3.7 Stem cells and regenerative medicine 281

3.7 Stem cells and regenerative medicine 281

ESSENTIALS There is a great and unmet need for treatments that will deliver re- storative solutions to patients with diseases hitherto considered ir- reparable. Advances in human pluripotent stem cell biology and gene-​editing technology offer unprecedented opportunities for both drug discovery and translational therapies that will likely herald a new chapter of regenerative and personalized medicine. Requirements for regenerative therapy A prerequisite for any regenerative therapy is the generation of scal- able and enriched numbers of defined cell types appropriate to the target condition. Preclinical work-​up requires demonstration of sustained stem-​cell mediated functional recovery in appropriate models of injury. General principles include the need for ensuring appropriate distribution, connectivity, survival, and functional inte- gration of stem cells in the context of injury, without the hazards of tumour generation or immune rejection. Efficacy demonstrated in early phase II trials needs to be extended to the demonstration of sustained clinical benefit in definitive phase III studies. How might the promise of stem cells be realized? Consideration of three major target conditions for regenerative medicine—​Parkinson’s disease, heart failure, and diabetes mellitus—​ emphasizes distinct and common challenges that must be overcome in order to realize the stem cell promise. Novel approaches to induce pluripotency from differentiated somatic cells and targeted genetic manipulation of stem cell populations, along with new insights de- rived from improved understanding of human pluripotent stem cell biology and increased recognition of endogenous stem cells, offers a range of mechanisms through which stem cells may be therapeutic. In addition to classic cell/​tissue replacement approaches, creation of disease models using human, potentially patient-​specific, pluripotent stem cell systems, and linked high-​throughput cell drug screening plat- forms offers hope for accelerated discovery of new targets and medicines. When will stem cell treatments become available? This will vary from disease to disease. The history of haematological stem cell medicine, from which much of the template of regenerative medicine is borrowed, suggests an incremental and combinatorial approach to treatment. Introduction Regenerative medicine is not a new discipline: the 1990 Nobel Prize in Physiology and Medicine to Joseph Murray and Donnall Thomas was in recognition of pioneering kidney and bone marrow trans- plantations undertaken in the 1950s. The surge of renewed interest has been catalysed by recent and rapid advances in human stem cell biology and gene technology, which offer the prospect of the development of novel reparative strategies for a host of diseases hitherto considered irreparable. These include diabetes mellitus, neurodegenerative diseases, and heart failure. Stem cells The human body is organized into discrete but interrelated organs and tissues that each contain differentiated or specialized functional cells. Stem cells are defined as cells that possess three functional characteristics: an immature phenotype, self-​renewal capacity, and the ability to differentiate into one or more functional or specialized derivatives (Fig. 3.7.1). The first or earliest stem cell is the embryonic stem cell (ESC) that arises from the epiblast (Fig. 3.7.2). Embryonic stem cells are pluripotent cells capable of generating all cell types in the body and can be considered as transient stem cells. During development and through adulthood other stem cells emerge that display progres- sively more restricted phenotypical range and can be considered tissue-​ or organ-​specific. Endogenous tissue-​specific stem cells are multipotent, with a differentiation repertoire normally confined to those cells of the tissue of origin. They persist through adulthood and are responsible for regenerating tissues with a rapid cell turn- over, such as the gastrointestinal tract epithelium, skin epidermis, and haematopoietic system. Stem cells have also been identified in relatively quiescent tissues including the heart and central nervous system, where their precise functional role has yet to be determined. Technical advances enabling long-​term ex vivo culture of human-​ derived pluripotent and adult tissue-​specific stem cells, along with increased recognition of endogenous adult stem cells and the pos- sibility of directed reprogramming and targeted gene editing, have generated intense excitement in the experimental and therapeutic potential of human stem cell biology. 3.7 Stem cells and regenerative medicine Alexis J. Joannides, Bhuvaneish T. Selvaraj, and Siddharthan Chandran

282 SECTION 3  Cell biology (a) (b) Pluripotent stem cell Multipotent tissue stem cell Transit-amplifying progenitor Terminally differentiated functional progeny Embryonic stem cells Fetal tissue stem cells Adult tissue stem cells iPS cells Safety Cell yield Plasticity Histocompatibility potential Ethical acceptability Fig. 3.7.1  Stem cells and their sources. (a) All stem cells, irrespective of developmental stage, share two fundamental properties: self-​renewal and differentiation to progressively lineage-​restricted cell types, ultimately generating terminally differentiated, functional progeny. (b) Human stem cells can be derived from embryonic, fetal, or adult tissue. Each has its relative merits and drawbacks, and choosing the most appropriate source largely depends on the requirements of each specific experimental or therapeutic context.

3.7  Stem cells and regenerative medicine 283 Historical perspective It has long been known that the cells in certain tissues are constantly replaced, but it is only recently that we have come to realize the number of these areas, the many ways by which a balance is achieved between cell production and loss, and particularly the speed of the renewal process. The remarkable behaviour of many cell popula- tions raises not only histological but biochemical questions which are yet unanswered. The concept of tissue stem cells emerged from the pioneering work of Charles Leblond, James Till, and Ernest McCullogh in the mid-​20th century. Leblond developed the technique of autoradi- ography which led to the identification of continuous cellular self-​ renewal in certain tissues and culminated in the description of what we now know as stem cell-​mediated renewal in spermatogenesis. Till and McCullogh independently proposed a similar model for haematopoiesis. During the 1960s they identified ‘spleen colony-​ forming cells’, which were able to reconstitute the haematopoietic system of a lethally irradiated animal host, and together with Louis Siminovitch went on to demonstrate their self-​renewal capacity by serial transplantation. Subsequent advances in stem cell biology have enabled iden- tification, propagation and directed differentiation of stem cells from a variety of adult tissues, including bone marrow stroma, skin epidermis, and brain (Fig. 3.7.3). Parallel pioneering studies on embryonic carcinoma cells derived from teratocarcinomas led to the isolation of embryonic stem cells from mouse blastocysts in 1981. Recognition of the fundamental advance of this finding and the enabling of the gene modification era led to the Nobel Prize in Medicine 2007. Successful isolation of human ESC cells, coupled with the discovery of a comparatively simple technique to induce pluripotency from adult differentiated cells, opened up the field of regenerative stem cell-​applied biology to include the possibility of generating patient-​specific cells or tissues through induced pluripotency. John Gurdon and Shunya Yamanaka were awarded the 2012 Nobel Prize for Medicine for showing that mature cells can be reprogrammed to become pluripotent. Complementing progress in stem cell generation, recent advances in targeted gene-​ editing technology have significantly increased the repertoire of stem cell-​based therapeutic possibilities. What can human stem cells offer
regenerative medicine? Experimental and therapeutic opportunities are the short an- swer. Regenerative medicine can be summarized as treatments (cell and drug based) that seek to restore structure and function following injury (Fig. 3.7.4a). Stem cells can achieve this goal in a variety of ways, direct and indirect (Fig. 3.7.4b). Perhaps the simplest and most intuitive therapeutic contribution is through cell replacement of lost or damaged cells. Cultured autologous keratinocytes for skin loss is a well-​established current example of cell-​based therapy. Cell replacement therapies for Parkinson’s disease and type 1 diabetes represent future therapeutic targets. Cell-​based therapies have also been demonstrated to have benefi- cial effects independent of their differentiation potential, such as the immunomodulatory effects of mesenchymal stem cells in the treatment of graft versus host disease. Beyond stem cells as direct therapy, human stem cells offer com- plementary opportunities to study human development and model disease, as well as providing a unique resource for drug discovery and testing. Such insights are likely to lead to novel disease-​modifying and regenerative therapies, and ultimately provide the largest clin- ical dividend. ESC Oct4 Nestin Vimentin Nanog Fetal NSC Adult MSC iPSC Fig. 3.7.2  Human stem cells grown in vitro. Representative light microscopy and immune micrograph pictures of (from left to right) embryonic stem cells (ESC), fetal-​derived neural stem cells (NSC), adult skin-​derived mesenchymal stem cells (MSC), and induced pluripotent stem cells (IPSC).

