Accepted Manuscript Differentiation of Pluripotent Stem Cells for Regenerative Medicine Ke Li, Yan Kong, Mingliang Zhang, Fei Xie, Peng Liu, Shaohua Xu PII:
S0006-291X(16)30186-3
DOI:
10.1016/j.bbrc.2016.01.182
Reference:
YBBRC 35299
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 28 January 2016 Accepted Date: 30 January 2016
Please cite this article as: K. Li, Y. Kong, M. Zhang, F. Xie, P. Liu, S. Xu, Differentiation of Pluripotent Stem Cells for Regenerative Medicine, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2016.01.182. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Differentiation of Pluripotent Stem Cells for Regenerative Medicine
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Ke Li a,b*, Yan Kong c, Mingliang Zhang a,b, Fei Xie d, Peng Liu a,b, Shaohua Xu a,b
Gladstone Institutes, San Francisco, California 94158, USA
b
University of California, San Francisco, San Francisco, California 94158, USA
c
Department of Biochemistry and Molecular Biology, School of Medicine, Southeast
d
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University, Nanjing, Jiangsu Province, 210009, China.
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a
Department of Medicine, University of California, San Francisco, San Francisco,
California 94143, USA
*Corresponding author: Ke Li, Ph.D.
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Gladstone Institute of Cardiovascular Disease 1650 Owens Street
San Francisco, CA, 94158
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Phone: 415-734-2828
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Email: [email protected]
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ABSTRACT
A long-standing goal in regenerative medicine is to obtain scalable functional cells on
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demand to replenish cells lost in various conditions, including relevant diseases, injuries, and aging. As an unlimited cell source, pluripotent stem cells (PSCs) are invaluable for regenerative medicine, because they have the potential to give rise to any cell type in an
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organism. For therapeutic purposes, it is important to develop specific approach to directing PSC differentiation towards desired cell types efficiently. Through directed
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differentiation, PSCs could give rise to scalable, clinically relevant cells for in vivo transplantation, as well as for studying diseases in vitro and discovering drugs to treat them. Over the past few years, significant progress has been made in directing differentiation of PSCs into a variety of cell types. In this review, we discuss recent progress in directed differentiation of PSCs, clinical translation of PSC-based cell
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replacement therapies, and remaining challenges.
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Keywords:
Pluripotent stem cells, Directed differentiation, Regenerative medicine, Cell therapy,
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Embryonic development, Small molecules
Abbreviations: PSC
pluripotent stem cell
iPSC
induced pluripotent stem cell
ESC
embryonic stem cells
FGF
fibroblast growth factor 2
KGF
keratinocyte growth factor
BMP
bone morphogenic protein
ENP
early-stage neuroepithelial progenitors
SCZ
Schizophrenia
ASD
autism spectrum disorder
RPE
retinal pigment epithelium
AMD
age-related macular degeneration
SHH
sonic hedgehog
AFE
anterior foregut endoderm
GMP
good manufacturing practices
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1. Introduction
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transforming growth factor
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TGF
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Many devastating diseases are rooted in cellular deficiency, including diabetes,
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neurodegenerative disorders, spinal cord injury, and heart failure. These diseases are all caused by a loss of one or more critical cell populations or their functions. A longstanding goal of regenerative medicine is to obtain a sufficient number of functional cells on demand to replenish the cells lost in disease. This goal may be achieved with pluripotent stem sells (PSCs), which have two important features that make them invaluable in regenerative medicine: they have the potential to give rise to any cell type in the body, and they have the ability to self-renew long-term. These features make it 3
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possible to obtain large numbers of homogeneous PSCs with long-term expansion [1, 2]. With direct differentiation, PSCs could give rise to a large number of clinically relevant cells that could be used for in vivo transplantation, as well as for studying diseases in
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vitro and discovering drugs to treat them.
Significant progress has been made in directed differentiation of PSCs into a variety of
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cell types. One of the most powerful strategies for this directed differentiation is to harness the signaling pathways that control embryonic development [3]. By
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understanding the requirements for embryonic development, researchers can establish robust differentiation protocols for every cell type in the body. Another powerful strategy is to facilitate directed differentiation of PSCs with small molecules [4, 5]. Small molecules are widely used to modulate cell fate and probe mechanisms, because they have many advantages over protein factors and genetic manipulation. For example, small
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molecules are more cost-effective than protein factors, and they can potently manipulate intracellular signaling pathways. In addition, small molecules can provide rapid and
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reversible temporal control, and their effects can be fine-tuned by varying their concentrations and combinations. In this review, we will focus on recent progress made
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in the directed differentiation of PSCs, clinical translation of PSC-based cell replacement therapies, and remaining challenges.
2. Differentiation of PSCs into Neural Ectoderm Derivatives
The induction of neural ectoderm from PSCs is considered the “default” pathway, because neural ectoderm develops in PSCs-cultures that contain no serum or other inducers; however, it does depend on fibroblast growth factor (FGF) signals
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endogenously produced by differentiating PSCs [6].
