Culturing Embryonic and Adult-Derived Stem Cells: Introduction and Key Applications. Amy Laws, Ph.D. November 2008

Transcription

1 Culturing Embryonic and Adult-Derived Stem Cells: Introduction and Key Applications Amy Laws, Ph.D. November 2008

2 Outline Introduction to stem cells Embryonic stem cells Alternatives to embryonic stem cells Introduction to human embryonic stem cell culture Adult stem cells Introduction to adult stem cell culture and differentiation

3 What Does It Mean To Be a Stem Cell? Stem cells are the foundation for every organ, tissue, and cell in the human body.

4 Different Types of Stem Cells Sources of cells Types of stem cells Embryonic self-renew differentiate into all tissue types Adult found in tissue self- renew differentiate into cells of the same lineage Progenitor derived from stem cells can not self-renew only differentiate into cells of the same lineage

5 Embryonic and Adult Stem Cells Embryonic Totipotent Can form any tissue, including placenta Pluripotent Can form any tissue in the embryo but not the placenta Multipotent Can form multiple cell types within a particular tissue, organ or physiological system Adult Schematic adapted from Wobus, A. M. and Boheler, K. R., Physiol. Rev. 85: (2005).

6 Potential Uses for Stem Cells Schematic adapted from

7 Current and Future Clinical Stem Cell Applications Blood disease Hematopoietic stem cells have been used for bone marrow transplantation for over 20 years. Spinal cord injury Geron is awaiting FDA approval to begin clinical trial with hesc-derived oligodendrocyte progenitor cells. Type II diabetes treatment Restore glucose-responsive insulin-secreting cells either by transplantation of stem cell-derived cells or reprogramming of existing cells.

8 Characteristics of Embryonic Stem (ES) Cells Undifferentiated/non-committed Self renewal Pluripotency Schematic adapted from Fischbach and Fischbach, J. Clin. Invest. 114: (2004).

9 How Were ES cells First Isolated? ES cells were first derived in 1981 from a MOUSE embryo Nature 292: (1981); Proc Natl Acad Sci USA 78: (1981) Isolation of human ES cells by James Thomson, et al. in 1998 Science 282: (1998) Protocols for hes cell culture were optimized from mouse ES cells and other embryonic stem cells culturing practices. hes cells were isolated by transferring the inner cell mass of a 3-5 day old embryo onto a mouse fibroblast feeder layer.

10 Potential Alternatives to ES Cells Somatic cell nuclear transfer (SCNT) The nucleus from an adult cell is transferred to an enucleated egg (the nucleus was removed) Advantages over ES cells No embryo derived cells Well characterized system in mice No Federal funding restrictions Potential for generating stem cells from any individual Challenges Limited success in primates Human egg donation Labor intensive Induced pluripotent stem cells (ips) Human cells are infected with genes that make them behave like hes cells Advantages over ES cells No embryo derived cells Adult cells only No Federal funding restrictions Potential for generating stem cells from any individual Challenges VERY new technology (2007) The infection process makes the genes integrate randomly into the DNA. (Potential for cancer in clinical applications.)

11 ips Cells for Studying Human Disease Potential for studying human disease Basic biology and drug screening ips cells generated from patients with a variety of genetic diseases (Park, et al. Cell 143:1 [2008]; Dimos, et al. Science 231:1218 [2008]) Amyotrophic lateral sclerosis (ALS) Parkinson disease (PD) Huntington disease (HD) Juvenile-onset, type 1 diabetes mellitus (JDM)

12 Key Challenges with hes Cell Culture Lack of defined culturing environment with a standard protocol Spontaneous differentiation Scaling up cultures Efficient transfection of hes cells Simulating physiological conditions in vitro Time required for full characterization of new culturing condition: in vitro and in vivo Difficulty in comparing data from different laboratories

13 A Decade of Developments in hes Cell Culture Environments Started with mes cell culture conditions MEF to human feeders, hes cell-derived feeders BD Matrigel Matrix/ECMs with MEF-CM ECMs + hes cell media (with soluble growth factors in media to control differentiation) BIO (GSK3 inhibitor wnt pathway) High bfgf Noggin +/- bfgf Activin A TGFβ ECMs + Defined media (with some animal components) ECMs + Animal-component free defined media Ultimate Goal = Completely Animal-Component Free Defined culture environment