284 SECTION 3  Cell biology 1905—first successful keratoplastry using cadaveric corneal tissue 1954—first successful kidney transplant between monozygotic twins 1957—first successful bone marrow transplant by intravenous infusion 1981—autologous keratinocytes used to treat third-degree burns 1990—transplantation of primary fetal tissue for Parkinson’s disease 1997—autologous limbal stem cells used for corneal grafting 1998—trials of autologous chondrocyte implantation in knee injury 2004—trials of autologous bone marrow infusion following myocardial infarction 2000—first successful pancreatic islet cell transplant from cadaveric donor tissue 2002—trials of autologous mesenchymal stem cells for graft vs host disease 1954—demonstration of continuous tissue self- renewal using autoradiography 1962—cloning by somatic cell nuclear transfer in Xenopus laevis 1963—discovery of endogenous stem-cell mediated self-renewal 1970—isolation and expansion of bone marrow stromal cells 1975—isolation and expansion of epidermal keratinocytes 1981—isolation and expansion of embryonic stem cell lines from mouse blastocysts 1992—isolation and expansion of neural stem cells from the embryonic and adult brain 2006—generation of induced pluripotent stem cells from adult tissue 1997—mammalian cloning in sheep by somatic cell nuclear transfer 1998—generation of embryonic stem cell lines from human blastocysts 2007—generation of primate embryonic stem cell lines by somatic cell nuclear transfer 1900 1950 1970 1990 2000 2010 Future prospects Implantation of β-cells, cardiomyocytes, and dopaminergic neurones from pluripotent cells Implantation of stem cell-derived retinal pigment epithelium for macular degeneration Oligodendrocyte precursor transplantation for spinal cord injury and multiple sclerosis Tissue protection trials for stroke, motor neurone disease, and Alzheimer’s disease Development of drug compounds from stem- cell-based screening Stem-cell-based immunotherapy for cancer and infectious diseases Stem-cell-derived organ transplantation Stem cell biology Regenerative medicine Short term Medium to long term 2014—clinical trials of human embryonic stem cell-derived β-cell in type I diabetes 2009—generation of integration-free induced pluripotent stem cells 2013—reproducible targeted gene editing using CRISPR/Cas9 system 2016—clinical improvement following cultured bone marrow cell injection in heart failure 2012—transplantation of embryonic stem-cell derived retinal pigment epithelium cells for AMD Fig. 3.7.3  Timeline of key advances and future prospects in stem cell biology and regenerative medicine.

3.7  Stem cells and regenerative medicine 285 Inadequate endogenous stem-cell-mediated repair Normal tissue Disease tissue Cell loss/dysregulation (a) (b) Pluripotent stem cells Multipotent tissue stem cells Target cell type Exogenous cell replacement Tissue protection Promoting endogenous repair Damaged tissue In vitro disease modelling and drug testing ± gene editing Fig. 3.7.4  Therapeutic principles of stem cell-​based treatments. (a) Organ function is dependent on a dynamic equilibrium between the extent of pathological injury (from any cause) and the extent of self-​repair from endogenous tissue stem cells (which is highly variable between organs). An imbalance leads to progressive tissue damage and/​or loss, ultimately resulting in organ impairment and functional decompensation. (b) Stem cell-​based interventions can be directed towards multiple points in disease progression. Stem cells and progenitors, delivered as single cells or within a tissue scaffold may have a disease-​modifying effect independent of differentiation potential through trophic support or immunomodulatory properties, while differentiated progeny can be used to replace lost cells. In addition, in vitro stem cell-​based studies can lead to the development of drug compounds for mobilizing endogenous stem cells and shifting the organ equilibrium towards self-​repair.

