Cell Production

 

Columbia University Stem Cell Core beating cardiomyocytes

 

The Cell Production section of the core facility offers maintenance and supply of human ESC/iPSC; derivation and characterization of hiPSC from fibroblasts or blood; differentiation of mESC into cortical neurons; and differentiation of human pluripotent stem cells into functional, mature, highly purified progenies (including neuronal, cardiac, hepatic and retinal pigment epithelium).

Pluripotent Stem Cell Line Expansion and Banking

We have expanded and stored a number of human ESC and iPSC lines. Please check with us if you want to know more about these lines. All our lines are carefully tested for Mycoplasma contamination and karyotyped on a regular basis. Investigators who wish to share their lines with the core are very welcome to contact us.

Please remember:

hiPSC Derivation From Fibroblasts

This service takes about 3-4 months to complete and includes:

  • Expansion, Mycoplasma testing, cryopreservation of fibroblast culture
  • Reprogramming using Sendai virus (CytoTune®-iPS 2.0 Sendai Reprogramming Kit, Life Technologies)
  • iPSC colony picking (12 colonies when possible)
  • Expansion of 3 iPSC colonies
  • Mycoplasma testing
  • Flow cytometry analysis for pluripotency markers TRA-1-60, SSEA4, Oct4 and Nanog

We accept either live or frozen donor cell stocks for iPSC derivation. Donor cells should be at low passage (less than 10) and proliferating. We can also provide fibroblast derivation from a biopsy.

Donor cells for iPSC derivation must have been tested negative for Mycoplasma. Only Mycoplasma-free cells will be used for reprogramming. Mycoplasma testing will be repeated in the core facility.

When possible, we usually pick 12 colonies, then freeze down 9 and keep 3 colonies for expansion. Users will receive 3 colonies of live cells per donor line, but it is also possible to ask us to freeze them down. We will also give you all the other colonies we picked.

Important: Some donor cells fail to get reprogrammed. If we don’t obtain colonies after the 1st attempt at reprogramming, we will try a 2nd time. If it still doesn’t work, we will stop trying. Price will be negotiated at this time.

If you wish to derive iPSCs from other cell types, please enquire about this service. We have successfully derived iPSC from myoblasts.

Quantity discount: Derivation service fees will be reduced through quantity discount.

  • 5-9 samples - 5% discount
  • 10-49 samples - 10% discount
  • 50 or more samples - 25% discount

hiPSC Derivation From Blood

This service takes about 3-4 months to complete and includes:

  • Expansion of erythroblasts
  • Mycoplasma testing
  • Reprogramming using Sendai virus (CytoTune®-iPS 2.0 Sendai Reprogramming Kit, Life Technologies)
  • iPSC colony picking (12 colonies when possible)
  • Expansion of 3 iPSC colonies
  • Mycoplasma testing
  • Flow cytometry analysis for pluripotency markers TRA-1-60, SSEA4, Oct4 and Nanog

Collect 4ml or 8ml blood into BD Vacutainer CPT Cell Preparation tubes with Sodium Citrate or into EDTA or heparinized tubes to Ficoll extract PBMC (peripheral blood mononuclear cells). Store the tube upright at room temperature (18-25C). Bring the tube to the core ASAP (within 2 hours of collection for best results).

Mycoplasma testing will be done in the core facility. When possible, we usually pick 12 colonies, then freeze down 9 and keep 3 colonies for expansion. Users will receive 3 colonies of live cells per donor line, but it is also possible to ask us to freeze them down. We will also give you all the other colonies we picked.

Characterization and Quality Control of Pluripotent Stem Cells

New iPSC lines can be transferred directly to the user or expanded and characterized in the core. We can also help you confirm the pluripotency status of your cells.

Characterization services include:

  • In vitro differentiation into the 3 germ layers: staining for α-Smooth Muscle Actin (SMA, mesoderm), AFP and Foxa2 (endoderm), βIII Tubulin (ectoderm)
  • Mycoplasma testing by PCR
  • G-band karyotyping or Microarray-based Comparative Genomic Hybridization (aCGH). For these, we use the services of an external company, Cell Line Genetics, Inc.

Differentiation of Human Pluripotent Stem Cells

We provide differentiation of human pluripotent stem cells into functional, mature, highly purified progenies (including neuronal, cardiac, hepatic and retinal pigment epithelium). We also provide differentiation of mESC into cortical neurons.

  • Motor Neuron Differentiation From Human Pluripotent Stem Cells: Using published protocol, we routinely generate highly pure populations of human spinal motor neurons expressing typical MN markers (HB9, Isl1/2, VachT) and exhibiting typical neuronal morphology with branching neuritis. We are also working on generating MN-reporter lines using CRISPR-Cas9. We already generated a number of iPSC lines from ALS patients, now available for distribution at RUCDR Infinite Biologics at Rutgers University or via our core.
  • Cortical Neuron Differentiation From Human Pluripotent Stem Cells: Using published protocols based on 3D cultures, we routinely generate highly pure populations of human PSC-derived cortical neuroepithelium containing a multilayered structure recapitulating human corticogenesis and exhibiting electrical activity. We also can generate functional cortical neurons from mESC.
  • Dopaminergic Neuron Differentiation From Human Pluripotent Stem Cells: Using published protocols, we routinely generate highly pure populations of midbrain DA neurons, expressing midbrain DA neuron markers as Lmx1, FoxA2, and TH.
  • Retinal Pigment Epithelium Cell (RPE) Differentiation From Human Pluripotent Stem Cells: Using published protocols combining the use of Noggin (a BMP4 inhibitor) and SB431542 (a TGFb inhibitor) to drive neural induction, and the subsequent addition of Activin A and Nicotinamide to improve RPE yield, we routinely generate pigmented monolayer of highly pure RPE cells.
  • Cardiac Differentiation From Human Pluripotent Stem Cells: We routinely generate highly pure populations of cardiomyocytes, both as EBs or adherent monolayer cultures, by using cytokines and small molecules that regulate key pathways of the cardiac development. By combining specific molecule selection, timing of administration and molecule dosage adjustment, the differentiation efficiency is optimized for every individual hPSC line. Terminal differentiation is identified by evaluation of the number, size and amplitude of contracting areas and by staining for marker such as cTNT, MLC2v, MLC2a.
  • Skeletal Muscle Differentiation From Human Pluripotent Stem Cells: Using published protocols and kits, we generate highly pure populations of skeletal muscle cells expressing markers as myogenin, desmin, and dystrophin. Their functionality is shown in assays like glucose uptake.
  • Hepatocyte Differentiation From Human Pluripotent Stem Cells: Using published protocols, we routinely generate highly pure populations of hepatocytes expressing various markers as albumin, SLC10a1, Cyp3a7 and Cyp3a4.