Stem Cell Workflow: Control cell destiny from start to finish

Simplify and improve your workflow with the primary cell type you’re working with. You’ll have consistently healthy viable cells at the end of each step, shorten your workflow timeline, and have more time to innovate instead. The AVATAR enables precision hypoxia and pressure control, enabling optimization of your total stem cell workflow from fibroblast to iPSC to motor & CNS-type neurons and even cardiomyocytes.

 

Stem Cell Applications Using AVATAR:

  • iPSC Generation – Fibroblast Reprogramming
  • Neural Induction – iPSC to Neural Stem/Progenitor Cell
  • Neuronal Differentiation – iPSC to Motor Neuron
  • Neuronal Differentiation – iPSC to CNS-type Neuron
  • Cardiomyocyte Differentiation – iPSC to Cardiomyocyte
  • Late-stage Cardiomyocyte Maturation

iPSC Generation

Fibroblast Reprogramming

 

Using the AVATAR system, researchers have identified that culturing under hypoxia and pressure during fibroblast reprogramming consistently increases iPSC colony generation (roughly 5X) relative to yields observed using a standard CO2 incubator. These findings highlight the importance of hypoxia and atmospheric pressure in the culture microenvironment during iPSC generation.

Primary human dermal fibroblasts were reprogrammed via transfection with a self-replicating RNA vector containing transcription factors SOX2, KLF4, OCT4, GLIS1, and c-MYC, and cultured in a range of oxygen concentrations and pressures using the AVATAR system.

Fibroblasts reprogrammed in hypoxic and pressurized culture conditions increased the number of iPSC colonies formed across all 3 time points relative to standard CO2 incubator (Normoxia) or hypoxia alone. N = 3 independent reprogrammings. Error bars are S.E.M.

Maintain Stemness

Expression of stemness markers Nanog, Oct4, and Sox2 increased 2-fold under hypoxic and pressurized conditions compared to standard or hypoxia alone.

AVATAR culture settings were next used to promote ‘stemness’ by inducing expression of Nanog, Oct4, Sox2. A combination of hypoxia and pressure (1% O2 and +2 PSI) maintained tightly-compacted colonies as compared to hypoxia alone and standard CO2 incubator. Expression of stemness markers Nanog, Oct4, and Sox2 increased 2-fold under hypoxic and pressurized conditions than standard or hypoxia alone (qPCR).

Differentiation

  Neural Induction: iPSC to Neural Stem/Progenitor Cells

The ability to derive patient-specific neuronal cell types has proven to be a critical tool for human developmental studies, drug discovery, and regenerative medicine. The ability to direct the differentiation of iPSCs into neuronal cell lineages has enabled investigators to develop models for a variety of neurodegenerative diseases. However, there remains an urgent need to be able to produce these phenotypically mature cell types more efficiently and consistently in vitro, as  current methods for deriving neuronal cell types are inherently time consuming with high donor-to-donor variability in efficiency.

  Motor Neuron Differentiation

The ability to derive patient-specific neuronal cell types has proven to be a critical tool for human developmental studies, drug discovery, and regenerative medicine. The ability to direct the differentiation of iPSCs into neuronal cell lineages has enabled investigators to develop models for a variety of neurodegenerative diseases. However, there remains an urgent need to be able to produce these phenotypically mature cell types more efficiently and consistently in vitro, as  current methods for deriving neuronal cell types are inherently time consuming with high donor-to-donor variability in efficiency.

  CNS-type Neuron Differentiation

Step 4 – Targeted Differentiation

AVATAR culture settings were tuned to promote differentiation of iPSCs into Neural Progenitor Cells (NPCs) by inducing expression of early neuronal markers PAX6 and NESTIN.

Validating through targeted NPC gene expression, mRNA-seq revealed up-regulation of genes involved in neural differentiation and neural progenitor cell maintenance.

mRNA-seq of neo-natal iPSCs (left) cultured in AVATAR reveals up-regulation of genes involved in neural differentiation and neural progenitor cell maintenance and down regulation in pluripotency genes. Following qPCR further compliments mRNA-seq data for the indicated genes in the right panel.

Step 5 – Functional cells

Early expression of Synapsin 1 (SYN1) was seen by day 14 of neuron differentiation in the AVATAR, validating targeted protein expression for motor neuron maturation compared to those grown in standard conditions.

Step 2 – Expand

Post-transfection with AVATAR also resulted in an increase in reprogramming efficiency. A 5X increase in iPSC colony formation was observed by day 19 using pressurized and hypoxic conditions.

  • Standard
  • AVATAR Setting #1

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