Monthly Archives: June 2022

Cell Tissue Res. Author manuscript; available in PMC 2018 May 22.

Published in final edited form as:

PMCID: PMC5963504

NIHMSID: NIHMS967727

1Department of Neuroscience, University of Wisconsin-Madison, Madison, WI 53705, USA

2Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA

1Department of Neuroscience, University of Wisconsin-Madison, Madison, WI 53705, USA

1Department of Neuroscience, University of Wisconsin-Madison, Madison, WI 53705, USA

2Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA

The astonishing progress in the field of stem cell biology during the past 40 years has transformed both science and medicine. Neural stem cells (NSCs) are the stem cells of the nervous system. During development they give rise to the entire nervous system. In adults, a small number of NSCs remain and are mostly quiescent; however, ample evidence supports their important roles in plasticity, aging, disease, and regeneration of the nervous system. Because NSCs are regulated by both intrinsic genetic and epigenetic programs and extrinsic stimuli transduced through the stem cell niche, dysregulation of NSCs due to either genetic causes or environmental impacts may lead to disease. Therefore, extensive investigations in the past decades have been devoted to understanding how NSCs are regulated. On the other hand, ever since their discovery, NSCs have been a focal point for cell-based therapeutic strategies in the brain and spinal cord. The limited number of NSCs residing in the tissue has been a limiting factor for their clinical applications. Although recent advancements in embryonic and induced pluripotent stem cells have provided novel sources for NSCs, several challenges remain. In this special issue, leaders and experts in NSCs summarize our current understanding of NSC molecular regulation and the importance of NSCs for disease modeling and translational applications.

The term stem cells first appeared in the scientific literature in 1868 by the German biologist Ernst Haeckel (Haeckel, 1868). In his writings (Haeckel, 1868), stem cells had two distinct meanings: one is the unicellular evolutionary origin of all multicellular organisms, and the other is the fertilized egg giving rise to all other cell types of the body. The latter definition has evolved into the modern definition of stem cells - cells that can divide to self-renew and to differentiate into other cell types in tissues and organs (Li and Zhao, 2008, Ramalho-Santos and Willenbring, 2007).

The behavior and fate of stem cells are strongly influenced by their specific anatomical locations and surrounding cell types, called the stem cell niche. The niche provides physical support to host or anchor stem cells, and supplies factors to maintain and regulate them (Li and Zhao, 2008). Stem cells are also regulated by intrinsic signaling cascades and transcriptional mechanisms, some of which are common among all stem cells, and others that are unique to specific types. Some of the best known regulators include TGF-, BMP, Smad, Wnt, Notch, EGF fibroblast growth factors (Jobe, et al., 2012, Li and Zhao, 2008). Therefore, stem cells are regulated by complex mechanisms in both temporal- and context-specific manners to maintain their unique characteristics. Understanding stem cell regulation gives us the opportunity to explore mechanisms of development, as well as disorders resulting from their dysfunction.

During development, the central nervous system (CNS) is generated from a small number of neural stem cells (NSCs) lining the neural tube (Kriegstein and Alvarez-Buylla, 2009). A great deal of experimental evidence has demonstrated that radial glia, the NSCs during mammalian CNS development, undergo both symmetric divisions to expand the NSC pool, and asymmetric divisions to give rise to intermediate progenitors (IPCs) and the differentiated cell types. The three major cell types in the CNS arise from NSCs in a temporally defined sequence, with neurons appearing first, followed by astrocytes, and then oligodendrocytes (Okano and Temple, 2009). The technical advancement of live imaging and genomic tools have allowed for the identification of human-specific NSC populations (e.g. outer radial glia, or oRG) located at the outer subventricular zone (SVZ) (Gertz, et al., 2014). These oRG are essential for cortical expansion to achieve the large size of the human cortex. Single-cell genomic technologies have identified specific oRG markers that might be used for further characterization of these cells (Liu, et al., 2016, Pollen, et al., 2014). Investigating the regulatory mechanisms governing the self-renewal and fate specification of NSCs, especially human-specific developmental features, has significantly contributed to our understanding of human brain development and developmental diseases. In addition, this knowledge also has helped scientists refine protocols for pluripotent stem cell differentiation into specific nervous system cell types for both therapeutic goals and disease modeling.