14 Historical hes Cell Culturing Conditions hes cells are typically cultured on mouse embryonic fibroblast (MEF) feeder layers. or irradiate

15 Limitations of Mouse Embryonic Fibroblast (MEF) Feeder Layer Systems MEF Issues Labor intensive to maintain two cell types Variation of different MEF lots Contamination Potential contamination of animal pathogens from mouse feeders is a major concern when trying to move into therapeutic applications Downstream Manipulation Colonies that form on feeder layers are compact and difficult to genetically manipulate and transfect Transfection efficiency of hescs is low. Quenching can occur from the feeder layers Difficult to isolate DNA / RNA due to potential cross-contamination from feeder layers

16 Limitations of Mouse Embryonic Fibroblast (MEF) Feeder Layer Systems (Cont d) Standardization There is no widely accepted standard protocol which could result in major issues when moving into the therapeutic arena Difficulty in comparing data from different laboratories exists due to the absence of standard hesc culture practices Cells undergo spontaneous differentiation on a MEF feeder layer which makes comparisons from culture to culture difficult

17 Feeder-free hes Cell Culture First documented in 2001 (Xu, et al. Nature Biotech. 24:185) BD Matrigel Matrix-coated surface used with mouse embryonic fibroblast feeder layer conditioned media (MEF-CM) Multiple media conditions and defined media have been used successfully in combination with BD Matrigel Matrix-coated surface for culturing hes cells

18 BD Matrigel Matrix: a reconstituted basement membrane basal lamina = basement membrane BD Matrigel Matrix = reconstituted basement membrane Figure: Molecular Biology of the Cell (3rd Edition).

19 Interaction of Cells with Basal Lamina ECM The ECM interacts with cells via cell surface receptors such as integrins Reservoir for growth factors Substrate for cell attachment and spreading, contact guidance for cell migration, and a scaffold for building tissues Influences morphology of cells May be associated with particular patterns of cell differentiation and proliferation Figure: Nature Reviews Cancer 3:422 (2003).

20 BD Matrigel Matrix: a reconstituted basement membrane Purified preparation from EHS mouse tumors Composition: Laminin ~ 60% Collagen IV ~ 30% Entactin ~ 8% Heparan sulfate proteoglycan (perlecan) Growth factors (e.g., PDGF, EGF, TGF-β) Matrix metalloproteinases Not a defined substrate

21 A Complete Culturing Environment for Human ESCs Media + Surfaces = Complete Cell Environments BD Biosciences, StemCell Technologies, and the WiCell Research Institute have established a strategic collaboration to develop optimized, feeder-independent cell culture environments for hes cell research. mtesr 1 Maintenance Medium from StemCell Technologies BD Matrigel hesc-qualified Matrix from BD Biosciences

22 BD Matrigel hesc-qualified Matrix Optimized surface for hes cell culture Qualified as mtesr 1-compatible 5 ml vial can aliquot and store Coats six well BD Falcon Multiwell Plates Available at bdbiosciences.com/stemcellsource 1. Xu, C., et al., Feeder-free growth of undifferentiated human embryonic stem cells, Nature Biotechnology 19:971-4 (2001). 2. Xu, C., et al., Immortalized fibroblast-like cells derived from human embryonic stem cells support undifferentiated cell growth Stem Cells 22: (2004). 3. Ludwig, T.E., et al., Derivation of human embryonic stem cells in defined conditions, Nature Biotechnology 24:185-7 (2006).

