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Pubmed Article
Microencapsulation technology: a powerful tool for integrating expansion and cryopreservation of human embryonic stem cells.
PLoS ONE
PUBLISHED: 02-04-2011
The successful implementation of human embryonic stem cells (hESCs)-based technologies requires the production of relevant numbers of well-characterized cells and their efficient long-term storage. In this study, cells were microencapsulated in alginate to develop an integrated bioprocess for expansion and cryopreservation of pluripotent hESCs. Different three-dimensional (3D) culture strategies were evaluated and compared, specifically, microencapsulation of hESCs as: i) single cells, ii) aggregates and iii) immobilized on microcarriers. In order to establish a scalable bioprocess, hESC-microcapsules were cultured in stirred tank bioreactors.The combination of microencapsulation and microcarrier technology resulted in a highly efficient protocol for the production and storage of pluripotent hESCs. This strategy ensured high expansion ratios (an approximately twenty-fold increase in cell concentration) and high cell recovery yields (>70%) after cryopreservation. When compared with non-encapsulated cells, cell survival post-thawing demonstrated a three-fold improvement without compromising hESC characteristics.Microencapsulation also improved the culture of hESC aggregates by protecting cells from hydrodynamic shear stress, controlling aggregate size and maintaining cell pluripotency for two weeks.This work establishes that microencapsulation technology may prove a powerful tool for integrating the expansion and cryopreservation of pluripotent hESCs. The 3D culture strategy developed herein represents a significant breakthrough towards the implementation of hESCs in clinical and industrial applications.
Authors: Kuldip Sidhu, Jaemin Kim, Methichit Chayosumrit, Sophia Dean, Perminder Sachdev.
Published: 03-09-2012
ABSTRACT
Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, >100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.
22 Related JoVE Articles!
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Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP
Authors: Kun-Yong Kim, Eriona Hysolli, In-Hyun Park.
Institutions: Yale School of Medicine.
Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.
Stem Cell Biology, Issue 62, Human iPS cells, iPSCs, Reprogramming, Retroviral vectors and Pluripotency
3804
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Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells
Authors: Justyna Jozefczuk, Katharina Drews, James Adjaye.
Institutions: Max Planck Institute for Molecular Genetics.
In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial. The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8. To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells. Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgfβ1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16. For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs. Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.
Stem Cell Biology, Issue 64, Molecular Biology, Developmental Biology, mouse embryonic fibroblasts (MEFs), human embryonic stem cells (hESCs), human induced pluripotent stem cells (hiPSCs), Activin A -conditioned medium (CM), cell culture
3854
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Generation of Myospheres From hESCs by Epigenetic Reprogramming
Authors: Sonia Albini, Pier Lorenzo Puri.
Institutions: Sanford-Burnham Institute for Medical Research, IRCCS Fondazione Santa Lucia.
Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.
Bioengineering, Issue 88, Tissues, Cells, Embryonic Structures, Musculoskeletal System, Musculoskeletal Diseases, hESC, epinegetics, Skeletal Myogenesis, Myosphere, Chromatin, Lentivirus, Infection
51243
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A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue
Authors: Brandon C. Shelley, Geneviève Gowing, Clive N. Svendsen.
Institutions: Cedars-Sinai Medical Center.
A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as “chopping” that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.
Neuroscience, Issue 88, neural progenitor cell, neural precursor cell, neural stem cell, passaging, neurosphere, chopping, stem cell, neuroscience, suspension culture, good manufacturing practice, GMP
51219
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Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells
Authors: Deborah Merrick, Hung-Chih Chen, Dean Larner, Janet Smith.
Institutions: University of Birmingham.
Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors. Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.
Cellular Biology, Issue 43, Skeletal muscle stem cell, embryonic tissue culture, apoptosis, growth factor, proliferation, myoblast, myogenesis, satellite cell, skeletal muscle differentiation, muscular dystrophy
2051
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Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors
Authors: Jesse J. Hubbard, Spencer K. Sullivan, Jason A. Mills, Brian J. Hayes, Beverly J. Torok-Storb, Aravind Ramakrishnan.