In adult brains, NSCs are reduced and become restricted to specific brain regions. In rodents, both NSCs and ongoing neurogenesis have been widely documented in the SVZ of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus (DG) of the hippocampus (Kempermann, et al., 2015). In humans, experimental evidence has supported ongoing neurogenesis in the hippocampus (Eriksson, et al., 1998, Spalding, et al., 2013). The confirmation of mammalian adult neurogenesis in the 1990s was one of the most exciting moments in science in the 21st century. Not only did it overthrow the prevailing dogma suggesting no neurons were made in the adult brain, but also it hinted that these adult NSCs could be utilized for neural repair in disease and following injury. Forty years later, we have learned a lot about NSCs. In the adult rodent SVZ, neurogenesis has been shown to be important for olfactory function and olfactory learning (Alonso, et al., 2006). During development, a subset of slowly-dividing NSCs are set aside to be the NSCs of the SVZ in the postnatal and adult brain (Fuentealba, et al., 2015, Furutachi, et al., 2015). The majority of neurogenic radial glia, however, become astrocytes and ependymal cells at the end of embryonic neurogenesis (Noctor, et al., 2004). A subset of these astrocytes persist as NSCs in specialized niches in the adult brain and continuously generate neurons that functionally integrate into restricted brain regions (Doetsch, 2003). In the hippocampus, radial glia-like stem cells of the SGZ make newborn neurons throughout life (Goritz and Frisen, 2012). These newborn neurons integrate into the circuity of the DG, contributing to behaviors such as pattern separation (Aimone, et al., 2011) and spatial learning (Dupret, et al., 2008), as well as hippocampus-associated learning, memory, and executive functions (Kempermann, Song and Gage, 2015).

Significant effort has been devoted into understanding the regulation of adult neurogenesis. As a result, we now know that many extrinsic stimuli and intrinsic mechanisms can affect this process. Mouse genetic studies have clearly demonstrated the important role of transcriptional regulation of NSCs through intrinsic genetic mechanisms (Hsieh and Zhao, 2016). Some examples include SOXC family proteins [Kavyanifar et al, in this issue (Kavyanifar, et al., 2018)], Bmi-1 (Molofsky, et al., 2003), Sox2 (Ferri, et al., 2004, Graham, et al., 1999), PTEN (Bonaguidi, et al., 2011), and Notch [Zhang et al, in this issue (Zhang, et al., 2018)]. In addition epigenetic regulation by DNA methylation pathways (e.g. Mbd1, Mecp2, Dnmt, Tet) (Noguchi, et al., 2015, Smrt, et al., 2007, Tsujimura, et al., 2009, Zhang, et al., 2013, Zhao, et al., 2003), chromatin remodeling (e.g. BAF, BRG1) (Ninkovic, et al., 2013, Petrik, et al., 2015, Tuoc, et al., 2017), and noncoding RNAs (Liu, et al., 2010)[Anderson and Lim, in this issue (Anderson and Lim, 2018)] play important roles. Many growth factors, signaling molecules, and neurotransmitters have been shown to regulate neurogenesis (Kempermann, Song and Gage, 2015). Catavero et al [in this issue (Catavero, et al., 2018)] review the role of GABA circuits, signaling, and receptors in regulating development of adult born cells, as well as the molecular players that modulate GABA signaling. Because progenitors with multipotent differentiation potentials have been found in brain regions without active neurogenesis (Palmer, et al., 1997), it is hypothesized that these progenitors might be manipulated to become neuron-competent in vivo so that they can contribute to brain generation [Wang et al, in this issue (Wang and Zhang, 2018)].

