hpsc Growth Medium DXF Dr. Lorna Whyte

Transcription

1 hpsc Growth Medium DXF Dr. Lorna Whyte

2 Training from Heidelberg

3 Overview Background: Stem Cells Introduction: Human Pluripotent Stem Cells (hpsc) vs. Adult Stem Cells Promise of PSC Research hpsc Growth Medium DXF & related products Properties of the hpsc-gm DXF Short Protocol of hpsc culture / Application Note Competitors / Comparison of product properties

4 Human Cells in Culture Transformed: Tumor cells / established cell lines Non-transformed: stem cells primary cells embryonic stem cells adult stem cells fetal cells postnatal/ adult cells D I F F E R E N T I A T I O N

5 Stem Cells A Stem Cell requires two properties: Unlimited Self-Renewal The ability to go through an unlimited number of cell division cycles and remain undifferentiated Potency The capacity to differentiate into several specialized cell types

6 Potency Muscle Cells Blood Cells Neural Cells

7 Stem Cells cell division Symmetric Cell Division Asymmetric Cell Division Stem Cell Stem Cell Stem Cell Progenitor Cell

8 What are Progenitor Cells? A Progenitor Cell requires two properties: Multipotency The capacity to differentiate into some specialized cell types Limited Self-Renewal The ability to go through cell division cycles and remain undifferentiated Progenitor Cells are in between stem cells and fully differentiated cells.

9 Stages of Potency Totipotent Can construct a complete organism Pluripotent Can differentiate into nearly all cells Multipotent Can differentiate only to a closely related family of cells

10 Differentiation Stem Cell Self-Renewal Self-Renewal & Differentiation Self-Renewal Progenitor Cell Differentiation Differentiated Cells

11 Stages of Potency Totipotent Fertilized Egg (Zygote) and Morula Pluripotent Embryonic Stem Cell Multipotent Adult Stem Cell

12 Adult vs Embryonic Stem Cells The use of adult stem cells is not as controversial as embryonic stem cells (No destruction of an embryo) Correct differentiation of embryonic stem cells is very difficult and embryonic stem cells can cause a teratoma Adult stem cell treatments have been successfully used for many years (leukemia or in veterinary medicine) In some instances adult stem cells can be obtained from the intended recipient, the risk of rejection is then non-existent Many nations currently have bans on embryonic stem cell research or the production of new embryonic stem cells lines

13 Characteristics of Adult Stem Cells Can be isolated from many different parts of the body (e.g. bone marrow, cord blood, placenta) Non-transformed, not immortalized, have a normal karyotype (similar to primary cells) Can be expanded in cell culture in vitro Undifferentiated, have the capacity to differentiate into a restricted number of cell types ( lineage-restricted multipotency ) Adult stem cells don t have the ethical and scientific problems of embryonic stem cells

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15 Pluripotent Stem Cells vs. Adult Multipotent Stem Cells Stem Cell (in general) Unspecialized cell with the ability to self-renew and to differentiate into multiple terminally mature cell types. Pluripotent Stem Cell Cells with indefinite life span by sustained selfrenewal. Can differentiate into all cell types of the adult body (pluripotency). Do not exist in the adult body, but can be cultured indefinitely in vitro. Examples: embryonic stem cells, ips cells. Adult Multipotent Stem Cell Unspecialized cells found in differentiated tissues, e.g. in bone marrow, brain, skeletal muscle, liver, or skin. Able to self-renew in vivo without differentiation for the lifetime of an organism. Unable to selfrenew for long periods of time in vitro. Can differentiate into a restricted number of specialized cell types (multipotency). Examples: mesenchymal stem cells, hematopoietic stem cells.

16 Human Pluripotent Stem Cells * Pluripotent stem cells (PSC) embryonic stem cells - from inner cell mass of the blastocyst (ESC) induced pluripotent stem cells (ips) Pluripotency the ability of a cell to differentiate into all terminally mature cell types of the body * Cellular reprogramming by overexpression of Oct4 / Sox2 / Klf4 / c-myc transcription factors

17 Human Embryonic Stem Cells Human Embryonic Stem Cells are derived from the inner cell mass of a preimplantation embryo at 5 days post-fertilization (= blastocyst) Pluripotent Can give rise to all cell types of the body (from all 3 germ layers: endoderm, mesoderm, ectoderm), even after prolonged periods in culture however they cannot differentiate to extraembryonic tissue! Immortal (in theory) Unique ability to self-renew indefinitely while pluripotency and normal karyotype is retained represent a potential unlimited source of starting material for a wide array of applications Drawbacks Ethical concerns and law-restrictions

18 Induced Pluripotent Stem Cells (ipsc) Adult differentiated cells (e.g. fibroblasts) that have been genetically reprogrammed to an embryonic stem cell-like state Mouse ipscs, first reported in 2006 Human ipscs, first reported in late 2007 Demonstrate important characteristics of pluripotent stem cells expression of stem cell markers capable of generating cells characteristic of all 3 germ layers Meet the defining criteria for pluripotent stem cells but there are also profound genetic and epigenetic differences! not known if ipscs and embryonic stem cells differ in clinically significant ways Advantages of Adult Stem Cells over Embryonic Stem Cells Ethical concerns and law-restrictions valid for hesc do not apply for human ips cells.