Institutions: Fred Hutchinson Cancer Research Center, The Children's Hospital of Philadelphia, The Children's Hospital of Philadelphia.
This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.
Cellular Biology, Issue 92, Induced pluripotent stem cells, iPSC, iPSC generation, human, HDAC inhibitors, histone deacetylase inhibitors, reprogramming, episomes, integration-free
52009
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Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions
Authors: Jason P. Awe, Agustin Vega-Crespo, James A. Byrne.
Institutions: University of California, Los Angeles (UCLA), University of California, Los Angeles (UCLA).
Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic genes remaining in actively transcribed regions of the genome, limit their potential for use in human therapeutic applications1. Additionally, non-human antigens derived from stem cell reprogramming or differentiation into therapeutically relevant derivatives preclude these hiPSCs from being used in a human clinical context2. In this video, we present a procedure for reprogramming and analyzing factor-free hiPSCs free of exogenous transgenes. These hiPSCs then can be analyzed for gene expression abnormalities in the specific intron containing the lentivirus. This analysis may be conducted using sensitive quantitative polymerase chain reaction (PCR), which has an advantage over less sensitive techniques previously used to detect gene expression differences3. Full conversion into clinical-grade good manufacturing practice (GMP) conditions, allows human clinical relevance. Our protocol offers another methodology—provided that current safe-harbor criteria will expand and include factor-free characterized hiPSC-based derivatives for human therapeutic applications—for deriving GMP-grade hiPSCs, which should eliminate any immunogenicity risk due to non-human antigens. This protocol is broadly applicable to lentiviral reprogrammed cells of any type and provides a reproducible method for converting reprogrammed cells into GMP-grade conditions.
Stem Cell Biology, Issue 93, Human induced pluripotent stem cells, STEMCCA, factor-free, GMP, xeno-free, quantitative PCR
52158
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Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells
Authors: Martina Kučerová-Levisohn, Jordana Lovett, Armin Lahiji, Roxanne Holmes, Juan Carlos Zúñiga-Pflücker, Benjamin D. Ortiz.
Institutions: City University of New York, University of Toronto.
The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.
Immunology, Issue 92, mouse, embryonic stem cells, in vitro differentiation, OP9 cells, Delta-like 1 (Dll-1) ligand, Notch, hematopoiesis, lymphocytes, T cells
52119
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Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector
Authors: Andreia Gianotti Sommer, Sarah S. Rozelle, Spencer Sullivan, Jason A. Mills, Seon-Mi Park, Brenden W. Smith, Amulya M. Iyer, Deborah L. French, Darrell N. Kotton, Paul Gadue, George J. Murphy, Gustavo Mostoslavsky.
Institutions: Boston University School of Medicine, Children's Hospital of Philadelphia, Children's Hospital of Philadelphia.
Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5. We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies. Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.
Stem Cell Biology, Issue 68, Induced pluripotent stem cells (iPSCs), peripheral blood mononuclear cells (PBMCs), reprogramming, single excisable lentiviral vector, STEMCCA
4327
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Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)
Authors: Allison M. Bock, David Knorr, Dan S. Kaufman.
Institutions: University of Minnesota, Minneapolis, University of Minnesota, Minneapolis.
We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.
Stem Cell Biology, Issue 74, Bioengineering, Biomedical Engineering, Medicine, Physiology, Anatomy, Cellular Biology, Molecular Biology, Biochemistry, Hematology, Embryonic Stem Cells, ESCs, ES Cells, Hematopoietic Stem Cells, HSC, Pluripotent Stem Cells, Induced Pluripotent Stem Cells, iPSCs, Luciferases, Firefly, Immunotherapy, Immunotherapy, Adoptive, stem cells, differentiation, NK cells, in vivo imaging, fluorescent imaging, turboFP650, FACS, cell culture
50337
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Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations
Authors: Claire N. Medine, Baltasar Lucendo-Villarin, Wenli Zhou, Christopher C. West, David C. Hay.
Institutions: University of Edinburgh.
Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical studies are substantial, therefore it is essential that more predictive toxicology screens are developed and deployed early on in drug development (Greenhough et al 2010). Human hepatocytes represent the current gold standard model for evaluating drug toxicity, but are a limited resource that exhibit variable function. Therefore, the use of immortalised cell lines and animal tissue models are routinely employed due to their abundance. While both sources are informative, they are limited by poor function, species variability and/or instability in culture (Dalgetty et al 2009). Pluripotent stem cells (PSCs) are an attractive alternative source of human hepatocyte like cells (HLCs) (Medine et al 2010). PSCs are capable of self renewal and differentiation to all somatic cell types found in the adult and thereby represent a potentially inexhaustible source of differentiated cells. We have developed a procedure that is simple, highly efficient, amenable to automation and yields functional human HLCs (Hay et al 2008 ; Fletcher et al 2008 ; Hannoun et al 2010 ; Payne et al 2011 and Hay et al 2011). We believe our technology will lead to the scalable production of HLCs for drug discovery, disease modeling, the construction of extra-corporeal devices and possibly cell based transplantation therapies.
Developmental Biology, Issue 56, Stem Cells, hESC, Development, Endoderm, Liver, Hepatocyte, Endocrine Function, Exocrine Function
2969
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Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction
Authors: Xuejun H. Parsons, Yang D. Teng, James F. Parsons, Evan Y. Snyder, David B. Smotrich, Dennis A. Moore.
Institutions: San Diego Regenerative Medicine Institute, Xcelthera, Harvard Medical School, Division of SCI Research, VA Boston Healthcare System, Sanford-Burnham Medical Research Institute, La Jolla IVF.
There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.
Neuroscience, Issue 56, stem cell, human embryonic stem cell, human, neuronal progenitor, neuron, human pluripotent cell, neuronal differentiation, small molecule induction, cell culture, cell therapy
3273
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Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction
Authors: Xuejun H. Parsons, Yang D. Teng, James F. Parsons, Evan Y. Snyder, David B. Smotrich, Dennis A. Moore.
Institutions: San Diego Regenerative Medicine Institute, Xcelthera, Harvard Medical School, VA Boston Healthcare System, Sanford-Burnham Medical Research Institute, La Jolla IVF.
To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.
Developmental Biology, Issue 57, human embryonic stem cell, human, cardiac progenitor, cardiomyocytes, human pluripotent cell, cardiac differentiation, small molecule induction, cell culture, cell therapy
3274
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Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells
Authors: Pengbo Zhang, Ninuo Xia, Renee A. Reijo Pera.
Institutions: Stanford University School of Medicine, Stanford University School of Medicine.
Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson’s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.
Neuroscience, Issue 91, dopaminergic neuron, substantia nigra pars compacta, midbrain, Parkinson’s disease, directed differentiation, human pluripotent stem cells, floor plate
51737
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Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells
Authors: Nadja Zeltner, Fabien G. Lafaille, Faranak Fattahi, Lorenz Studer.
Institutions: Sloan-Kettering Institute for Cancer Research, The Rockefeller University.
Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.
Neuroscience, Issue 87, Embryonic Stem Cells (ESCs), Pluripotent Stem Cells, Induced Pluripotent Stem Cells (iPSCs), Neural Crest, Peripheral Nervous System (PNS), pluripotent stem cells, neural crest cells, in vitro differentiation, disease modeling, differentiation protocol, human embryonic stem cells, human pluripotent stem cells
51609
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Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis
Authors: Kevin G. Chen, Rebecca S. Hamilton, Pamela G. Robey, Barbara S. Mallon.
Institutions: National Institutes of Health, National Institutes of Health.
Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.
Stem Cell Biology, Issue 89, Pluripotent stem cells, human embryonic stem cells, induced pluripotent stem cells, cell culture, non-colony type monolayer, single cell, plating efficiency, Rho-associated kinase, Y-27632, transfection, transduction
51519
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Enrichment and Purging of Human Embryonic Stem Cells by Detection of Cell Surface Antigens Using the Monoclonal Antibodies TG30 and GCTM-2
Authors: Juan Carlos Polanco, Bei Wang, Qi Zhou, Hun Chy, Carmel O'Brien, Andrew L. Laslett.
Institutions: CSIRO.