A great amount of literature has documented how physiological activities and enriched environment influences adult neurogenesis (Kempermann, Song and Gage, 2015). However, as summarized by Eisinger and Zhao [in this issue (Eisinger and Zhao, 2018)], the genes and gene network involved in these changes within NSCs have not been systematically analyzed at genome wide levels. Adult neurogenesis is also influenced by diseases including epilepsy (Parent and Lowenstein, 1997), stroke (Zhang and Chopp, 2016), depression (Dranovsky and Hen, 2006, Kempermann, et al., 2003), and injury [(Morshead and Ruddy, in this issue (Morshead and Ruddy, 2018) in this issue). Thodeson et al [in this issue (Thodeson, et al., 2018)] further summarize the contribution and dysregulation of adult neurogenesis in epilepsy and discuss how we can translate these findings to human therapeutics by using patient-derived neurons to study monogenic epilepsy-in-a-dish.

Aging affects every individual and is a major risk factor for many diseases. One of the strongest negative regulators of adult neurogenesis is aging. Both intrinsic and extrinsic components regulate the limitations of NSC proliferation and function (Moore and Jessberger, 2017, Seib and Martin-Villalba, 2015). In this issue, Mosher and Schaffer (Mosher and Schafer, 2018) and Ruddy and Morshead (Morshead and Ruddy, 2018) examine factors such as secreted signals, cell contact- dependent signals, and extracellular matrix cues that control neurogenesis in an age-dependent manner, and define these signals by the extrinsic mechanism through which they are presented to the NSCs. Smith et al [in this issue (Smith, et al., 2018)] discuss how age-related changes in the blood, such as blood-borne-factors, and peripheral immune cells, contribute to the age-related decline in adult neurogenesis in the mammalian brain.

Despite the extensive knowledge we have gained regarding adult neurogenesis, critical questions remain. For example, the control of the functional integration of new neurons remains a mystery. It has been shown that adult NSC-differentiated newborn neurons exhibit a critical period for sensitivity to external stimuli (Bergami, et al., 2015), and a heightened sensitivity to seizures (Kron, et al., 2010). It remains unclear how new neurons choose their connections. Jahn and Bergami [in this issue (Jahn and Bergami, 2018)] further discuss the critical period and its regulators during adult newborn neuron development.

Understanding the extrinsic and intrinsic regulation of adult NSCs and their newborn progeny, and their response to both positive and negative stimuli will further illuminate their role in disease, injury, stress, and brain function.

Human pluripotent stem cells (PSCs), including human embryonic stem cells (ESCs) and induced PSCs (iPSCs), offer a model system to reveal cellular and molecular events underlying normal and abnormal neural development in humans. ESCs are pluripotent cells derived from the inner cell mass of blastocyst stage preimplantation embryos, which were first isolated from mouse by Evans and Kaufman in 1981 (Evans and Kaufman, 1981) and later from humans by James Thompson in 1998 (Thomson, et al., 1998). Human ESCs are invaluable in the study of early embryonic development, allowing us to identify critical regulators of cell commitment, differentiation, and adult cell reprogramming (Dvash, et al., 2006, Ren, et al., 2009). iPSCs are reprogrammed from somatic cells by forced expression of stem cell genes and have the characteristics of ESCs (Okita, et al., 2007, Yu, et al., 2007). The development of iPSC technology has allowed us access to cells of the human nervous system through reprogramming of patient-derived cells, revolutionizing our ability to study human development and diseases.

To generate neural cells from either ESCs or iPSCs, the first step is neural induction. Through actions of a number of activators and inhibitors of cell signaling pathways, this process yields neural epithelial cell-like NSCs and then intermediate neural progenitors, resembling embryonic development. Despite many advances, a major hurdle of neural differentiation is lineage control. Using a standard dorsal forebrain neural differentiation protocol, most neural progenitors obtained are forebrain excitatory progenitors that produce mostly forebrain glutamatergic excitatory neurons. However, the purity and layer-specific composition of these progenitors, as well as neurons, vary significantly from experiment to experiment, cell line to cell line, and lab to lab. In addition, differentiation into specific types of neurons with high purity has always been a challenging goal. Much effort has been devoted into improving the efficiency of dopaminergic neuron and GABAergic neuron differentiation with great success (Hu, et al., 2010). However, the brain has many other types of neurons. Vadodaria et al [in this issue (Vadodaria, et al., 2018)] discuss how to generate serotonergic neurons, a type of neuron highly relevant to psychiatric disorders. To better understand the molecular control of human PSC and NSC differentiation, where protocols result in a large amount of cellular heterogeneity in identity and response, analysis must be done at the level of single cells. Harbom et al [in this issue (Harbom, et al., 2018)] summarizes how new state-of-the-art single-cell analysis methods may help to define differentiation from pluripotent cells.