In practice, to specify neural
lineages from PSCs, researchers mainly use three distinct approaches: embryoid-body formation, co-culture on neural-inducing feeders, and directed neural induction. Dr.
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Lorenz Studer’s lab made a breakthrough in differentiating human PSCs into neural cells by developing the dual-SMAD inhibition approach. This approach promotes the neuroectodermal differentiation of human PSCs by suppressing mesoderm and endoderm
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differentiation. Studer’s lab achieved a rapid and efficient neural differentiation from human PSCs by using SB431542 to inhibit the transforming growth factor (TGF)-
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β/Activin/Nodal pathway, which is crucial for endoderm differentiation, and Noggin to inhibit the bone morphogenic protein (BMP) signaling pathway, which is crucial for mesoderm differentiation [7]. This strategy induces early-stage neuroepithelial progenitors (ENPs) in a monolayer fashion with more than 80% purity. Importantly, these
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ENPs are highly responsive to regionalization cues, and could efficiently generate neuronal subtypes relevant to that region, thus represent a promising multipotent population for clinical therapy. However, this population could not be maintained in vitro
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as they spontaneously convert into a late-stage neural progenitor populations. Therefore, development of an efficient approach that could capture this stage-specific population
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would be of great interest for basic research and clinical purpose.
Despite the directed generation of neural progenitor cell types, induced pluripotent stem cells (iPSCs)-derived terminally differentiated cell types, such as neurons, offer a powerful system for studying diseases in vitro and discovering drugs to treat them. Schizophrenia (SCZ) and autism spectrum disorder (ASD) are genetically and phenotypically complex disorders of neural development. Dysregulation of synaptic
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function and structure have been strongly implicated in both neuropsychiatric disorders [8, 9]. However, little is known about the pathophysiology of synapses in patient neurons, as well as the genes that lead to synaptic deficits in human. To this end,
Dr. Hongjun
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Song and his colleagues generated induced pluripotent stem cells (iPSCs) from four members of a family in which a frameshift mutation disrupted in schizophrenia 1 (DISC1) co-segregated with major psychiatric disorders. They then differentiated these iPSCs into
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neurons. With this approach, they found that mutant DISC1 dysregulates expression of genes related to synapses and psychiatric disorders in human forebrain neurons, and
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consequently causes deficits in synaptic-vesicle release and transcriptional dysregulation in these neurons. Their findings provide new insight into the molecular mechanisms underlying psychiatric disorders [10]. Similarly, in iPSC studies of syndromic ASD, researchers showed decreased synaptic connectivity with fewer synapses. They also
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found immature synapses in neurons differentiated from iPS cells of syndromic ASD patients. iPSC-derived neurons from patients with Timothy syndrome, a syndromic form of ASD, exhibited changes in the expression of genes enriched in ASD and upregulated
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in ASD postmortem brains [11]. These studies support a central role for synapse formation and function in SCZ and ASD. As a good model for mechanistic study, the
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paradigm of iPSC-derived terminally differentiated cell types, such as neurons, is useful for exploration of the underlying mechanism, and subsequently development of specific treatments for specific diseases, such as SCZ and ASD.
Among all the neural ectoderm derivatives, retinal cells have drawn the greatest attention from researchers in regenerative medicine because they bear tremendous therapeutic value for restoring lost vision. Dr. Masayo Takahashi’s group has done remarkable
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pioneering work in retinal differentiation and clinical application. In 2005, Ikeda et al. developed a step-wise procedure to generate putative photoreceptors and retinal pigment epithelial (RPE) cells from rodent and primate ESCs with Wnt and Nodal antagonists
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(Dkk1 and LeftyA) [12]. In a follow up study, Hirami et al. derived retinal progenitor cells from both mouse and human iPSCs with similar protocol, and further differentiated these progenitor cells into retinal photoreceptor cells with retinoic acid (RA) and taurine
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[13]. Furthermore, Osakada et al. reported an alternative chemical approach, in which a combination of small molecules, CKI-7 (a casein kinase I inhibitor), SB-431542, and Y-
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27632 could induce the differentiation of human embryonic stem cells (hESCs) and hiPSCs into retinal progenitor cells without using recombinant protein factors [14]. A few years later, Maruotti et al. developed an improved, growth-factor-free condition for efficient differentiation of RPE cells from hPSCs. Through high-throughput screening,
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they identified two small molecules chetomin and nicotinamide, applying of which converted about 50% hPSCs into RPE cells [15]. Recently, Dr. Takahashi’s team attempted to transplant human iPSC-derived RPE cells back into a patient with age-
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related macular degeneration (AMD), a disease causes severe visual impairment [16]. This pioneering work represents a significant step forward toward using iPSCs in clinical
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applications.