286 SECTION 3  Cell biology Current therapeutic applications of stem cells Not infrequently in medical discovery, application of scientific in- novation precedes biological or mechanistic understanding. Stem cells are no exception. Within the translational arena, stem cell transplantation has been performed (even unknowingly) for over a century (Fig. 3.7.3). Eduard Zirm carried out the first successful keratoplasty in 1905, while E. Donnall Thomas performed the first successful bone marrow transplantation by intravenous infusion in 1957. Haemopoietic stem cell transplantation for haematological disease is now routine procedure (see Chapter 22.8.2). Rheinwald and Green’s success in using autologous, ex vivo, ex- panded human keratinocytes for treating patients with third-​degree burns in 1981 established an important proof of concept for stem cell-​based therapy. The potential of regenerative therapy is apparent from the process of autologous epidermal grafting. Keratinocytes, although relatively quiescent in vivo, can be expanded exponentially in culture with a doubling time of 16 to 18 hours, achieving a 10 000-​ fold expansion over a 2-​ to 3-​week period. Sufficient cell numbers can thus be obtained from very small full-​thickness skin biopsies, making it possible to treat patients with large area skin loss where split-​thickness skin grafts are not feasible. Together with autologous chondrocyte implantation for articular cartilage defects, this is a fast-​growing area that is now mainstream. Use of limbal epithelial stem cells for corneal disease is a further example of an emerging clinical application of autologous adult stem cells. Finally, combination of autologous material with the disciplines of material science and biotissue engineering raises the imminent prospect of ex vivo tissue organogenesis. Use of tissue-​ engineered bladder augmentation for neurogenic bladders is an exciting advance and likely to herald wider application (see later). Current barriers to clinical application Clinical application of stem cells has now become routine practice in haematology, plastic surgery, orthopaedics, and ophthalmology. However, beyond these areas the promise of using stem cells in mainstream therapies remains anticipated and unrealized, and raises many issues. Although comprehensive and detailed analysis of individual disease requirements is beyond the scope of this chapter, some general themes for clinical application of stem cell-​based re- generative medicine emerge (Fig. 3.7.5). These include: 1. Identifying the correct stem cell source 2. Generating appropriate numbers of specialized cells and valid- ating sustained in vivo function in injury models 3. Establishing the infrastructure and correct trial design methodo­ logy to rigorously clinically evaluate putative regenerative therapies These separate areas are considered by way of illustration and in ref- erence to three principal target medical conditions that could benefit from regenerative medicine:  neurodegenerative diseases such as Parkinson’s disease, cardiac failure, and type 1 diabetes mellitus. Identifying the correct human stem cell source Accepting the need for human material, this is in many ways an issue of determining the appropriate developmental stage of stem cells. Adult tissue-​specific stem cells possess some advantage, being poten- tially autologous, often readily accessible, as well as being ethically less controversial (see Fig. 3.7.1b). However, their limited prolifera- tive capacity and restricted differentiation potential place significant practical constraints on their widespread utility. Although several studies have reported adult stem cell ‘transdifferentiation’ to other lineages, these findings have not always been reproducible and are likely accounted for by alternative explanations such as cell fusion in vivo or genetic transformation in vitro. Notwithstanding their intuitive attraction, some populations (e.g. neural stem cells) are inaccessible and would require invasive methods, with attendant risks, for harvesting. By contrast, embryonic and induced pluripo- tent stem cells are scientifically attractive on account of their unique ability to respond predictably to developmental cues, which together with their non​transformed nature and almost unlimited prolifera- tive capacity allow the realistic prospect of generating scaleable numbers of all cell types. A stem cell source should thus ideally combine the practical and ethical acceptability of adult stem cells with the biological poten- tial of embryonic or pluripotent stem cells. Successful somatic cell nuclear transfer (SCNT) in mammalian reproductive cloning dem- onstrated the conceptual feasibility of nuclear reprogramming to generate embryonic cells from an adult mammalian somatic cell source. Primate and human SCNT has since been demonstrated and independently confirmed, but a significant practical hurdle of SCNT is the need for large numbers of oocytes. An alternative approach proposes fusion of existing embryonic stem cell lines and dermal fibroblasts, and a key milestone in this field has been the demonstration that somatic (non​stem) cell repro- gramming can be induced by overexpression of a limited number of transcription factors in both adult mouse and human systems. The resulting induced pluripotent stem cells (iPS) show many of the characteristics of embryonic stem cells, and ongoing work on the underlying mechanisms of somatic reprogramming has improved the efficiency and reliability of the process. This approach to ‘re- programming’ opens up the possibility of an era of patient-​specific stem cell-​based studies and conceivably even personalized ‘stem cell’ medicine. Generating appropriate numbers of functional cell type(s) There are two overriding requirements: generation of the correct functional cell type without any contaminant undifferentiated stem cell(s). Although it is axiomatic that cells of appropriate regional identity and function are required for experimental or therapeutic applica- tion, directed differentiation has lagged behind advances in stem cell isolation and culture. Pluripotent stem cells possess a clear advantage over (non-​reprogrammed) adult stem cells, responding predictably to developmental signals and retaining imposed positional identity following transplantation. It is worth noting that most cell types in future regenerative therapies, including neuronal subtypes, pancre- atic islet β cells, and cardiomyocytes, physiologically emerge at a defined developmental stage and are not normally generated from resident adult stem cell populations. It is unclear whether any other stem cell populations have the in vitro or in vivo potential to generate these functional cell types. Nevertheless, applying insights borrowed from developmental principles of patterning and specification are

3.7  Stem cells and regenerative medicine 287 Derivation Expansion Ethical acceptability Differentiation Route, dose ± co- treatment Functional improvement Correct disease stage and subtype Develop accurate disease models Potentially autologous Scaleable proliferation while retaining plasticity Clinical grade conditions Homogeneous population Correct physiological function Preclinical studies Demonstrate functional recovery Bioengineering Clinical evaluation Patient selection Tissue scaffolds Encapsulation In vitro organogenesis Outcome measures Final product Genetic editing * Fig. 3.7.5  Criteria for clinical implementation of cell-​based therapy. The many challenges and requirements for cell-​based strategies can be classified into three fundamental steps (see text for a detailed discussion). Initially, the right cell type needs to be generated in sufficient numbers, in high purity and in the right form for therapy (step 1, shaded in yellow). Subsequently, putative therapies need to be tested in appropriate animal models of disease for both safety and efficacy, and criteria for patient phenotypes most likely to benefit from therapy should be established (step 2, shaded in pink). Finally, clinical cell therapy needs to be evaluated in the context of other existing therapies, and functional improvement should be monitored for a sufficient period to demonstrate benefits in disease morbidity and/​or mortality (step 3, shaded in blue).