The advancement in iPSC and gene editing technology has transformed the field of human disease modeling. As in many human disorders, especially neuropsychiatric disorders, mouse models have been useful. Yet there are several critical reasons why it is necessary to use human cells to define the underlying mechanisms that lead to human patient characteristics, particularly those affecting the nervous system. For example, in fragile X syndrome (FXS), the epigenetic silencing of the Fragile X Mental Retardation Gene 1 (FMR1) gene that causes FXS occurs only in humans. Mice engineered to mimic the human mutation in the FMR1 gene do not show the same methylation and silencing characteristics of the gene as in humans (Brouwer, et al., 2007). These results indicate that some epigenetic mechanisms in human and mice are different and preclude the ability to study epigenetic mechanisms of FMR1 silencing in mouse models of FXS (Bhattacharyya and Zhao, 2016). In this issue, Li and Shi discuss disease modeling using human PSC-differentiated neural progenitors (Li, et al., 2018), and Brito et al specifically focus on modeling autism spectrum disorder (Brito, et al., 2018).

The use of NSCs as a treatment strategy in CNS disease and injury has been tested for decades. Parkinsons disease specifically has gained the most momentum for potential therapeutic benefits (Studer, 2017); however, similar work has been performed in Huntingtons disease, stroke, and following spinal cord injury [for a review on this topic, see (Vishwakarma, et al., 2014)]. In some of these paradigms, NSCs are expected to differentiate into a specific cell type in the local CNS environment; in other cases, they are in a supportive role. In this issue, Kameda et al explores progress in using NSCs as a therapy following spinal cord injury (Kameda, et al., 2018).

While the development of PSC technologies has been a scientific breakthrough for future studies, there are limitations and risks that may be associated with their use. ESCs, iPSCs, and their differentiated NSCs are dividing cells. Either transplantation of NSCs or in vivo reprogramming of endogenous cells into NSCs could lead to tumorigenesis. In addition, reprogramming somatic cells into iPSCs results in a loss of some epigenetic signatures of disease and aging which are critical for disease modeling (Mertens, et al., 2015, Miller, et al., 2013, Ocampo, et al., 2016). In recent years, direct reprogramming of fibroblasts or other cell types into induced neurons (iN) has been developed (for review see (Mertens, et al., 2016)). Remarkably a growing number of studies have demonstrated that such direct reprogramming also can be effective in vivo. Wang et al [(Wang and Zhang, 2018) in this issue] will summarize recent progress of in vivo reprogramming into new neurons and present how this method can be used for spinal cord injury.

In cellular reprogramming, the cells targeted and the genetic factors used vary; however, the biggest difference is that some protocols push cells through a NSC stage, whereas others skip these stages (Gascon, et al., 2017, Guo, et al., 2014, Wang, et al., 2016). Bypassing this developmental stage has both pros and cons, and may lead to a completely novel path towards lineage commitment [discussed by Falk and Karow (Falk and Karow, 2018) in this issue].

NSCs are fascinating and promising cells because of their capability, flexibility, and multiplicity. Understanding how NSCs function provides important knowledge in development, adaptation, disease, regeneration, and rehabilitation of the nervous system. The studies of cortical development and adult neurogenesis using rodent models have contributed significantly to our knowledge about NSCs and will continually yield important new information, taking advantage of novel genetic and imaging technologies. However, using human NSCs provides us with a window to investigate human-specific aspects of development and disease mechanisms, which is potentiated by the fast advancement of stem cell and gene editing technologies. Challenges still remain regarding cell lineage control, in vivo NSC behavior, three dimensional cellular interactions, and preservation of epigenetic and aging marks.