3. Differentiation of PSCs into Mesoderm Derivatives
During embryonic development, mesoderm gives rise to the hematopoietic, bone, vascular, cardiac, and skeletal muscle lineages. Signals mediated through Wnt/β-catenin and TGF-β family members, including activin and BMPs, promote differentiation of
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mouse ESCs into mesoderm [17, 18]. Additionally, the early stages of mesoderm induction could be monitored by the upregulation of Flk-1, which is correlated with commitment to a mesoderm fate [19]. To induce cardiac mesoderm, Wnt/β-catenin
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signaling must first be activated to induce mesoderm, and then its inhibition is subsequently required to induce cardiac mesoderm [17, 18]. To induce hematopoietic
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mesoderm, a combination of Wnt, activin/Nodal, and BMP signaling is required [20].
Given that cardiovascular disease is the leading cause of death worldwide, hPSC-based
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cell therapy represents a promising approach for cardiac repair. Cardiomyocytes are the cells that make up the cardiac muscle. Over the years, researchers have made sufficient progress in cardiomyocyte differentiation and transplantation, which support that these efforts may translate to a clinical setting in the foreseeable future.
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To generate cardiac cells from PSCs, researchers mainly use two strategies; monolayerbased differentiation and embryoid body-based differentiation [21, 22]. Monolayer-based differentiation is based on the sequential exposure of differentiating hPSCs to activin A
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and BMP4 [21], while embryoid body-based differentiation follows a more complex series of defined patterning signals [22]. Later, by activating Wnt signaling with
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CHIR99021 and then inhibiting it with IWP2 or IWP4, Lian et al. enabled efficient cardiac differentiation of hPSCs under growth-factor-free conditions [23]. Also in 2012, Minami et al. induced robust cardiac differentiation of hPSCs in a defined xeno-free medium using similar biphasic-modulation strategy. This method used Wnt activators (BIO and CHIR) at the early phase of cardiac differentiation and Wnt inhibitors
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(KY02111 and XAV939) at the late phase [24]. These studies have led to chemically defined conditions for cardiac differentiation of hiPSCs [25].
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Unfortunately, differentiated cells usually exhibit a low degree of maturation and are noticeably different from adult cardiomyocytes; they have immature sarcomere structure and high proliferation rates, and they express the fetal gene program [26]. Immature
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cardiomyocytes also transiently express features of pacemaker cells that may autonomously trigger cardiac contractions. Thus, developing methods to increase cell
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maturation will be a major focus towards clinically translating this technology.
Recently, some preclinical studies using PSC-based cell therapy for cardiac repair have been successful. For example, Shiba et al. reported robust survival of hESC-derived cardiomyocytes and the ability of these cells to both couple with host cells and suppress
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arrhythmias in injured hearts [27]. Nunes et al. developed a platform to mature hPSCderived cardiomyocytes, which combines three-dimensional (3D) cell culture with electrical stimulation. They found that their platform could generate 3D, aligned cardiac
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tissues with more mature cardiomyocytes [28].
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4. Differentiation of PSCs into Endoderm Derivatives
Endoderm-derived organs, such as pancreas, liver, lung, and intestine, are notable targets for regenerative therapy. Thus, we need to understand the pathways that regulate the induction and specification of this germ layer. High levels of activin/Nodal signaling efficiently induce definitive endoderm in cultures of human and mouse ESCs [29, 30, 31]. During mouse and human embryonic development, once induced, endoderm forms an
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epithelial sheet that undergoes specification to distinct regions known as foregut, midgut, and hindgut [32]. This specification is partially controlled by factors secreted by surrounding mesoderm-derived tissues. To de novo generate endoderm-derived lineages
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in vitro, embryo development is commonly recapitulated in the induction strategies. Here, we summarize recent progress in generating two important endoderm lineages for
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regenerative therapies.
4.1 Pancreatic Cells
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D'Amour et al. devised a stepwise approach to induce the definitive endoderm into pancreatic cells expressing endocrine hormones [33]. They found that combining RA with inhibition of sonic hedgehog (SHH) could specify definitive endoderm to a pancreatic fate. Although the endocrine-like cells generated with this approach can
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synthesize endocrine hormones, the insulin-expressing cells are similar to fetal β-cells; they release C-peptide in response to multiple secretory stimuli, but only minimally to glucose. Later, Kroon et al. optimized D'Amour’s protocol to generate pancreatic
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endoderm from hESCs. After transplantation into mice, the pancreatic endoderm could
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generate glucose-responsive insulin-secreting β-like cells in vivo [34].
Recently, Douglas Melton Lab, James Johnson Lab and Timothy Kieffer Lab succeeded in generating functional human pancreatic β-like cells in vitro from hPSCs [35, 36]. These cells expressed key markers of mature human pancreatic β cells and displayed glucose-stimulated insulin secretion similar to that of human islets. Furthermore, transplantation of these β-like cells ameliorated hyperglycemia in diabetic mice.