288 SECTION 3  Cell biology likely to be critical of the generation of region-​specific cell types re- gardless of age or source of origin of stem cell. In addition to the conventional application of soluble factors in two-​dimensional culture, the development of advanced tissue engineering approaches can potentially enable better definition of the extracellular microenvironment. Thus, novel techniques such as micropatterning, biomaterial scaffolds, and bioprinting have the po- tential to further enhance cell differentiation potential, yield, and long-​term survival. Minimizing the risk of tumorigenic ‘rogue’ cells is a major obs- tacle. Potential approaches include a combination of positive and negative ex vivo selection techniques, predifferentiation, insertion of inducible (preferably pluripotency-​associated) ‘suicide’ genes, or use of oncolytic viruses. Such methods will require custom- ized developments particular to individual stem cells. Although standard practice in a laboratory setting, clinical application will require further refinement. While teratoma incidence has been well controlled in recent cell-​based trials, the increased risk of leukaemia following gene therapy for X-​linked severe combined immunodeficiency highlights the need for robust long-​term evalu- ation in a clinical setting. Donor cell developmental stage The final stem cell-​derived product can be either a progenitor popu- lation or ex vivo predifferentiated cells. Predifferentiation has the additional challenge of further controlled differentiation steps with complex protocols utilizing a combination of soluble factors and biological scaffolds, and using specialized selection techniques to isolate a possibly rarer differentiated subtype (e.g. separating dif- ferentiating mature β cells from islet progenitors from other endo- crine cell types). The importance of the local cellular niche in cell fate subspecification also adds to the complexity of ex vivo differenti- ation. However, predifferentiation may prove necessary, particularly where the potential pathological host environment may otherwise impose inappropriate differentiation cues upon implanted progen- itors. For example, the inflammatory environment in spinal cord demyelination models has been shown to promote astrocyte spe- cification from neural precursors, while prior ex vivo oligodendro- cyte lineage specification enables effective exogenous remyelination. Predifferentiation is also a method to reduce the risk of uncontrolled in vivo proliferation. Gene editing as a means of introducing novel cellular functions Stem cell manipulation ex vivo beyond directed differentiation can offer additional opportunities for introducing novel functions that are not usually present in the unmodified cell type. While traditional methods for targeted gene editing with homologous recombination have been costly and inefficient, recent advances in this area have made the scaleable use of genetically-​edited stem cells a therapeutic possibility. New techniques for gene targeting include the use of transcrip- tion activator-​like effector nucleases (TALENs—​nucleases fused to a synthetic protein binding to a specific DNA base sequence), and clustered regularly interspaced short palindromic repeats (CRISPR) together with CRISPR-​associated proteins (Cas). For example, the Type II CRISPR/​Cas9 system enables a double strand break in the genome by the Cas9 nuclease at a specific locus determined by complementary RNA sequences (crRNA and tracrRNA which can be combined into a single chimeric sgRNA molecule). While the host cell predominantly repairs the double strand break by non​homologous end joining, its highly error-​prone nature leads to disruption of the target gene, resulting in a gene knock-​out. As introduction of a double strand break at the target locus can also increase the efficiency of homologous recombination, introducing donor DNA containing a novel sequence provides the opportunity for recombination with cleaved sequences via homology directed repair, thus resulting in a gene knock-​in or correction. A key advan- tage of the CRISPR/​Cas9 system is the ability to change the target specificity by altering the RNA sequence, which is cheap and rapidly synthesized compared to synthesis of a new protein. Potential therapeutic possibilities of such technologies include the correction of genetic defects in stem cells (multipotent or iPS) which can then be autologously transplanted back into their donor. More novel targets include conferring host resistance to infectious disease (e.g. CCR5 receptor deletion for resistance to HIV), somatic gene transfer in case of cystic fibrosis, and cancer immunotherapy using engineered T cells (e.g. chimeric antigen receptor-​modified T cells in acute lymphoblastic leukaemia). Recent progress using animal models have shown potentials of in vivo gene therapy using CRISPR-​Cas9. Adeno associated virus mediated delivery of CRISPR-​Cas9 to remove mutated exon 23 of Dystrophin gene in mice with Duchene muscular dystrophy resulted in a partial restoration of muscle function. Much wider application for CRISPR-​Cas9 in gene therapy is its potential in correcting the disease-​causing genetic mutation. Disease-​causing phenotypes were rescued in mouse models of a hereditary liver disease, tyrosinemia, by homology directed repair mediated gene correction in hepatocytes. Although the efficacy of the repair was low, the gene corrected cells conferred a positive growth advantage resulting in functional rescue. However, the benefits of the wider therapeutic repertoire offered by gene editing is in part offset by the need for a higher burden of biological safety. Efficacy of gene correction, issues including DNA target sequence specificity and exclusion of off-​target gene editing will need to be demonstrated prior to clinical use. Nonetheless, the use of gene-​editing technologies to generate genetically-​bespoke cell populations for in vitro disease modelling and drug develop- ment is likely to precede their application in cell-​based therapies (see later). Particular tissue types Nervous tissue The ability to programme or direct neuroectodermal differentiation from human embryonic stem cells, and by extension iPS cells, has progressed more rapidly compared with other lineages. This reflects in part the ‘default’ nature of neural induction from pluripotent stem cells when grown in simplified conditions with limited extrinsic signalling. There are several neural differentiation protocols with the po- tential for scaleable derivation of neural stem cells under clinical grade conditions. Methods of derivation and/​or enrichment in- clude utilizing stage-​specific cell surface markers (including CD133, Notch, and β1-​integrin) for neural progenitor selection. While fur- ther differentiation of neural progenitors into astrocytes (for use in neuroprotective approaches) is relatively straightforward, the

3.7  Stem cells and regenerative medicine 289 generation of the whole range of functional region-​specific neuronal subtypes remains problematic. This is a major challenge for regenera- tive neurology given that regional identity subserves distinct physio- logical function(s) and thus absolute precision of spatial identity is a prerequisite for functional restitution. Midbrain dopaminergic and spinal cord motor neuron differentiation from human pluripotent stem cells are arguably the most advanced, and the former is primed for clinical trials in Parkinson’s disease. Derivation of other neuronal subtypes, however, has been less successful. A combination of devel- opmentally based approaches along with use of positive selection exploiting region-​specific surface markers is likely to overcome this hurdle. Cardiac tissue Attempts to generate cardiomyocytes from adult cells has been prob- lematic, and initial reports suggesting that bone marrow stromal cells and skeletal muscle satellite cells could ‘transdifferentiate’ into cardiomyocytes proved to be flawed. Early protocols utilizing pluri- potent stem cells were dependent on spontaneous differentiation or coculture with visceral endoderm cells or conditioned medium with relatively low efficiency, highlighting the need for a more ra- tional, developmentally rooted approach to differentiation. Over the last decade this approach has been highly successful, and the serial application of activin-​A and BMP-​4 has been demonstrated to re- producibly generate high yields of functional cardiomyocytes for use in preclinical studies. In parallel, the existence of defined surface markers to identify cardiac progenitors (e.g. Flk1+ CXCR4+ ) permits prospective identification and isolation for clinical use. Pancreatic tissue Cadaveric islet cell transplantation offers proof of concept of cell-​ based therapy for type 1 diabetes. The fundamental requirements are islet β-​cell generation displaying physiological glucose-​stimulated insulin secretion. Early studies on hESCs demonstrated very low rates of differen- tiation to islet β cells and several purported examples later emerged as probable culture artefacts. The multiple serial differentiation stages between pluripotent cells and β-​cells (including definitive endoderm, posterior foregut, and islet precursor) has made de- velopmentally guided protocols challenging to develop. The first report of functional β-​cell derivation from hES cells using a devel- opmental approach via sequential differentiation over an 18-​day period was reported in 2006. Since then several protocols for multi- stage differentiation have been reported, utilizing defined develop- mental cues such as activin-​A and retinoic acid. However, further protocol optimization is required to identify key steps utilizing the minimum number of factors and stages. Furthermore, although glucose-​stimulated insulin secretion has been demonstrated in generated β-​cells using glucose stimulation assays, differences have been noted when comparing such responses with primary human β-​cells. These findings could be consistent with an immature β-​cell phenotype, highlighting the need for correlation with functional outcomes (i.e. reversal of diabetes) from preclinical and clinical studies (see later). Validating stem cell-​mediated functional recovery Even when challenges in obtaining scaleable numbers of appro- priate specialized cell types have been overcome, significant barriers to clinical application remain. Foremost is the need to demonstrate, in appropriate experimental systems, restoration of lost function. Notwithstanding the reasonable view that for certain diseases (un- treatable and fatal, e.g. motor neuron disease) a lower burden of mechanistic proof is required before commencing experimental clinical trials, it remains a fundamental tenet of drug or cell medi- cine development that prior demonstration of behavioural recovery is necessary. Although welcome evidence from animal studies of in vivo stem cell-​mediated function is emerging, robust and sustained restoration of lost function remains elusive. This problem reflects in part the limitations of experimental systems in accurately modelling human disease. The challenge of restoring lost function varies according to dis- ease and the organ(s) involved, and is most severe in regenerative neurology where reconnection of circuitry is required over and above restoration of macroscopic structure. Due to the syncytial and pacing nature of myocardium, a prerequisite for regenerative car- diology is a method that ensures electrophysiological synchroniza- tion on cell implantation. By contrast, stem cell-​based therapeutics for type 1 diabetes is comparatively straightforward; restoration of endocrine function does not require homotopic transplantation, and cadaver-​derived transplantation of islet β cells into the hepatic portal vein has established proof of concept for a cell replacement strategy. Other issues Survival, engraftment, and connectivity Sustained functional integration represents the holy grail of regen- erative medicine, and is largely unmet. Clinical context matters, but some general principles can be rehearsed. Donor cell survival, acute and chronic, requires a primed host environment as well as immuno- logical mismatching to be overcome. Achieving long-​term integra- tion requires a permissive host environment. Combined approaches with, for example, immunomodulatory treatment, are one way not just to manage ongoing disease activity but also to limit donor cell vulnerability to immune attack. A common problem for treatment of autoimmune-​mediated disease is to protect the implanted cell population, including autologous material, from the host immuno- logical response. Ensuring appropriate connectivity is likely to require supple- mentary approaches (e.g. brain and spinal cord injury is associ- ated with an inhibitory glial scar that behaves as a physical and biochemical barrier to axonal growth). Approaches under clinical trial to permit appropriate axonal regrowth include cotreatment with enzymes targeting the inhibitory extracellular proteoglycan matrix, and excision of the glial scar combined with a nerve graft bridge. In cardiac repair a functionally integrated cardiac syn- cytium with appropriate excitation–​contraction coupling is a min- imal requirement without the risk of potentially fatal arrhythmias. Indeed, a recent primate study demonstrating remuscularization of myocardial infarcts by human ESC-​derived cardiomyocytes following intramyocardial delivery also noted the development of non​fatal ventricular arrhythmias. By contrast, studies reporting restoration of left ventricular function after human bone marrow cell infusion have failed to show histological integration or sus- tained improvement, with short-​term benefits most likely due to trophic support.