Human embryonic stem cells (hESC) can self-renew indefinitely in vitro, and with the appropriate cues can be induced to differentiate into potentially all somatic cell lineages. Differentiated hESC derivatives can potentially be used in transplantation therapies to treat a variety of cell-degenerative diseases. However, hESC differentiation protocols usually yield a mixture of differentiated target and off-target cell types as well as residual undifferentiated cells. For the translation of differentiated hESC-derivatives from the laboratory to the clinic, it is important to be able to discriminate between undifferentiated (pluripotent) and differentiated cells, and generate methods to separate these populations. Safe application of hESC-derived somatic cell types can only be accomplished with pluripotent stem cell-free populations, as residual hESCs could induce tumors known as teratomas following transplantation. Towards this end, here we describe a methodology to detect pluripotency associated cell surface antigens with the monoclonal antibodies TG30 (CD9) and GCTM-2 via fluorescence activated cell sorting (FACS) for the identification of pluripotent TG30Hi-GCTM-2Hi hESCs using positive selection. Using negative selection with our TG30/GCTM-2 FACS methodology, we were able to detect and purge undifferentiated hESCs in populations undergoing very early-stage differentiation (TG30Neg-GCTM-2Neg). In a further study, pluripotent stem cell-free samples of differentiated TG30Neg-GCTM-2Neg cells selected using our TG30/GCTM-2 FACS protocol did not form teratomas once transplanted into immune-compromised mice, supporting the robustness of our protocol. On the other hand, TG30/GCTM-2 FACS-mediated consecutive passaging of enriched pluripotent TG30Hi-GCTM-2Hi hESCs did not affect their ability to self-renew in vitro or their intrinsic pluripotency. Therefore, the characteristics of our TG30/GCTM-2 FACS methodology provide a sensitive assay to obtain highly enriched populations of hPSC as inputs for differentiation assays and to rid potentially tumorigenic (or residual) hESC from derivative cell populations.
Stem Cell Biology, Issue 82, Stem cells, cell surface antigens, antibodies, FACS, purging stem cells, differentiation, pluripotency, teratoma, human embryonic stem cells (hESC)
50856
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High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry
Authors: Subarna Bhattacharya, Paul W. Burridge, Erin M. Kropp, Sandra L. Chuppa, Wai-Meng Kwok, Joseph C. Wu, Kenneth R. Boheler, Rebekah L. Gundry.
Institutions: Medical College of Wisconsin, Stanford University School of Medicine, Medical College of Wisconsin, Hong Kong University, Johns Hopkins University School of Medicine, Medical College of Wisconsin.
There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle “in a dish” for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.
Cellular Biology, Issue 91, human induced pluripotent stem cell, flow cytometry, directed differentiation, cardiomyocyte, IRX4, TNNI3, TNNT2, MCL2v, MLC2a
52010
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From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs
Authors: Jin Zhang, Ivan Khvorostov, Michael Teitell.
Institutions: University of California, Los Angeles.
This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells.
Cellular Biology, Issue 16, human embryonic stem cell (hESC), mouse embryonic fibroblast (MEF), matrigel, conditioned-media, feeder cell, pluripotency
722
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From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel
Authors: Jin Zhang, Ivan Khvorostov, Michael Teitell.
Institutions: University of California, Los Angeles.
This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated.
Cellular Biology, Issue 16, human embryonic stem cell (hESC), mouse embryonic fibroblast (MEF), matrigel, conditioned-media, feeder cell, pluripotency
832
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Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures
Authors: William S. Turner, Kara E. McCloskey.
Institutions: University of California, Merced.
Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.
Cellular Biology, Issue 68, Human Embryonic Stem Cells, Cell Culture, Cell Isolation, Oct, Cell Purification, MEF Removal, SSEA-4
3951
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From MEFs to Matrigel 2: Splitting hESCs from MEFs onto Matrigel
Authors: Ivan Khvorostov, Jin Zhang, Michael Teitell.
Institutions: University of California, Los Angeles.
This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF plates to feeder cell-free Matrigel plates.
Cellular Biology, Issue 16, human embryonic stem cell (hESC), mouse embryonic fibroblast (MEF), matrigel, conditioned-media, feeder cell, pluripotency
831
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