23 Undifferentiated hes Cell Colony MEF feeder layer BD Matrigel hesc-qualified Matrix BD Matrigel hesc-qualified Matrix hes media MEF-conditioned media mtesr 1 Compact and dense H9 colonies on MEF feeders Spread-out and monolayer-like colonies on BD Matrigel Plates

24 Markers of Undifferentiated hes Cells on MEF-CM & mtesr 1 MEF-CM mtesr 1 OCT4 + Hoechst33342 H9 on BD Matrigel hesc-qualified Matrix

25 Undifferentiated Marker Expression FACS Analysis Isotype control Oct-3/4 Isotype control SSEA-4 98% OCT4 positive cells 94% SSEA4 positive cells H9 cells grown on BD Matrigel hesc-qualified Matrix in mtesr 1 for 5 passages

26 Comparison of BD Matrigel Matrix vs. other ECM Proteins BD Matrigel Matrix is equivalent or better than most single extracellular matrices (ECMs) tested: Laminin equivalent to BD Matrigel Matrix (Xu, et al. 2001) Laminin, collagen, fibronectin, and vitronectin were combined for optimal ECM complex for culturing hes cells (Ludwig et al. 2006) Fibronectin and collagen combination is required to match the performance of BD Matrigel Matrix (Lu, et al. 2006) Pure ECM often much more expensive to use ECM combination requires more steps and time to coat

27 Alternative Surface for ES Cell Culture BD Laminin/Entactin Complex High Concentration Major component of basement membrane in Engelbreth-Holm-Swarm (EHS) mouse tumors

28 Comparison of Different Surfaces and Media by Immunofluorescence MEF-CM mtesr TM 1 BD Laminin/Entactin Complex High Concentration BD Matrigel hesc-qualified Matrix OCT-4 expression in H9 cells

29 Embryoid Body (EB) Formation from hes Cells BD Laminin/Entactin Complex High Concentration BD Matrigel hesc-qualified Matrix Phase contrast image of Embryoid bodies formed by H9 cells grown on different surfaces.

30 Neurons and Cardiomyocytes from H9-derived Embryoid Bodies Cardiomyocytes GATA-4 Neurons Nestin EBs derived from H9 cells cultured on BD Laminin/Entactin Complex High Concentration for 32 passages in mtesr 1 media

31 Summary of hes Cell Culture Conditions Traditional method Mouse Embryonic Fibroblast (MEF) feeder layer Use MEF feeder layer as a substrate that provides essential growth and attachment factors Alternative feeder layer method human foreskin fibroblast feeder layer Undefined media and surface components, but no animal-derived factors Feeder-free hesc culture BD Matrigel or extracellular matrix (ECM) proteins are used as a substrate May use conditioned media from MEFs or a defined media Complete hesc environment BD Matrigel hesc-qualified Matrix + mtesr 1 Maintenance Medium for Human Embryonic Stem Cells Pre-qualified system saves significant time and resources No need to test lots of BD Matrigel Matrix to determine if they will sustain undifferentiated growth of hescs The media is completely defined

32 Embryonic vs. Adult Stem Cells Embryonic Stem Cells Pluripotent Relatively easy to grow in culture Adult Stem Cells Multipotent Difficult to isolate, purify and maintain in the undifferentiated state

33 Characteristics of Adult Stem Cells Adult stem cells are found in many tissues They are undifferentiated cells found among differentiated cells. Their primary role in the body is to maintain and repair the tissue in which they are found. Adult stem cells are multipotent, not pluripotent Pluripotent: can differentiate into any cell type in the embryo Multipotent: can differentiate into a subset of cell types, but NOT a complete organism Adult stem cells may exhibit plasticity

34 Adult Stem Cell Plasticity Plasticity is the ability of stem cells from one adult tissue to generate the differentiated types of another tissue.

35 Adult stem cells Hematopoietic Mesenchymal Neuronal

36 Hematopoietic Stem Cells (HSCs) Source: bone marrow Differentiation pathways Ex. T cell, B cell, Erythrocyte Culture conditions Maintenance of undifferentiated HSCs Ex. stem cell factor (SCF), IL-3, IL-6 Differentiation Ex. EPO, G-CSF

37 Mesenchymal Stem Cells (MSCs) Source: umbilical vein, bone marrow, adipose tissue, human embryonic stem cells Differentiation Pathways Ex. osteogenic, chondrogenic, adipogenic Culture Conditions Maintenance of undifferentiated MSCs Culture surface: Tissue culture (TC)-treated cellware Better yield on BD Falcon TC Flasks (Sotiropoulou, et al. Stem Cells 24:462 [2006]) Differentiation Ex. FGF, EGF, ITS+ Premix, TGF-β, Hydrocortisone