290 SECTION 3  Cell biology Overcoming immune rejection Stem cells allow the development of novel approaches, beyond classic immune suppression, to manage immune mismatch. Personalized cells, masking strategies, and derivation from predetermined tissue-​ matched banks of cell lines are all rational methods under study. Although a conceptually attractive method, generation of autolo- gous stem cell lines for each patient would be impractical and cost prohibitive to implement for common conditions. However, a study focusing on the United Kingdom population has estimated that as few as 10 hESC lines homozygous for common HLA haplotypes (which could be derived by iPS, SCNT, or parthenogenetic ES cells) could achieve complete HLA matching for 38%, and a beneficial match for 67% of cases. Microencapsulation in a permeable substance such as alginate or poly-​l-​ornithine is another option for creating an immunological protective barrier, with promising results in human islet transplant- ation trials. This approach is confined to grafts that do not require cell–​cell contact for function and subcutaneously implanted encapsu- lated stem cell-​derived β-​cells are in phase II trials for type I diabetes. Whether long-​term immunotherapy is necessary is unknown in the context of some stem cell-​based interventions (e.g. within the relative immune privilege of the brain). Indirect evidence sup- porting such an idea comes from the demonstration of successful and early withdrawal of antirejection drugs after dopaminergic fetal neuroblast transplantation for Parkinson’s disease. Route and location of delivery Distribution of cell therapy poses very different challenges com- pared with small molecules and macromolecules—​again context matters. In some cases, such as β-​cell replacement, donor cell func- tion is largely independent of location. Conceptually there is no compelling reason for glucose sensitive insulin secreting cells to be located within the pancreas. By contrast, precise focal targeting is required for neurological disorders and cardiac failure. The problem is compounded in diseases characterized by multifocal pathology. Stereotactic implantation is comparatively straightforward for site-​specific disorders such as Parkinson’s disease or spinal cord injury, but unfeasible for diffuse and multifocal disorders, such as Alzheimer’s disease and multiple sclerosis, respectively. Recent studies that highlight the property of stem cell ‘patho-​ tropism’ or ‘homing’ to sites of injury in response to cytokine/​ chemokine gradients offer a means to circumvent this long-​standing conceptual obstacle to cell-​based therapies for a range of disorders. Analysis by in situ hybridization of Y chromosomes of female heart transplants into male recipients provides some evidence for extracardiac origin of cardiac cells, although these were predomin- antly endothelial cells. Several experimental studies have also shown homing of peripherally delivered cells to the injured heart and brain. However, the significance of limited homing is unclear. Estimates from experimental and clinical studies suggest less than 5% cardiac retention 2 h after infusion of bone marrow-​derived cells. This may, in part, explain why clinical trials in cellular cardiomyoplasty have not thus far shown long-​term benefit. Reproducibility and scale Regardless of the precise method deployed to generate a functional cell type from stem cells, widespread clinical application requires scale and targeted delivery. In many ways this is essentially indis- tinguishable from standard pharmaceutical practice, which requires upscaling and automation. Ultimately, protocol effectiveness will need to be user independent, with adoption of mass production techniques sufficient to generate scaleable production of cells. Aside from logistical and manufacturing issues, the variability of cell lines needs to be addressed. No two lines are the same with regard to epi- genetic, molecular, or immunological factors, or indeed ease of dif- ferentiation to a given germ layer and its cellular derivatives. The potential for personalized cell lines both complicates and potentially resolves these issues. In summary, successful regeneration is an incremental process that begins with in vitro generation of uniform and scaleable num- bers of correct cell type, followed by in vitro and ultimately in vivo demonstration of appropriate distribution, connectivity, survival, and function. Translational considerations: Testing novel regenerative therapeutics An overlooked area in regenerative medicine is the critical import- ance of patient selection and optimal trial design to ensure correct evaluation of novel reparative therapies. Identifying patients with the right disease is an obvious point, but not as simple as it may appear. With the emergence of genetically stratified trials for men- delian disease (such as Huntington’s disease), and the increased rec- ognition of molecular subtypes affecting disease progression (such as in malignant gliomas), it is likely that genetically selective study cohorts for sporadic disease will follow. Furthermore, it is also es- sential that any patients studied are at the appropriate stage of dis- ease for the proposed intervention be studied. To do otherwise is likely to introduce noise, account for type 2 errors, and contribute to inconsistent results in early phase clinical trials. Efficacy demonstrated in early phase II trials needs to be extended to the demonstration of sustained clinical benefit and safety in de- finitive phase III studies, including the inclusion of sham treatment as a robust control where feasible. Recognizing the limitations of preclinical animal studies, it is often necessary to enter the clinic in advance of understanding the mechanism of efficacy. Indeed, creative trial design and outcome measures should be sought to allow early trials to not only test effi- cacy but also to inform on putative mechanism of action. This is il- lustrated by the experience to date of cell replacement in Parkinson’s disease, cellular cardiomyoplasty, and the use of adult mesenchymal stem cells in graft-​versus-​host disease (see next). Neurological repair Although more than 250 cell transplantations involving Parkinson’s disease patients have been undertaken, it is only recently that the importance of patient selection has emerged. Historically, and not unexpectedly for a novel treatment, cell transplantation was under- taken in patients with comparatively advanced disease who had become refractory to conventional treatments. Unexpected re- sults from a randomized study have since led to the re-​evaluation of the role of cell implantation and an emerging consensus is that comparatively early onset Parkinson’s disease is the ideal recipient of cell implantation therapy to minimize adverse events such as graft-​induced dyskinesias. Disability scores, the need for adjunctive pharmacological therapies, and functional imaging together provide

