem Cells (iPSC) Generation, Culture, and Differentiation to Lung Progenitor Cells [Methods Mol Biol.], Guidelines and Techniques for the Generation of Induced Pluripotent Stem Cells [Science Direct], Generation of human iPSCs from cells of fibroblastic and epithelial origin by means of the oriP/EBNA-1 episomal reprogramming system [Stem Cell Research & Therapy].

Looks like I’ve got some reading to do…

Laboratory, Project

Project Meeting

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We had a detailed project planning and ethics clearance review meeting on Monday. It was heartening to hear that the ethics clearance documentation is almost ready for formal submission.

These are some of the processes we are expecting to undertake:

  • Cell culture of fibroid cells (optimisation of culture methods for 2D and 3D environments, cellular response and proliferation testing)
  • Co-culture of fibroid cells with other cell lines
  • Genetic profiling of primary fibroid cells
  • Immortalisation of primary fibroid cells via established commercial kit (e.g. Applied Biological Materials (ABM) cell immortalisation kits)
  • Reprogramming of primary cells to generate induced pluripotent stem cells (iPSCs) via established commercial kit (e.g. Epi5™ Episomal iPSC Reprogramming Kit available via Thermo Fisher)
  • Cell culture of reprogrammed or immortalised fibroid cells (optimisation of culture methods for 2D and 3D environments, cellular response and proliferation testing)
  • Genetic profiling of reprogrammed or immortalised fibroid cells and cells lines (if successful)
  • Fixing and staining of cells
  • Light and confocal microscopy of cultured cells
  • Timelapse video of cultured cells

Other potential processes include:

  • Green Fluorescent Protein (GFP) cell tagging [UTAS]
  • Scanning Electron Microscopy (SEM) of cultured cells [UTAS]
  • Transmission Electron Microscopy (TEM) of cultured cells [UTAS]
  • Histopathology of cultured 3D structures [UTAS or QUT]
  • Development of Gastruloids, Organoids or Neurospheres (self-organised 3D cell masses) [UTAS]

I was delighted to hear that my desires for creating aggregates of cells via the production of gastruloids or organoids was not outside the domain of possibility. Jo-Maree has produced neurospheres (balls of neural stem cells) previously, so there may be scope (and hopefully time) to experiment with cell clusters.

Gastuloids are of particular interest to me as they are cell clusters that display features of early embryo development. Staining and fluorescence imaging (apart from being visually stunning) enables the visualisation of tissue organisation as shown in this figure and caption from the Nature publication ‘Multi-axial self-organization properties of mouse embryonic stem cells into gastruloids’:

Tissue organisation in gastruloids

Tissue organization in gastruloids a, Gastruloids formed from Sox1GFP;BramCherry (SBR) line and stained for Sox2 expression (Sox1GFP and SOX2 signals are displayed in green and magenta, respectively). White arrowheads indicate tubular SOX2/Sox1-positive neural structures. Red arrowheads point to the presumptive digestive tube. b, WISH on 8-µm transverse cryosections of gastruloids at 144 h AA using Sox2 and Meox1 antisense probes, counter-stained with Nuclear Fast Red. Sox2-positive cells localized predominantly in a compact dorsal domain, whereas Meox1 signals were found in two bilateral domains. The domain of expression of each gene is outlined with white dashed lines. c, Haematoxylin and eosin staining of transverse paraffin sections of different gastruloids at 120 h AA, showing the diversity of cell types and several levels of tissue organization. d, Gastruloids formed from Sox1GFP;BramCherry ESCs were fixed and stained at 168 h AA for OLIG2 (top, white), PAX3 (middle, red) and PAX7 (bottom, red). Scale bars as indicated. c, d, Gastruloids formed from Sox1GFP; BramCherry ESCs collected at 168 h AA and stained for SOX17 (magenta, c) or CDX2 (magenta, d). Scale bars as indicated.

Gastruloids are created from embryonic stem cells, although iPSC cells (cells that have been reprogrammed into a stem cell like state) have also been used. As such, if I have success with reprogramming my fibroid cells to iPSC cells, I could use them to make, and learn more about, gastruloids.

During our discussion, we decided it would be a good idea to include an optional alternative to the fibroid cells – just in case there is an issue with contamination or the freezing/thawing process. While I have isolated skin cells (fibroblasts) from hair follicles and skin grafts previously as part of the HSE Project at QUT, we decided on skin scrapings.   This approach was selected as will enable the isolation of skin cells, is not too invasive and is well established within the School of Medicine.

Once I’ve included this information including the protocol, I should be ready to submit the final ethics clearance document.

Beccari, L., Moris, N., Girgin, M., Turner, D.A., Baillie-Johnson, P., Cossy, A.C., Lutolf, M.P., Duboule, D. and Arias, A.M., 2018. Multi-axial self-organization properties of mouse embryonic stem cells into gastruloids. Nature, 562(7726), pp.272-276.