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To develop an efficient differentiation protocol, Dr. Douglas Melton’s lab performed comprehensive compound screening. Finally, their protocol takes 4–5 weeks and involves a unique combination of sequential culture steps with factors that affect signaling in
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many pathways, including signaling by Wnt, activin, hedgehog, EGF, TGF-β, thyroid hormone, and RA, and γ-secretase [35]. The labs of Dr. James Johnson and Timothy Kieffer improved their previous protocol in several ways, including substituting
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cytokines with small molecules and adding vitamin C during steps 2–4. Addition of vitamin C at this stage reduced mRNA expression of NGN3, a master regulator of
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pancreatic endocrine cells. This suppression is important during the early stages of differentiation, because premature induction of NGN3 in pancreatic endoderm cells primes the cells toward populations enriched with polyhormonal cells, which are typical immature endocrine–pancreatic cells [36]. Besides, Ding lab has made big breakthrough
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in generating mouse and human pancreatic cells from fibroblasts by transdifferentiation
diabetes.
4.2 Lung Cells
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[37, 38]. These preclinical studies provided solid foundation for cell therapy of type I
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Lung is composed of many epithelial lineages that arise from the anterior foregut endoderm. Soon after gastrulation, definitive endoderm folds to form the gut tube, which is patterned along the anterior-posterior and dorsal-ventral axes through paracrine signals from the surrounding mesoderm [39]. These signals specify the lung field that will bud from the ventral side of the anterior foregut to form the primitive lung bud. Then, it will give rise to the highly arborized respiratory tree. By E16.5 in mice, primary branching of
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the lung is complete. Then, cell differentiation continues through the early postnatal period to eventually generate all cell types that compose the mature lung [40].
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Green et al. found that treatment of definitive endoderm with inhibitors of BMP and Nodal signaling specifies a population of cells with an anterior foregut–endoderm (AFE) identity. These cells can be directed to an early lung-endoderm fate by timed treatment
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with signaling factors required for lung development, including Wnt, FGF, BMP, EGF, and keratinocyte growth factor (KGF) [41]. In 2012, Longmire et al. and Mou et al.
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made a breakthrough in inducing differentiated lung cells from ESCs and hiPSCs [42, 43]. They both used a stepwise application of inhibitors and activators of Wnt, BMP, and FGF, which are critical for proper specification of lung epithelial cells, to induce lungendoderm formation. In addition, they both found a stepwise process through the stages of lung development: formation of AFE, specification of lung-endoderm progenitors,
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determination of proximal and distal lung progenitors, and differentiation and maturation into proximal and distal lung-epithelial cells. Later, Huang et al. improved the efficiency
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of lung- endoderm differentiation from hPSCs by optimizing the induction of AFE from definitive endoderm via sequentially inhibiting BMP and Wnt signaling while
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simultaneously inhibiting TGF-β signaling [44, 45].
5. Challenges for Clinical Applications
Although progress has been made in using direct differentiation to support clinical translation in the foreseeable future, considerable challenges still remain. For example, PSCs have the potential to form tumors, raising concerns for their safety in clinical settings. A possible solution to this challenge is to purify the desired cell type and
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exclude cells that are not fully differentiated. Another challenge is the ability to scalably produce hPSC derivatives using xeno-free reagents and defined culture media with good manufacturing practices (GMPs). Small molecules could be a powerful tool for
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improving differentiation protocols and reducing the cost of scalable production and adherence to GMPs, because they are usually less expensive and more scalable than protein factors. Finally, patient selection is a concern in using this type of cell therapy in
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clinical applications. To overcome this concern, appropriate patients must be selected
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based on their specific phenotypes as defined by physiological and genetic approaches.
6. Closing Remarks
Direct differentiation of PSCs holds great promise for regenerative medicine. A powerful strategy for directed differentiation of PSCs involves a step-wise process that mimics
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embryonic development. Small molecules could precisely modulate the signaling pathways that are crucial for this process. Recent progress in directed differentiation provides not only new insights into basic developmental processes, but also new
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approaches for regenerative medicine. However, some challenges still remain. Solving safety concerns, increasing cell maturity, and creating scalable production methods that
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adhere to GMPs will be major focuses on the path towards clinical translation of this form of therapy.
Conflict of interest
The authors declare that they have no conflicts of interest.
Statement of human and animal rights
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This article does not contain any studies with human or animal subjects performed by any of the authors.
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Acknowledgments We would like to thank Crystal Herron of Gladstone Institutes for scientific editing of
this manuscript. Due to space limitations, we apologize to all scientists whose research
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could not be discussed and cited in this review.
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Highlights: 1. PSCs could give rise to all cell types in an organism. 2. PSCs could give rise to clinically relevant cells by directed differentiation.