19 Promise of human PSC Research Developmental biology Identify the factors that affect embryonic function and development in vivo and establish treatments for cell-based diseases Drug screening and toxicity testing Generate unlimited numbers of pure and standardized populations of a particular cell type (e.g. mature cardiac myocytes) and use them to identify potential drugs or evaluate toxicity of drug candidates prior to clinical trials Regenerative medicine and therapeutic cell transplants Treat cell-based diseases by transplanting new replacement cells to the source of disease or injury Treat a wide range of degenerative diseases (Parkinson s, diabetes, spinal cord injury, heart failure) with PSC-derived replacement cells or organs / organoids

20 Potential Uses of Stem Cells

21 PromoCell hpsc Medium and Associated Products Product hpsc Growth Medium DXF (500 ml Basal Medium + 2 ml SupplementMix) hpsc Dissociation Buffer DXF hpsc ECM DXF, 20x conc. Optional: ROCKi Y (PK-CA ) or Thiazovivin (PK-CA ) Description Expansion of human pluripotent stem cells in a chemicallydefined, xeno-free environment Supports cell clump passage as well as single cell subculture For coating of culture vessels Needed for thawing cryo-hpsc and for subculture of single cells hpsc-dissociation Buffer DXF + hpsc ECM DXF complete DXF culture system for hpsc hpsc ECM DXF (extracellular matrix) is recommended, but the PromCell culture system is also compatible with other established ECMs (some customers need that)

22 Typical hpsc Culture using hpsc-gm DXF Low-density ips, 1 day after passage (1:3): still slightly irregularly-shaped and loosely organized colonies. ips colonies 4-5 days after passage: tightly packed cells and smooth colony edges.

23 Short Protocol: hpsc Culture i. Coat culture vessel with appropriate ECM (e.g. hpsc ECM DXF) ii. iii. iv. Prepare the complete hpsc Medium DXF Plate the cells - existing proliferating cultures: perform clump passage - single cell passage or cryopreserved cells: add Y or Thiazovivin to the medium Perform medium change after 24 hours (w/o Y or Thiazovivin) v. Cell expansion: perform daily media changes until the single colonies touch each other (~ 4-7 days) vi. vii. Subculture with hpsc Dissociation Buffer DXF - either by clump passage - or by single cell passage Replate or use for your experiments Detailed AppNote available!

24 Properties of PromoCell hpsc-gm DXF Chemically defined and xeno-free (DXF) Formulation with very low protein content Complete DXF culture system (feeder-free) Free of ANY substances purified from human or animal origin (USP!) only anorganic/synthetic, recombinant and plant sources tightly controlled and highly standardized culture process Physiologically low in growth factors (USP!) enhanced cloning efficiency and pluripotency Extensively tested with hips and hesc tested with human ips cells tested with the most widely used hesc lines

25 Application Tests with the PromoCell hpsc Culture System DXF hesc: Typical colony morphology (right) and immunofluorescence pluripotency marker panel (below) after 5 passages in the PromoCell hpsc DXF Culture System: positive for Oct- 4/Sox-2/SSEA-4/Tra-1-60/Tra-1-81 and negative for SSEA-1. hips: Robust expression of the pluripotency markers Oct-3/4 and SSEA-4 by >95% of the cells after 17 passages in the PromoCell hpsc DXF Culture System.

26 Competitors: Stemgent (USA) / Miltenyi hesc NutriStem XF / FF Culture Medium Xeno-free, no animal components, feeder-free conditions Low growth factor content (bfgf and TGF-β1) Maintains pluripotency, normal morphology, karyotype, and differentiation potential of human ES and ips cells over long term culture Coating required: Mouse ECM ( Matrigel, Geltrex ) ECM not defined nor animal-free!

27 Competitors: STEMCELL Technologies (I) mtesr 1 / TeSR 2 mtesr 1: the most widely-published feeder-free culture medium for pluripotent stem cells TeSR 2: new and improved version of mtesr 1, with added value of being free of animal proteins Both systems are defined, feeder-independent media for maintenance and culture of undifferentiated hpscs, but nonphysiologically high in growth factors Coating required: Mouse ECM ( Matrigel, Geltrex ) ECM not defined nor animal-free! Very expensive

28 Competitors: STEMCELL Technologies (II) TeSR -E8 : Highly defined, low protein, feeder-free culture medium For maintenance of human ES cells and human ips cells Based on the E8 formulation published by Chen et. al. Simplified, low protein formulation, contains supraphysiologically high concentrations of rh-bfgf and rh-tgf-β1 Surface coating required (Matrigel hesc-qualified Matrix or defined, xeno-free ECM)

29 Competitors: Life Technologies/Gibco Essential 8 Medium Xeno-free, feeder cell-free medium originally developed by Chen et al. (E8-formulation); non-physiologically high concentrations of growth factors For the growth and expansion of human pluripotent stem cells Supports ipsc growth for > 50 passages without any signs of karyotypic abnormalities and maintains the ability of ipscs to differentiate into all three germ line lineages ECM-coating: human Vitronectin (= defined and xeno-free)

30 Overview: Competitors vs. PromoCell PromoCell Stemcell Technologies/ Gibco Stemcell Technologies Stemcell Technologies Biol. Industries/ Stemgent - Miltenyi hpsc-gm DXF E8 - Medium TeSR-1 TeSR-2 hesc Nutristem Chemically defined Xeno-free Free of human & animal-derived substances Physiologically low in growth factors * ECM defined and xeno-free * High growth factor concentrations lead to adverse effects in performance!

31 The End

32 Thank You!