3.7  Stem cells and regenerative medicine 291 reasonable metrics of efficacy. The recognition of such clinical parameters and variation has led to the development of larger scale, multisite studies for definitive evaluation of cell transplantation in Parkinson’s disease. The importance of identifying the right cohort for the pro- posed intervention can be further illustrated in neurological medi- cine with regard to multiple sclerosis. Patients with early active relapsing–​remitting disease require disease-​modifying therapy (immunomodulatory), whereas those with advanced progressive disease characterized by significant neurodegeneration require neuroprotection and repair. Cardiac repair Regardless of cell type, clinical indication and the timing of inter- vention matter (e.g. the needs of acute versus chronic ischaemia differ from that of end-​stage heart failure). For example, stem cell therapy for non​ischaemic heart failure (e.g. dilated cardiomyop- athy) in phase II studies has been associated with more consistent functional improvement compared to stem cell trials in ischaemic heart disease. Furthermore, in contrast to diabetes or Parkinson’s disease, where the mechanism of efficacy is known, this is cur- rently less understood in cardiomyoplasty. It follows therefore that standardization of endpoints should necessarily focus on functional (ventricular ejection fraction) and patient disability scores. In this respect, consideration of disease trajectory is important. For ex- ample, a recent randomized study of intracardiac injection of ex- panded bone marrow cells in patients with advanced heart failure (NYHA III-​IV) has demonstrated a 37% reduction in adverse clin- ical events at one year. This is likely to be of clinical value given the baseline prognosis of the condition, despite no significant changes being noted in left ventricular function. Diabetes The Edmonton experience has been instrumental in providing proof of concept of islet transplantation and has also revealed that insulin independence, the ultimate goal, is short-​lived. An understanding of the mechanisms of normal islet β-​cell self-​renewal and of the fate of transplanted islets is needed to take forward further transplantation studies. However, trials to date demonstrate that those with ‘brittle’ diabetes and recurrent hypoglycaemia appear to benefit the most, regardless of insulin independence, illustrating the value of cohort subselection. The outcomes of ongoing phase II trials using subcuta- neously implanted encapsulated β-​cells are likely to inform further translational work in this area. Regulatory considerations Irrespective of source, stem cell culture and expansion need to fulfil several mandatory criteria for therapeutic application. Although clinical keratinocyte protocols presently use bovine serum and feeder cells, future stem cell therapies will need to conform to good manufacturing practice conditions, which are most likely to stipu- late exclusive use of chemically defined and human-​derived com- ponents. Currently, many culture (and differentiation) protocols require animal products or unknown factors present in conditioned media or proprietary supplements. Regardless of the precise details, stem cell-​based therapies will ultimately need to conform to inter- nationally agreed guidelines laid down by regulatory bodies such as the United States Food and Drug Administration (FDA) and the European Medicines Evaluation Agency (EMEA). Future prospects Tissue replacement and solid organ transplantation Using stem cells to generate solid organs is an important goal in regenerative medicine. Ex vivo organogenesis represents both an engineering and a biological challenge. The use of appropriate scaf- folds for cells to grow and differentiate is one approach that has yielded some success. Tissue-​engineered autologous bladders from urothelial and muscle cells seeded on a collagen–​polyglycolic acid matrix have been successfully used in patients requiring cystoplasty, and some biological and synthetic tissue scaffolds have since been developed which will inform future clinical trials. A key challenge in generating tissue replacements for tubular or- gans for clinical evaluation is determining optimal cell-​scaffold com- binations and cell seeding to achieve successful epithelialization. Aside from urology, this an area of ongoing work for organs such as the gastrointestinal tract and trachea. Use of a natural organ scaffold has been suggested as a potential solution for more complex organs. Building on previous studies using decellularized heart valve grafts, successful recolonization of a completely decellularized heart (with an extracellular matrix and vascular structure) with cardiac and endothelial cells has been dem- onstrated, with some evidence of pump function. A second challenge is achieving the right architecture when the organ is composed of multiple cell types (e.g. despite the success and life-​saving nature of autologous keratinocyte grafts, reconstruc- tion of sweat glands, hair follicles, and melanocytes has not been achieved). This challenge is particularly relevant to bioengineering an artificial kidney, arguably the organ with the highest demand. The kidney’s characteristic anatomical and topographical nephron arrangement develops from a specific reciprocal induction process between the ureteric bud and the metanephrogenic mesenchyme—​ replicating this in vitro is still a long way from being achieved. Stem cell repair independent of differentiation
potential Stem cells can be therapeutic by two mechanisms: firstly, by supple- menting (exogenous) and secondly by enhancing endogenous re- pair. Although exogenous repair through cell/​tissue replacement is conceptually straightforward, the promotion of endogenous repair and tissue protection is an area of active research that may ultimately deliver the larger clinical gain. Using stem cells therapeutically for properties independent of their ability to be differentiated into a specific cell type is comple- mentary to the classic view of stem cells as a means of replacing lost cells. This notion proposes that stem cells that display unexpected properties, including immunoregulation, pathotropism, and the ability to function as cellular ‘mini-​pumps’, can be harnessed to pro- mote tissue protection and endogenous repair. Stem cells as cellular immunomodulators have already entered the clinic and are undergoing clinical trials in various disease con- texts. In 2004, le Blanc and colleagues reported striking remission of severe treatment-​refractory graft-​versus-​host disease following