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3. Recapitulating embryo development is a useful strategy for directed differentiation.
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4. Small molecules could facilitate directed differentiation of PSCs.
S0006-291X(16)30186-3
DOI:
10.1016/j.bbrc.2016.01.182
Reference:
YBBRC 35299
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 28 January 2016 Accepted Date: 30 January 2016
Please cite this article as: K. Li, Y. Kong, M. Zhang, F. Xie, P. Liu, S. Xu, Differentiation of Pluripotent Stem Cells for Regenerative Medicine, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2016.01.182. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Differentiation of Pluripotent Stem Cells for Regenerative Medicine
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Ke Li a,b*, Yan Kong c, Mingliang Zhang a,b, Fei Xie d, Peng Liu a,b, Shaohua Xu a,b
Gladstone Institutes, San Francisco, California 94158, USA
b
University of California, San Francisco, San Francisco, California 94158, USA
c
Department of Biochemistry and Molecular Biology, School of Medicine, Southeast
d
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University, Nanjing, Jiangsu Province, 210009, China.
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a
Department of Medicine, University of California, San Francisco, San Francisco,
California 94143, USA
*Corresponding author: Ke Li, Ph.D.
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Gladstone Institute of Cardiovascular Disease 1650 Owens Street
San Francisco, CA, 94158
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Phone: 415-734-2828
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Email: [email protected]
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ABSTRACT
A long-standing goal in regenerative medicine is to obtain scalable functional cells on
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demand to replenish cells lost in various conditions, including relevant diseases, injuries, and aging. As an unlimited cell source, pluripotent stem cells (PSCs) are invaluable for regenerative medicine, because they have the potential to give rise to any cell type in an
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organism. For therapeutic purposes, it is important to develop specific approach to directing PSC differentiation towards desired cell types efficiently. Through directed
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differentiation, PSCs could give rise to scalable, clinically relevant cells for in vivo transplantation, as well as for studying diseases in vitro and discovering drugs to treat them. Over the past few years, significant progress has been made in directing differentiation of PSCs into a variety of cell types. In this review, we discuss recent progress in directed differentiation of PSCs, clinical translation of PSC-based cell
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replacement therapies, and remaining challenges.
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Keywords:
Pluripotent stem cells, Directed differentiation, Regenerative medicine, Cell therapy,
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Embryonic development, Small molecules
Abbreviations: PSC
pluripotent stem cell
iPSC
induced pluripotent stem cell
ESC
embryonic stem cells
FGF
fibroblast growth factor 2
KGF
keratinocyte growth factor
BMP
bone morphogenic protein
ENP
early-stage neuroepithelial progenitors
SCZ
Schizophrenia
ASD
autism spectrum disorder
RPE
retinal pigment epithelium
AMD
age-related macular degeneration
SHH
sonic hedgehog
AFE
anterior foregut endoderm
GMP
good manufacturing practices
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1. Introduction
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transforming growth factor
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TGF
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Many devastating diseases are rooted in cellular deficiency, including diabetes,
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neurodegenerative disorders, spinal cord injury, and heart failure. These diseases are all caused by a loss of one or more critical cell populations or their functions. A longstanding goal of regenerative medicine is to obtain a sufficient number of functional cells on demand to replenish the cells lost in disease. This goal may be achieved with pluripotent stem sells (PSCs), which have two important features that make them invaluable in regenerative medicine: they have the potential to give rise to any cell type in the body, and they have the ability to self-renew long-term. These features make it 3
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possible to obtain large numbers of homogeneous PSCs with long-term expansion [1, 2]. With direct differentiation, PSCs could give rise to a large number of clinically relevant cells that could be used for in vivo transplantation, as well as for studying diseases in
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vitro and discovering drugs to treat them.
Significant progress has been made in directed differentiation of PSCs into a variety of
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cell types. One of the most powerful strategies for this directed differentiation is to harness the signaling pathways that control embryonic development [3]. By
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understanding the requirements for embryonic development, researchers can establish robust differentiation protocols for every cell type in the body. Another powerful strategy is to facilitate directed differentiation of PSCs with small molecules [4, 5]. Small molecules are widely used to modulate cell fate and probe mechanisms, because they have many advantages over protein factors and genetic manipulation. For example, small
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molecules are more cost-effective than protein factors, and they can potently manipulate intracellular signaling pathways. In addition, small molecules can provide rapid and
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reversible temporal control, and their effects can be fine-tuned by varying their concentrations and combinations. In this review, we will focus on recent progress made
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in the directed differentiation of PSCs, clinical translation of PSC-based cell replacement therapies, and remaining challenges.
2. Differentiation of PSCs into Neural Ectoderm Derivatives
The induction of neural ectoderm from PSCs is considered the “default” pathway, because neural ectoderm develops in PSCs-cultures that contain no serum or other inducers; however, it does depend on fibroblast growth factor (FGF) signals
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endogenously produced by differentiating PSCs [6].