We thank Klaus Unsicker for his encouragement and support and Jutta Jger for her help with invitations, and communications with authors and reviewers. This work was supported by grants from the US National Institutes of Health (R01MH078972, R56MH113146, R21NS098767, and R21NS095632 to X.Z, U54HD090256 to the Waisman Center), University of Wisconsin (UW)-Madison Vilas Trust (Kellett Mid-Career Award to X.Z.) and UW-Madison and Wisconsin Alumni Research Foundation (WARF to X.Z.), Jenni and Kyle Professorship (to X.Zhao), a Sloan Research Fellowship (to D.L.M.), a Junior Faculty Grant from the American Federation for Aging Research (to D.L.M.), and startup funds from UW-Madison School of Medicine and Public Health, WARF, and the Neuroscience Department (to D.L.M.).

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Neural stem cells: developmental mechanisms and disease modeling

Stem cell technology will transform medical practice. While stem cell research has already elucidated many basic disease mechanisms, the promise of stem cellbased therapies remains largely unrealized. In this review, we begin with an overview of different stem cell types. Next, we review the progress in using stem cells for regenerative therapy. Last, we discuss the risks associated with stem cellbased therapies.

There are three major types of stem cells as follows: adult stem cells (also called tissue-specific stem cells), embryonic stem (ES) cells, and induced pluripotent stem (iPS) cells.

A majority of adult stem cells are lineage-restricted cells that often reside within niches of their tissue of origin. Adult stem cells are characterized by their capacity for self-renewal and differentiation into tissue-specific cell types. Many adult tissues contain stem cells including skin, muscle, intestine, and bone marrow (Gan et al, 1997; Artlett et al, 1998; Matsuoka et al, 2001; Coulombel, 2004; Humphries et al, 2011). However, it remains unclear whether all adult organs contain stem cells. Adult stem cells are quiescent but can be induced to replicate and differentiate after tissue injury to replace cells that have died. The process by which this occurs is poorly understood. Importantly, adult stem cells are exquisitely tissue-specific in that they can only differentiate into the mature cell type of the organ within which they reside (Rinkevich et al, 2011).

Thus far, there are few accepted adult stem cellbased therapies. Hematopoietic stem cells (HSCs) can be used after myeloablation to repopulate the bone marrow in patients with hematologic disorders, potentially curing the underlying disorder (Meletis and Terpos, 2009; Terwey et al, 2009; Casper et al, 2010; Hill and Copelan, 2010; Hoff and Bruch-Gerharz, 2010; de Witte et al, 2010). HSCs are found most abundantly in the bone marrow, but can also be harvested at birth from umbilical cord blood (Broxmeyer et al, 1989). Similar to the HSCs harvested from bone marrow, cord blood stem cells are tissue-specific and can only be used to reconstitute the hematopoietic system (Forraz et al, 2002; McGuckin et al, 2003; McGuckin and Forraz, 2008). In addition to HSCs, limbal stem cells have been used for corneal replacement (Rama et al, 2010).

Mesenchymal stem cells (MSCs) are a subset of adult stem cells that may be particularly useful for stem cellbased therapies for three reasons. First, MSCs have been isolated from a variety of mesenchymal tissues, including bone marrow, muscle, circulating blood, blood vessels, and fat, thus making them abundant and readily available (Deans and Moseley, 2000; Zhang et al, 2009; Lue et al, 2010; Portmann-Lanz et al, 2010). Second, MSCs can differentiate into a wide array of cell types, including osteoblasts, chondrocytes, and adipocytes (Pittenger et al, 1999). This suggests that MSCs may have broader therapeutic applications compared to other adult stem cells. Third, MSCs exert potent paracrine effects enhancing the ability of injured tissue to repair itself. In fact, animal studies suggest that this may be the predominant mechanism by which MSCs promote tissue repair. The paracrine effects of MSC-based therapy have been shown to aid in angiogenic, antiapoptotic, and immunomodulatory processes. For instance, MSCs in culture secrete hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1), and vascular endothelial growth factor (VEGF) (Nagaya et al, 2005). In a rat model of myocardial ischemia, injection of human bone marrow-derived stem cells upregulated cardiac expression of VEGF, HGF, bFGF, angiopoietin-1 and angiopoietin-2, and PDGF (Yoon et al, 2005). In swine, injection of bone marrow-derived mononuclear cells into ischemic myocardium was shown to increase the expression of VEGF, enhance angiogenesis, and improve cardiac performance (Tse et al, 2007). Bone marrow-derived stem cells have also been used in a number of small clinical trials with conflicting results. In the largest of these trials (REPAIR-AMI), 204 patients with acute myocardial infarction were randomized to receive bone marrow-derived progenitor cells vs placebo 37 days after reperfusion. After 4 months, the patients that were infused with stem cells showed improvement in left ventricular function compared to control patients. At 1 year, the combined endpoint of recurrent ischemia, revascularization, or death was decreased in the group treated with stem cells (Schachinger et al, 2006).