292 SECTION 3  Cell biology intravenous infusion of allogeneic mesenchymal stem cells, an in- novative approach that was undertaken in advance of definitive experimental proof of concept. Similar findings have since been re- ported in preclinical studies on animal models of autoimmune dis- ease including multiple sclerosis, Crohn’s disease, and rheumatoid arthritis. These studies highlight the potential value of stitching to- gether two increasingly recognized properties of stem cells—​ability to traffic to sites of injury and to recalibrate a dysregulated hos- tile immune system—​in the context of inflammatory or immune-​ mediated disease. Alternatively, stem cells can be used as cellular vehicles for the delivery of protective or reparative factors, which may be pro- duced by default or by genetic overexpression. Growth factors have been shown to have a beneficial effect in several neurological dis- eases including Parkinson’s disease and motor neuron disease. In this regard accumulating evidence suggests that some of the more promising results from stem cell trials in cardiac repair cannot be accounted for by graft-​derived cell/​tissue replacement but rather by graft-​derived trophic-​mediated support. More recently, stem cells have also been used as a means of enzyme replacement in metabolic diseases. Implanted neural stem cells have been shown to prolong survival in an animal model of Sandhoff’s disease through a variety of mechanisms, and display synergy with oral medication. This study further highlights the multifaceted action(s) of stem cells with cell replacement, anti-​inflammatory, and enzyme replacement properties all implicated as contributory to efficacy. Endogenous repair, disease modelling,
and drug discovery Endogenous repair The promotion of endogenous repair is an intuitive and attractive long-​term regenerative strategy. Recognition of adult stem cells in organs hitherto considered incapable of self-​renewal—​brain and heart—​has only fuelled such a proposition. The evidence for en- dogenous niche-​resident adult neural stem cells is irrefutable, not- withstanding the disputed ‘multipotentiality’ of widely distributed oligodendrocyte precursor cells. Increasingly persuasive studies also appear to confirm the presence of endogenous cardiac progenitor/​ stem cells and a recent mammalian report provides strong evidence for endogenous cardiac repair that occurs after injury but not age-​ related loss. Other findings that suggest endogenous replacement of islet β cells raise the prospect of parallel and complementary strat- egies to cell implantation in patients with diabetes with some intact β-​cell tissue. An outstanding question is whether limited numbers of stem cells in restricted niches are relevant to organ repair given that damage is often extensive and geographically distant. Furthermore, the physiological role of such cells as well as their response to in- jury is unknown. Nevertheless, these cells and their progeny pro- vide a rational cellular target for pharmacological compounds to activate, mobilize, and thus promote cell-​mediated repair (Fig. 3.7.6). A  complementary cell-​based approach could seek to isolate endogenous—​typically slow cycling—​stem cells and reimplant them to the injured organ after ex vivo expansion. Such a strategy is well established for haematological stem cell therapy in the context of malignancy. Direct in vivo reprogramming to facilitate endogenous repair Successful reprogramming of differentiated cells into pluripotent stem cells using four transcription factors have led to a second wave of cellular reprogramming where lineage-​restricted transcription factors are harnessed to convert one type somatic cell to another. Direct cellular reprograming has been successfully used to generate neurons, glial cells, hepatocytes, cardiomyocytes in vitro using com- bination of transcription factors that control corresponding cell fate and development. This concept provides an attractive proposition in endogenous tissue repair by regenerating affected cells in the damaged organ by switching fate of the locally residing support cells. Recent studies in rodent models have demonstrated the therapeutic potential of direct reprogramming, such as the direct conversion of hepatic myofibroblasts to hepatocytes in vivo by overexpression of four tran- scription factors which were subsequently capable of ameliorating chemically induced liver fibrosis. Similar strategies have also been demonstrated to convert gastric antrum cells to insulin producing ß cells in vivo. As with other proposed regenerative therapies, the major chal- lenge for clinical translation of these promising proof-​of-​concept studies will be demonstration of target-​specific delivery, clinical safety (particularly for viral-​based gene delivery) and long-​term efficacy. Disease modelling and drug discovery By virtue of their proliferation and differentiation potential, stem cells offer a unique experimental resource for drug discovery and in vitro disease modelling. These opportunities converge on im- proved understanding of disease pathogenesis, endogenous repair, and failure to repair normally, and thus together they provide clues to novel regenerative approaches (Fig. 3.7.6). Human stem cells and their derivatives provide a unique opportunity for disease model- ling and understanding genetic and/​or environmental influences of many human disorders. The scaleable and precise differentiation of human pluripotent stem cells into functional derivates can all be scaled into high-​ throughput and automated systems which have the potential to revolutionize drug discovery. Refinements to a human stem based platform system include incorporation of several complementary strategies including (1) generating iPS cells from patients with spe- cific (or unknown) gene mutations or polymorphisms and differen- tiating these to lineages affected in that disease (e.g. cystic fibrosis); (2)  modelling disease directly by genetic overexpression or gene inactivation or silencing, facilitated by the development of more powerful gene-​editing technologies; and (3) modulating environ- mental parameters to replicate disease conditions (e.g. hypergly- caemia in diabetes). Furthermore, development of advanced culture techniques such as microfluidics and 3D bioprinting can enable the evaluation of more complex cellular environments over a longer time period. Following on from an understanding of pathological mechan- isms, in vitro disease models can be used for drug evaluation and testing. In this respect, stem cells offer distinct advantages over cur- rent human sources used in drug screening, which include primary tissue (capable of only limited proliferation) and tumour cell lines (which have a grossly aneuploid genome). Evaluation of drug targets

Assay development High-throughput screening Clinical testing Secondary assays Gene editing Patient cell lines Environmental modulation Toxicology Pharmacokinetics Human variants Candidate drug Safety and efficacy * Lead compounds Fluorescent gene reporters Cell survival Colorimetric assays Physiological parameters * ** * ** * Fig. 3.7.6  Stem cells and drug discovery. A source of potentially unlimited numbers of non​transformed human cell types presents multiple opportunities in drug discovery and development. High-​throughput stem cell-​based screening can result in the identification of novel disease-​modifying compounds. Their safety and differential efficacy can subsequently be determined in secondary assays utilizing stem cell-​derived material, ultimately leading to the development of candidate drugs that can be evaluated through the conventional clinical trial route.