In practice, to specify neural
lineages from PSCs, researchers mainly use three distinct approaches: embryoid-body formation, co-culture on neural-inducing feeders, and directed neural induction. Dr.
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Lorenz Studer’s lab made a breakthrough in differentiating human PSCs into neural cells by developing the dual-SMAD inhibition approach. This approach promotes the neuroectodermal differentiation of human PSCs by suppressing mesoderm and endoderm
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differentiation. Studer’s lab achieved a rapid and efficient neural differentiation from human PSCs by using SB431542 to inhibit the transforming growth factor (TGF)-
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β/Activin/Nodal pathway, which is crucial for endoderm differentiation, and Noggin to inhibit the bone morphogenic protein (BMP) signaling pathway, which is crucial for mesoderm differentiation [7]. This strategy induces early-stage neuroepithelial progenitors (ENPs) in a monolayer fashion with more than 80% purity. Importantly, these
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ENPs are highly responsive to regionalization cues, and could efficiently generate neuronal subtypes relevant to that region, thus represent a promising multipotent population for clinical therapy. However, this population could not be maintained in vitro
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as they spontaneously convert into a late-stage neural progenitor populations. Therefore, development of an efficient approach that could capture this stage-specific population
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would be of great interest for basic research and clinical purpose.
Despite the directed generation of neural progenitor cell types, induced pluripotent stem cells (iPSCs)-derived terminally differentiated cell types, such as neurons, offer a powerful system for studying diseases in vitro and discovering drugs to treat them. Schizophrenia (SCZ) and autism spectrum disorder (ASD) are genetically and phenotypically complex disorders of neural development. Dysregulation of synaptic
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function and structure have been strongly implicated in both neuropsychiatric disorders [8, 9]. However, little is known about the pathophysiology of synapses in patient neurons, as well as the genes that lead to synaptic deficits in human. To this end,
Dr. Hongjun
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Song and his colleagues generated induced pluripotent stem cells (iPSCs) from four members of a family in which a frameshift mutation disrupted in schizophrenia 1 (DISC1) co-segregated with major psychiatric disorders. They then differentiated these iPSCs into
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neurons. With this approach, they found that mutant DISC1 dysregulates expression of genes related to synapses and psychiatric disorders in human forebrain neurons, and
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consequently causes deficits in synaptic-vesicle release and transcriptional dysregulation in these neurons. Their findings provide new insight into the molecular mechanisms underlying psychiatric disorders [10]. Similarly, in iPSC studies of syndromic ASD, researchers showed decreased synaptic connectivity with fewer synapses. They also
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found immature synapses in neurons differentiated from iPS cells of syndromic ASD patients. iPSC-derived neurons from patients with Timothy syndrome, a syndromic form of ASD, exhibited changes in the expression of genes enriched in ASD and upregulated
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in ASD postmortem brains [11]. These studies support a central role for synapse formation and function in SCZ and ASD. As a good model for mechanistic study, the
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paradigm of iPSC-derived terminally differentiated cell types, such as neurons, is useful for exploration of the underlying mechanism, and subsequently development of specific treatments for specific diseases, such as SCZ and ASD.
Among all the neural ectoderm derivatives, retinal cells have drawn the greatest attention from researchers in regenerative medicine because they bear tremendous therapeutic value for restoring lost vision. Dr. Masayo Takahashi’s group has done remarkable
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pioneering work in retinal differentiation and clinical application. In 2005, Ikeda et al. developed a step-wise procedure to generate putative photoreceptors and retinal pigment epithelial (RPE) cells from rodent and primate ESCs with Wnt and Nodal antagonists
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(Dkk1 and LeftyA) [12]. In a follow up study, Hirami et al. derived retinal progenitor cells from both mouse and human iPSCs with similar protocol, and further differentiated these progenitor cells into retinal photoreceptor cells with retinoic acid (RA) and taurine
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[13]. Furthermore, Osakada et al. reported an alternative chemical approach, in which a combination of small molecules, CKI-7 (a casein kinase I inhibitor), SB-431542, and Y-
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27632 could induce the differentiation of human embryonic stem cells (hESCs) and hiPSCs into retinal progenitor cells without using recombinant protein factors [14]. A few years later, Maruotti et al. developed an improved, growth-factor-free condition for efficient differentiation of RPE cells from hPSCs. Through high-throughput screening,
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they identified two small molecules chetomin and nicotinamide, applying of which converted about 50% hPSCs into RPE cells [15]. Recently, Dr. Takahashi’s team attempted to transplant human iPSC-derived RPE cells back into a patient with age-
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related macular degeneration (AMD), a disease causes severe visual impairment [16]. This pioneering work represents a significant step forward toward using iPSCs in clinical
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applications.