Embryonic stem cells are derived from the inner cell mass of the developing embryo during the blastocyst stage (Thomson et al, 1998). In contrast to adult stem cells, ES cells are pluripotent and can theoretically give rise to any cell type if exposed to the proper stimuli. Thus, ES cells possess a greater therapeutic potential than adult stem cells. However, four major obstacles exist to implementing ES cells therapeutically. First, directing ES cells to differentiate into a particular cell type has proven to be challenging. Second, ES cells can potentially transform into cancerous tissue. Third, after transplantation, immunological mismatch can occur resulting in host rejection. Fourth, harvesting cells from a potentially viable embryo raises ethical concerns. At the time of this publication, there are only two ongoing clinical trials utilizing human ES-derived cells. One trial is a safety study for the use of human ES-derived oligodendrocyte precursors in patients with paraplegia (Genron based in Menlo Park, California). The other is using human ES-derived retinal pigmented epithelial cells to treat blindness resulting from macular degeneration (Advanced Cell Technology, Santa Monica, CA, USA).

In stem cell research, the most exciting recent advancement has been the development of iPS cell technology. In 2006, the laboratory of Shinya Yamanaka at the Gladstone Institute was the first to reprogram adult mouse fibroblasts into an embryonic-like cell, or iPS cell, by overexpression of four transcription factors, Oct3/4, Sox2, c-Myc, and Klf4 under ES cell culture conditions (Takahashi and Yamanaka, 2006). Yamakana's pioneering work in cellular reprogramming using adult mouse cells set the foundation for the successful creation of iPS cells from adult human cells by both his team (Takahashi et al, 2007) and a group led by James Thomson at the University of Wisconsin (Yu et al, 2007). These initial proof of concept studies were expanded upon by leading scientists such as George Daley, who created the first library of disease-specific iPS cell lines (Park et al, 2008). These seminal discoveries in the cellular reprogramming of adult cells invigorated the stem cell field and created a niche for a new avenue of stem cell research based on iPS cells and their derivatives. Since the first publication on cellular reprogramming in 2006, there has been an exponential growth in the number of publications on iPS cells.

Similar to ES cells, iPS cells are pluripotent and, thus, have tremendous therapeutic potential. As of yet, there are no clinical trials using iPS cells. However, iPS cells are already powerful tools for modeling disease processes. Prior to iPS cell technology, in vitro cell culture disease models were limited to those cell types that could be harvested from the patient without harm usually dermal fibroblasts from skin biopsies. However, mature dermal fibroblasts alone cannot recapitulate complicated disease processes involving multiple cell types. Using iPS technology, dermal fibroblasts can be de-differentiated into iPS cells. Subsequently, the iPS cells can be directed to differentiate into the cell type most beneficial for modeling a particular disease process. Advances in the production of iPS cells have found that the earliest pluripotent stage of the derivation process can be eliminated under certain circumstances. For instance, dermal fibroblasts have been directly differentiated into dopaminergic neurons by viral co-transduction of forebrain transcriptional regulators (Brn2, Myt1l, Zic1, Olig2, and Ascl1) in the presence of media containing neuronal survival factors [brain-derived neurotrophic factor, neurotrophin-3 (NT3), and glial-conditioned media] (Qiang et al, 2011). Additionally, dermal fibroblasts have been directly differentiated into cardiomyocyte-like cells using the transcription factors Gata4, Mef2c, and Tb5 (Ieda et al, 2010). Regardless of the derivation process, once the cell type of interest is generated, the phenotype central to the disease process can be readily studied. In addition, compounds can be screened for therapeutic benefit and environmental toxins can be screened as potential contributors to the disease. Thus far, iPS cells have generated valuable in vitro models for many neurodegenerative (including Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis), hematologic (including Fanconi's anemia and dyskeratosis congenital), and cardiac disorders (most notably the long QT syndrome) (Park et al, 2008). iPS cells from patients with the long QT syndrome are particularly interesting as they may provide an excellent platform for rapidly screening drugs for a common, lethal side effect (Zwi et al, 2009; Malan et al, 2011; Tiscornia et al, 2011). The development of patient-specific iPS cells for in vitro disease modeling will determine the potential for these cells to differentiate into desired cell lineages, serve as models for investigating the mechanisms underlying disease pathophysiology, and serve as tools for future preclinical drug screening and toxicology studies.