38 Neural Stem Cells (NSCs) Source: brain cortices, differentiated from embryonic stem cells Differentiation pathways Ex. neuron, astroglial Culture conditions Maintenance for undifferentiated cells Neurospheres Media components: ex. EGF, FGF Differentiation Surface: poly-l-ornithine/laminin, BD PuraMatrix Peptide Hydrogel, BD Matrigel Matrix Media components: ex. FGF, BDNF

39 Analysis of hesc-derived self-renewing neural stem cells (NSC) by IF and FACS hesc Embryoid bodies Neural rosettes Neural stem cells Neurons Sox2 Nestin Ki67 Hoechst 7 days days days days C D Sox2 PE Sox2 PE E OCT3/4 Alexa Fluor 488 F Nestin Alexa Fluor 647 Ki67 Alexa Fluor % 39% Sox2: hesc, NSC Nestin: NSC Oct3/4: hesc Ki67: proliferation Hoechst: Cell nuclei Sox2 Alexa Fluor 647

40 Summary Embryonic stem cells Pluripotent Alternatives: SCNT and ips cells Multiple culture methods Feeder layer BD Matrigel Matrix Defined ECM Adult stem cells Multipotent Culture environment (surface and media) specific to cell type and differentiation pathway

41 References hes and ips Cells hes and ips cells Xu, et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nature 19:971 (2001). Ludwig, et al. Feeder-independent culture of human embryonic stem cells. Nat. Methods 3(8):637 (2006). Ludwig, et al. Derivation of human embryonic stem cells in defined conditions. Nat. Biotechnology 24(2):185 (2006). Takahashi, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:1 (2007). Yu, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318:1917 (2007). Dimos, et al. Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science 321:1218 (2008). Park, et al. Disease-specific induced pluripotent stem cells. Cell 134:1 (2008).

42 References Adult Stem Cells HSCs Petzer, et al. Self renewal of primitive human hematopoietic cells (long-term-culture-initiating cells) in vitro and their expansion in defined medium. PNAS 93:1470 (1996). Bhatia, et al. Quantitative analysis reveals expansion of human hematopoietic repopulating cells after short-term ex vivo culture. J. Exp. Med. 186:619 (1997). Kang, et al. A novel function of interleukin-10 promoting self-renewal of hematopoietic stem cells. Stem Cells 25:1814 (2007). Wilson & Trumpp Bone-marrow haematopoietic-stem-cell niches. Nat. Rev. Immunol. 6:93 Staal, et al (2008) WNT signalling in the immune system: WNT is spreading its wings. Nat. Rev. Immunol. 8:581 (2006). O Connell, et al. Sustained expression of microrna-155 in hematopoietic stem cells causes a myeloproliferative disorder. J. Exp. Med. 205:585 (2008). MSCs Pittenger, et al. Multilineage potential of adult human mesenchymal stem cells. Science 284:143 (1999). Lee, et al. Isolation of multipotent mesenchymal stem cells from umbilical cord blood. Blood 103:1669 (2004). Sotiropoulou, et al. Characterization of the optimal culture conditions for clinical scale production of human mesenchymal stem cells. Stem Cells 24:462 (2006). Uccelli et al. Mesenchymal stem cells in health and disease. Nat. Rev. Immunol. 8:726 (2008). NSCs Flanagan, et al. Regulation of human neural precursor cells by laminin and integrins. J. Neurosci. Res. 83:845 (2006). Malaterre, et al. c-myb is required for neural progenitor cell proliferation and maintenance of neural stem cell niche in adult brain. Stem Cells 26:173 (2008). Thornhoff, et al. Compatibility of human fetal neural stem cells with hydrogel biomaterials in vitro. Brain Res. 1187:42 (2008).

43 Acknowledgements Susan Qian Suparna Sanyal Deepa Saxena Jeff Partridge Jennifer Brown Christian Carson

44 Contact Information Amy Laws, Ph.D. Technical Support Representative tel: PuraMatrix is a registered trademark of 3DM Inc. StemCell Technologies and all other StemCell trademarks are the property of StemCell Technologies Inc. WiCell logo, mtesr,tesr and all other WiCell trademarks are the property of WiCell Research Institute. BD, BD logo, and BD Matrigel are trademarks of Becton, Dickinson and Company. 2008