294 SECTION 3  Cell biology can be approached through either a high-​throughput phenotyping-​ based screening approach (with subsequent deconvolution) or testing of candidate compounds with a disease-​modifying rationale. Simple and measurable outcomes, such as fluorescent gene reporters or cell survival over time, will be necessary for any drug-​based assay in order to allow sufficient scalability. In addition to disease mod- elling and discovery, stem cell-​derived lineages can be utilised for evaluation of drug toxicity. Conclusion Regenerative medicine, although in its infancy, will become of increasing importance in the face of the rising global challenge of diseases such as diabetes, neurodegeneration, and heart failure. Human stem cell biology is rapidly advancing, with significant pro- gress already made in technology enabling efficient pluripotent cell derivation, genetic manipulation, and directed differentiation. It is likely to lead to significant gains in understanding of disease mech- anisms and thus open new therapeutic opportunities—​cell and pharmacologically based—​both to modify disease course and to promote repair of the injured organ. Stem cells can be exploited directly and indirectly to promote re- pair. Specifically, stem cell-​based methods or insights seek to sup- plement and enhance, where appropriate, endogenous repair. Cell implantation strategies require the ability to generate large num- bers of defined functional cell populations appropriate to clinical need (e.g. pancreatic islet cells for diabetes or midbrain dopamin- ergic neuroblasts for Parkinson’s disease). In this regard human embryonic-​ or iPS-​derived populations offer significant advan- tages on account of their developmental competence. However, beyond generation of specific cell populations for replacement strategies, it is an oversimplification to view repair as simply recap- itulation of development given the distinct cellular architecture of adulthood complicated by injury-​related structural and biochem- ical changes. In addition to classic cell or tissue replacement, the evolving concept of ‘therapeutic stem cell plasticity’ offers add- itional methods through which stem cells may be useful for regen- erative medicine. Outside of drug discovery, these include utilizing stem cells to limit damage and promote tissue repair by acting as cellular vehicles to deliver trophic/​angiogenic factors or as cellular immunomodulators. Time to clinic is less easily predicted. This will vary and, as with any innovative treatment, there will be a trade-​off between justifi- able risk and benefit. The ability of human stem cells to both inform on and potentially treat devastating and frequently untreatable dis- orders provides cautious grounds for optimism that stem cells will accelerate the emergence of novel therapeutics for regenerative medicine. FURTHER READING Current cell-​based clinical applications Atala A, et al. (2006). Tissue-​engineered autologous bladders for pa- tients needing cystoplasty. Lancet, 367, 1241–​6. O’Connor NE, et al. (1981). Grafting of burns with cultured epithelium prepared from autologous epidermal cells. Lancet, i, 75–​8. Pellegrini G, et al. (1997). Long-​term restoration of damaged corneal sur- faces with autologous cultivated corneal epithelium. Lancet, 349, 990–​3. Wasiak J, et al. (2006). Autologous cartilage implantation for full thick- ness articular cartilage defects of the knee. Cochrane Database Syst Rev, 3, CD003323. Translation of stem cell-​based therapies Dimmeler S, et al. (2014). Translational strategies and challenges in regenerative medicine. Nat Med, 20, 814–​21. Knoepfler PS (2015). From bench to FDA to bedside: US regulatory trends for new stem cell therapies. Adv Drug Deliv Rev, 82, 192–​6. Simonson OE, et al. (2015). The safety of human pluripotent stem cells in clinical treatment. Ann Med, 47, 370–​80. Trounson A, DeWitt N (2016). Pluripotent stem cells progressing to the clinic. Nat Rev Mol Cell Biol, 17, 194–​200. Trounson A, McDonald C (2015). Stem cell therapies in clinical trials: progress and challenges. Cell Stem Cell, 17, 11–​22. Stem cell derivation and molecular biology Evans MJ, Kaufman MH (1981). Establishment in culture of pluripo- tential cells from mouse embryos. Nature, 292, 154–​6. Gurdon JB, Melton DA (2009). Nuclear reprogramming in cells. Science, 322, 1811–​15. Mali P, et  al. (2013). RNA-​guided human genome engineering via Cas9. Science, 339, 823–​6. Takahashi K, et al. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 131, 861–​72. Takahashi K, Yamanaka S (2016). A decade of transcription factor-​ mediated reprogramming to pluriplotency. Nat Rev Mol Cell Biol, 17, 183–​93. Thomson JA, et al. (1998). Embryonic stem cell lines derived from human blastocysts. Science, 282, 1145–​7. Yin H, et al. (2014). Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat Biotechnol, 32, 551–​3. Stem cell immunomodulation Le Blanc K, Ringden O (2006). Mesenchymal stem cells:  proper- ties and role in clinical bone marrow transplantation. Curr Opin Immunol, 18, 586–​91. Le Blanc K, et al. (2004). Treatment of severe acute graft-​versus-​host disease with third party haploidentical mesenchymal stem cells. Lancet, 363, 1439–​41. Naik S, et al. (2018). Two to tango: dialog between immunity and stem cells in health and disease. Cell, 175, 908–20. Pluchino S, et al. (2005). Neurosphere-​derived multipotent precursors promote neuroprotection by an immunomodulatory mechanism. Nature, 436, 266–​71. Zappia E, et al. (2005). Mesenchymal stem cells ameliorate experi- mental autoimmune encephalomyelitis inducing T-​cell anergy. Blood, 106, 1755–​61. Regenerative neurology Eriksson PS, et al. (1998). Neurogenesis in the adult human hippo- campus. Nature Med, 4, 1313–​17. Gill SS, et al. (2003). Direct brain infusion of glial cell line-​derived neurotrophic factor in Parkinson disease. Nature Med, 9, 589–​95. Lee JP, et al. (2007). Stem cells act through multiple mechanisms to benefit mice with neurodegenerative metabolic disease. Nature Med, 13, 439–​47. Lindvall O, et al. (1990). Grafts of fetal dopamine neurons survive and improve motor function in Parkinson’s disease. Science, 247, 574–​7.

3.7  Stem cells and regenerative medicine 295 Suzuki M, et al. (2007). GDNF secreting human neural progenitor cells protect dying motor neurons, but not their projection to muscle, in a rat model of familial ALS. PLoS ONE, 2, e689. Cardiomyocytes and cardiac repair Chong JJ, et al. (2014). Human embryonic stem cell-​derived cardiomyo­ cytes regenerate non-​human primate hearts. Nature, 510, 273–​7. Hsieh PC, et al. (2007). Evidence from a genetic fate-​mapping study that stem cells refresh adult mammalian cardiomyocytes after in- jury. Nature Med, 13, 970–​4. Laugwitz KL, et al. (2005). Postnatal isl1+ cardioblasts enter fully dif- ferentiated cardiomyocyte lineages. Nature, 433, 647–​53. Ott HC, et al. (2008). Perfusion-​decellularized matrix: using nature’s platform to engineer a bioartificial heart. Nature Med, 14, 213–​21. Patel AN, et al. (2016). Ixmyelocel-​T for patients with ischaemic heart failure: a prospective randomised double-​blind trial. Lancet, 387, 2412–​21. Schuldt AJ, et al. (2008). Repairing damaged myocardium: evaluating cells used for cardiac regeneration. Curr Treat Options Cardiovasc Med, 10, 59–​72. Pancreatic β cells and islet transplantation Calafiore R, et al. (2006). Microencapsulated pancreatic islet allografts into nonimmunosuppressed patients with type 1 diabetes: first two cases. Diabetes Care, 29, 137–​8. Kumar SS, et al. (2014). Recent developments in β-​cell differentiation of pluripotent stem cells induced by small and large molecules. Int J Mol Sci, 15, 23418–​47. Schulz, TC (2015). Concise review: manufacturing of pancreatic endo- derm cells for clinical trials in type I diabetes. Stem Cells Transl Med, 4, 927–​31. Shapiro AM, et al. (2006). International trial of the Edmonton protocol for islet transplantation. N Engl J Med, 355, 1318–​30. Other therapeutic possibilities Ajalloueian F, et al. (2018). Bladder biomechanics and the use of scaf- folds for regenerative medicine in the urinary bladder. Nat Rev Urol, 15, 155–​74. Drake MJ, et al. (2015). Application of gene-​editing technologies to HIV-​1. Curr Opin HIV AIDS, 10, 123–​7. Long C, et  al. (2016). Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science, 351, 400–​3. Moreau T, et  al. (2016). Large-​scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming. Nat Communications, 7, 11208. Perales MA, et al. (2015). Fast cars and no brakes: autologous stem cell transplantation as a platform for novel immunotherapies. Biol Blood Marrow Transplant, 22, 17–​22. Schwartz SD, et al. (2015). Human embryonic stem cell-​derived retinal pigment epithelium in patients with age-​related macular degener- ation and Stargardt’s macular dystrophy:  follow-​up of two open-​ label phase 1/​2 studies. Lancet, 385, 509–​16. Song G, et al. (2016). Direct reprogramming of hepatic myofibroblasts into hepatocytes in vivo attenuates liver fibrosis. Cell Stem Cell, 18, 797–​808.