Despite substantial improvements in therapy, cardiovascular disease remains the leading cause of death in the industrialized world. Therefore, there is a particular interest in cardiovascular regenerative therapies. The potential of diverse progenitor cells to repair damaged heart tissue includes replacement (tissue transplant), restoration (activation of resident cardiac progenitor cells, paracrine effects), and regeneration (stem cell engraftment forming new myocytes) (Codina et al, 2010). It is unclear whether the heart contains resident stem cells. However, experiments show that bone marrow mononuclear cells (BMCs) can repair myocardial damage, reduce left ventricular remodeling, and improve heart function by myocardial regeneration (Hakuno et al, 2002; Amado et al, 2005; Dai et al, 2005; Schneider et al, 2008). The regenerative capacity of human heart tissue was further supported by the detection of the renewal of human cardiomyocytes (1% annually at the age of 25) by analysis of carbon-14 integration into human cardiomyocyte DNA (Bergmann et al, 2009). It is not clear whether cardiomyocyte renewal is derived from resident adult stem cells, cardiomyocyte duplication, or homing of non-myocardial progenitor cells. Bone marrow cells home to the injured myocardium as shown by Y chromosome-positive BMCs in female recipients (Deb et al, 2003). On the basis of these promising results, clinical trials in patients with ischemic heart disease have been initiated primarily using bone marrow-derived cells. However, these small trials have shown controversial results. This is likely due to a lack of standardization for cell harvesting and delivery procedures. This highlights the need for a better understanding of the basic mechanisms underlying stem cell isolation and homing prior to clinical implementation.

Although stem cells have the capacity to differentiate into neurons, oligodendrocytes, and astrocytes, novel clinical stem cellbased therapies for central and peripheral nervous system diseases have yet to be realized. It is widely hoped that transplantation of stem cells will provide effective therapy for Parkinson's disease, Alzheimer's disease, Huntington's Disease, amyloid lateral sclerosis, spinal cord injury, and stroke. Several encouraging animal studies have shown that stem cells can rescue some degree of neurological function after injury (Daniela et al, 2007; Hu et al, 2010; Shimada and Spees, 2011). Currently, a number of clinical trials have been performed and are ongoing.

Dental stem cells could potentially repair damaged tooth tissues such as dentin, periodontal ligament, and dental pulp (Gronthos et al, 2002; Ohazama et al, 2004; Jo et al, 2007; Ikeda et al, 2009; Balic et al, 2010; Volponi et al, 2010). Moreover, as the behavior of dental stem cells is similar to MSCs, dental stem cells could also be used to facilitate the repair of non-dental tissues such as bone and nerves (Huang et al, 2009; Takahashi et al, 2010). Several populations of cells with stem cell properties have been isolated from different parts of the tooth. These include cells from the pulp of both exfoliated (children's) and adult teeth, the periodontal ligament that links the tooth root with the bone, the tips of developing roots, and the tissue that surrounds the unerupted tooth (dental follicle) (Bluteau et al, 2008). These cells probably share a common lineage from neural crest cells, and all have generic mesenchymal stem cell-like properties, including expression of marker genes and differentiation into mesenchymal cells in vitro and in vivo (Bluteau et al, 2008). different cell populations do, however, differ in certain aspects of their growth rate in culture, marker gene expression, and cell differentiation. However, the extent to which these differences can be attributed to tissue of origin, function, or culture conditions remains unclear.