3. Differentiation of PSCs into Mesoderm Derivatives
During embryonic development, mesoderm gives rise to the hematopoietic, bone, vascular, cardiac, and skeletal muscle lineages. Signals mediated through Wnt/β-catenin and TGF-β family members, including activin and BMPs, promote differentiation of
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mouse ESCs into mesoderm [17, 18]. Additionally, the early stages of mesoderm induction could be monitored by the upregulation of Flk-1, which is correlated with commitment to a mesoderm fate [19]. To induce cardiac mesoderm, Wnt/β-catenin
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signaling must first be activated to induce mesoderm, and then its inhibition is subsequently required to induce cardiac mesoderm [17, 18]. To induce hematopoietic
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mesoderm, a combination of Wnt, activin/Nodal, and BMP signaling is required [20].
Given that cardiovascular disease is the leading cause of death worldwide, hPSC-based
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cell therapy represents a promising approach for cardiac repair. Cardiomyocytes are the cells that make up the cardiac muscle. Over the years, researchers have made sufficient progress in cardiomyocyte differentiation and transplantation, which support that these efforts may translate to a clinical setting in the foreseeable future.
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To generate cardiac cells from PSCs, researchers mainly use two strategies; monolayerbased differentiation and embryoid body-based differentiation [21, 22]. Monolayer-based differentiation is based on the sequential exposure of differentiating hPSCs to activin A
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and BMP4 [21], while embryoid body-based differentiation follows a more complex series of defined patterning signals [22]. Later, by activating Wnt signaling with
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CHIR99021 and then inhibiting it with IWP2 or IWP4, Lian et al. enabled efficient cardiac differentiation of hPSCs under growth-factor-free conditions [23]. Also in 2012, Minami et al. induced robust cardiac differentiation of hPSCs in a defined xeno-free medium using similar biphasic-modulation strategy. This method used Wnt activators (BIO and CHIR) at the early phase of cardiac differentiation and Wnt inhibitors
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(KY02111 and XAV939) at the late phase [24]. These studies have led to chemically defined conditions for cardiac differentiation of hiPSCs [25].
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Unfortunately, differentiated cells usually exhibit a low degree of maturation and are noticeably different from adult cardiomyocytes; they have immature sarcomere structure and high proliferation rates, and they express the fetal gene program [26]. Immature
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cardiomyocytes also transiently express features of pacemaker cells that may autonomously trigger cardiac contractions. Thus, developing methods to increase cell
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maturation will be a major focus towards clinically translating this technology.
Recently, some preclinical studies using PSC-based cell therapy for cardiac repair have been successful. For example, Shiba et al. reported robust survival of hESC-derived cardiomyocytes and the ability of these cells to both couple with host cells and suppress
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arrhythmias in injured hearts [27]. Nunes et al. developed a platform to mature hPSCderived cardiomyocytes, which combines three-dimensional (3D) cell culture with electrical stimulation. They found that their platform could generate 3D, aligned cardiac
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tissues with more mature cardiomyocytes [28].
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4. Differentiation of PSCs into Endoderm Derivatives
Endoderm-derived organs, such as pancreas, liver, lung, and intestine, are notable targets for regenerative therapy. Thus, we need to understand the pathways that regulate the induction and specification of this germ layer. High levels of activin/Nodal signaling efficiently induce definitive endoderm in cultures of human and mouse ESCs [29, 30, 31]. During mouse and human embryonic development, once induced, endoderm forms an
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epithelial sheet that undergoes specification to distinct regions known as foregut, midgut, and hindgut [32]. This specification is partially controlled by factors secreted by surrounding mesoderm-derived tissues. To de novo generate endoderm-derived lineages
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in vitro, embryo development is commonly recapitulated in the induction strategies. Here, we summarize recent progress in generating two important endoderm lineages for
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regenerative therapies.
4.1 Pancreatic Cells
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D'Amour et al. devised a stepwise approach to induce the definitive endoderm into pancreatic cells expressing endocrine hormones [33]. They found that combining RA with inhibition of sonic hedgehog (SHH) could specify definitive endoderm to a pancreatic fate. Although the endocrine-like cells generated with this approach can
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synthesize endocrine hormones, the insulin-expressing cells are similar to fetal β-cells; they release C-peptide in response to multiple secretory stimuli, but only minimally to glucose. Later, Kroon et al. optimized D'Amour’s protocol to generate pancreatic
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endoderm from hESCs. After transplantation into mice, the pancreatic endoderm could
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generate glucose-responsive insulin-secreting β-like cells in vivo [34].
Recently, Douglas Melton Lab, James Johnson Lab and Timothy Kieffer Lab succeeded in generating functional human pancreatic β-like cells in vitro from hPSCs [35, 36]. These cells expressed key markers of mature human pancreatic β cells and displayed glucose-stimulated insulin secretion similar to that of human islets. Furthermore, transplantation of these β-like cells ameliorated hyperglycemia in diabetic mice.