There are several issues determining the long-term outcome of stem cellbased therapies, including improvements in the survival, engraftment, proliferation, and regeneration of transplanted cells. The genomic and epigenetic integrity of cell lines that have been manipulated in vitro prior to transplantation play a pivotal role in the survival and clinical benefit of stem cell therapy. Although stem cells possess extensive replicative capacity, immune rejection of donor cells by the host immune system post-transplantation is a primary concern (Negro et al, 2012). Recent studies have shown that the majority of donor cell death occurs in the first hours to days after transplantation, which limits the efficacy and therapeutic potential of stem cellbased therapies (Robey et al, 2008).

Although mouse and human ES cells have traditionally been classified as being immune privileged, a recent study used in vivo, whole-animal, live cell-tracing techniques to demonstrate that human ES cells are rapidly rejected following transplantation into immunocompetent mice (Swijnenburg et al, 2008). Treatment of ES cell-derived vascular progenitor cells with inter-feron (to upregulate major histocompatibility complex (MHC) class I expression) or in vivo ablation of natural killer (NK) cells led to enhanced progenitor cell survival after transplantation into a syngeneic murine ischemic hindlimb model. This suggests that MHC class I-dependent, NK cell-mediated elimination is a major determinant of graft survivability (Ma et al, 2010). Given the risk of rejection, it is likely that initial therapeutic attempts using either ES or iPS cells will require adjunctive immunosuppressive therapy. Immunosuppressive therapy, however, puts the patient at risk of infection as well as drug-specific adverse reactions. As such, determining the mechanisms regulating donor graft tolerance by the host will be crucial for advancing the clinical application of stem cellbased therapies.

An alternative strategy to avoid immune rejection could employ so-called gene editing. Using this technique, the stem cell genome is manipulated ex vivo to correct the underlying genetic defect prior to transplantation. Additionally, stem cell immunologic markers could be manipulated to evade the host immune response. Two recent papers offer alternative methods for gene editing. Soldner et al (2011) used zinc finger nuclease to correct the genetic defect in iPS cells from patients with Parkinson's disease because of a mutation in the -Synuclein (-SYN) gene. Liu et al (2011) used helper-dependent adenoviral vectors (HDAdV) to correct the mutation in the Lamin A (LMNA) gene in iPS cells derived from patients with HutchinsonGilford Progeria (HGP), a syndrome of premature aging. Cells from patients with HGP have dysmorphic nuclei and increased levels of progerin protein. The cellular phenotype is especially pronounced in mature, differentiated cells. Using highly efficient helper-dependent adenoviral vectors containing wild-type sequences, they were able to use homologous recombination to correct two different Lamin A mutations. After genetic correction, the diseased cellular phenotype was reversed even after differentiation into mature smooth muscle cells. In addition to the potential therapeutic benefit, gene editing could generate appropriate controls for in vitro studies.

Finally, there are multiple safety and toxicity concerns regarding the transplantation, engraftment, and long-term survival of stem cells. Donor stem cells that manage to escape immune rejection may later become oncogenic because of their unlimited capacity to replicate (Amariglio et al, 2009). Thus, ES and iPS cells may need to be directed into a more mature cell type prior to transplantation to minimize this risk. Additionally, generation of ES and iPS cells harboring an inducible kill-switch may prevent uncontrolled growth of these cells and/or their derivatives. In two ongoing human trials with ES cells, both companies have provided evidence from animal studies that these cells will not form teratomas. However, this issue has not been thoroughly examined, and enrolled patients will need to be monitored closely for this potentially lethal side effect.

In addition to the previously mentioned technical issues, the use of ES cells raises social and ethical concerns. In the past, these concerns have limited federal funding and thwarted the progress of this very important research. Because funding limitations may be reinstituted in the future, ES cell technology is being less aggressively pursued and young researchers are shying away from the field.

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The benefits and risks of stem cell technology - PMC