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To develop an efficient differentiation protocol, Dr. Douglas Melton’s lab performed comprehensive compound screening. Finally, their protocol takes 4–5 weeks and involves a unique combination of sequential culture steps with factors that affect signaling in
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many pathways, including signaling by Wnt, activin, hedgehog, EGF, TGF-β, thyroid hormone, and RA, and γ-secretase [35]. The labs of Dr. James Johnson and Timothy Kieffer improved their previous protocol in several ways, including substituting
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cytokines with small molecules and adding vitamin C during steps 2–4. Addition of vitamin C at this stage reduced mRNA expression of NGN3, a master regulator of
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pancreatic endocrine cells. This suppression is important during the early stages of differentiation, because premature induction of NGN3 in pancreatic endoderm cells primes the cells toward populations enriched with polyhormonal cells, which are typical immature endocrine–pancreatic cells [36]. Besides, Ding lab has made big breakthrough
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in generating mouse and human pancreatic cells from fibroblasts by transdifferentiation
diabetes.
4.2 Lung Cells
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[37, 38]. These preclinical studies provided solid foundation for cell therapy of type I
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Lung is composed of many epithelial lineages that arise from the anterior foregut endoderm. Soon after gastrulation, definitive endoderm folds to form the gut tube, which is patterned along the anterior-posterior and dorsal-ventral axes through paracrine signals from the surrounding mesoderm [39]. These signals specify the lung field that will bud from the ventral side of the anterior foregut to form the primitive lung bud. Then, it will give rise to the highly arborized respiratory tree. By E16.5 in mice, primary branching of
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the lung is complete. Then, cell differentiation continues through the early postnatal period to eventually generate all cell types that compose the mature lung [40].
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Green et al. found that treatment of definitive endoderm with inhibitors of BMP and Nodal signaling specifies a population of cells with an anterior foregut–endoderm (AFE) identity. These cells can be directed to an early lung-endoderm fate by timed treatment
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with signaling factors required for lung development, including Wnt, FGF, BMP, EGF, and keratinocyte growth factor (KGF) [41]. In 2012, Longmire et al. and Mou et al.
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made a breakthrough in inducing differentiated lung cells from ESCs and hiPSCs [42, 43]. They both used a stepwise application of inhibitors and activators of Wnt, BMP, and FGF, which are critical for proper specification of lung epithelial cells, to induce lungendoderm formation. In addition, they both found a stepwise process through the stages of lung development: formation of AFE, specification of lung-endoderm progenitors,
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determination of proximal and distal lung progenitors, and differentiation and maturation into proximal and distal lung-epithelial cells. Later, Huang et al. improved the efficiency
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of lung- endoderm differentiation from hPSCs by optimizing the induction of AFE from definitive endoderm via sequentially inhibiting BMP and Wnt signaling while
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simultaneously inhibiting TGF-β signaling [44, 45].
5. Challenges for Clinical Applications
Although progress has been made in using direct differentiation to support clinical translation in the foreseeable future, considerable challenges still remain. For example, PSCs have the potential to form tumors, raising concerns for their safety in clinical settings. A possible solution to this challenge is to purify the desired cell type and
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exclude cells that are not fully differentiated. Another challenge is the ability to scalably produce hPSC derivatives using xeno-free reagents and defined culture media with good manufacturing practices (GMPs). Small molecules could be a powerful tool for
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improving differentiation protocols and reducing the cost of scalable production and adherence to GMPs, because they are usually less expensive and more scalable than protein factors. Finally, patient selection is a concern in using this type of cell therapy in
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clinical applications. To overcome this concern, appropriate patients must be selected
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based on their specific phenotypes as defined by physiological and genetic approaches.
6. Closing Remarks
Direct differentiation of PSCs holds great promise for regenerative medicine. A powerful strategy for directed differentiation of PSCs involves a step-wise process that mimics
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embryonic development. Small molecules could precisely modulate the signaling pathways that are crucial for this process. Recent progress in directed differentiation provides not only new insights into basic developmental processes, but also new
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approaches for regenerative medicine. However, some challenges still remain. Solving safety concerns, increasing cell maturity, and creating scalable production methods that
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adhere to GMPs will be major focuses on the path towards clinical translation of this form of therapy.
Conflict of interest
The authors declare that they have no conflicts of interest.
Statement of human and animal rights
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This article does not contain any studies with human or animal subjects performed by any of the authors.
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Acknowledgments We would like to thank Crystal Herron of Gladstone Institutes for scientific editing of
this manuscript. Due to space limitations, we apologize to all scientists whose research
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could not be discussed and cited in this review.
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Highlights: 1. PSCs could give rise to all cell types in an organism. 2. PSCs could give rise to clinically relevant cells by directed differentiation.
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3. Recapitulating embryo development is a useful strategy for directed differentiation.
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4. Small molecules could facilitate directed differentiation of PSCs.
