Cardiovascular disease is a leading cause of death in the world, leading to an estimated 17 million deaths per year.49 As life expectancy in the developed world rises, so too do risk factors associated with chronic heart disease. It is estimated that there are approximately 800,000 new cases of acute myocardial infarction (AMI)50 annually. Heart failure occurs when there is significant deprivation of oxygen to cardiac tissue, which results in decreased cardiac output and function as a result of loss of cardiomyocytes, scar formation, and tissue remodeling.50 Identifying ways to regenerate or repair heart tissue will be key to developing effective treatment options for heart failure.
Since the mid-1990s, scientists have been investigating the potential of adult progenitor cells for use in heart regeneration. These early studies were triggered by the discovery that certain adult tissue-specific stem cells could be differentiated in vitro to become cardiac-like cells.51 This discovery led to many preclinical studies that assessed the ability of adult stem cells to repair or enhance cardiac function after various types of injury.
The cells reported to differentiate in vitro to cardiac-like cells in vivo are satellite cells, which are undifferentiated skeletal muscle myoblasts,52 which led to studies using autologous skeletal myoblasts surgically implanted into the heart muscle.53 Although these cells survived for short periods of time, they retained their intrinsic contractile properties and did not fully integrate into the cardiac tissue,54 which led to arrhythmias and gave little long-term significant benefit in overall heart function.
Some marrow-derived cell populations (lin−; c-kit+) were capable of differentiating to myocytes expressing cardiomyocyte markers such as Nkx2.5, Gata4, and MEF.55 These marrow-derived cells were shown to survive in infarcted hearts and were capable of differentiating into smooth muscle and endothelial cells but not cardiomyocytes in vivo.56 Additional studies demonstrated that other marrow-derived progenitor cell populations (endothelial progenitors, angioblasts, or CD34+ cells) were able to contribute to angiogenesis and neovascularization of the infarcted myocardium.57 This differed from more immature marrow progenitor populations, called side-population cells (Lin− c-kit+ Sca-1+), that not only contribute to neovascularization but also regenerate myocardium.58 These marrow-derived side-population cells homed to the border zone of infarction and resulted in improved left ventricular function.58 The cardiac regeneration capacity of the marrow-derived progenitor cells facilitated large-scale clinical studies using heterogeneous populations of bone mononuclear cells (MNCs), also called epithelial progenitor cells (EPCs), for cardiac repair in patients with AMI59,60 or ischemic cardiomyopathy.61 These studies showed only moderate improvements, and therefore led to further refinement of the selection criteria for marrow-derived cells (CD34+/CD133+) and changes in the route of administration (intracoronary injection) in the Regeneration by Intracoronary Infusion of Selected Population of Stem Cells in Acute Myocardial Infarction (REGENT) study, which still only showed modest success.62
The moderate success of marrow-derived MNCs spurred the investigation of other populations of adult progenitor cells, as well as new routes of administration. The investigations led to the discovery of MSCs, as well as endogenous cardiac-derived stem cells, that could differentiate into cardiomyocytes and endothelial cells in animal models.63 These findings led to clinical studies comparing marrow-derived MNCs versus the new MSCs used for intracoronary injection into patients with ischemic cardiomyopathy.64 These tests showed significant improvement in left ventricle ejection fraction in response to MSC treatment.
To date, the clinical success of cell therapy approaches for cardiac regeneration has been mixed. This is in contrast to the promising early preclinical studies that showed significant improvement in many different measures of cardiac function. This difference has been attributed to the differences between rodent cardiac injury models and human clinical pathology, the cell population administration route, the origin of the cell populations, and the limited number of cells injected.
ESCs, because of their pluripotency and unlimited ability to proliferate, have been the subject of extensive preclinical investigation for many tissues, particularly cardiac tissue repair.65,66 However, there has been less enthusiasm for hESC-derived cardiomyocytes for human cell therapy because their allogeneic nature requires concomitant immunosuppressive therapy and because ethical issues surround their derivation. Despite these challenges, clinical studies have begun to collect ESCs for cardiac differentiation with the intent of being used in a trial for AMI patients. However, as about any somatic cell can be used to generate embryonic stem-like iPSCs,67 with the capability to differentiate into cardiomyocytes, endothelial cells, and smooth muscle cells.68 Initial preclinical studies of murine iPSC-derived cardiomyocytes, injected into ischemic myocardium, led to rejection of transplanted cells by immune reaction as well as continuous proliferation that led to teratoma formation.69 Although iPSCs are attractive for their allogeneic potential they have potential disadvantages for human cell therapy based on their oncogenic nature, epigenetic memory, and maintenance of potency for other cells types.
Growing tissues in vitro for use in regenerative therapies has been investigated as another delivery method of cells for heart repair. This tissue engineering approach involves seeding cells onto scaffolds and growing them for later engraftment or for the generation of whole organs.70 In these systems, cells are transplanted with the scaffolding to the cardiac wall, which provides structural support and a better microenvironment for the migration of cells into the damaged myocardium.71
A common theme in most preclinical and clinical cell therapy-based studies is the demonstration of improvements in cardiac function that is not correlated to number of cells injected or their longevity after administration. This observation has led investigators to speculate that transplanted cells improve cardiac function through paracrine rather than structural effects. This model suggests that observed improvements in myocardial regeneration or vasculogenesis are a result of transplanted cells secreting molecules that are known to improve cardiovascular function after injury.72 The effects of paracrine factors include decreased inflammation, increased angiogenesis, induction of proliferation of cardiomyocytes or protection of existing ones, and activation of endogenous stem cells.73 Ischemic hearts subjected to secreted factors showed an increase in the expression of genes involved in cardiogenesis and a downregulation of cell-death markers, effects that contribute to survival of ischemic cardiomyocytes.74 The advantage of the paracrine model is the potential for the commercial development of paracrine factors that have proven potential for cardiac repair.
The prevalence of lung diseases, like the chronic obstructive pulmonary diseases (COPDs) that include asthma and emphysema, has increased dramatically over the last 50 years. Lung disease is expected to become the third leading cause of disease-related death in the world by 2020. New therapeutic approaches from regenerative medicine are being developed ranging from stem cell therapies to bioengineering of entire tissues of the respiratory system for transplantation. These approaches are based on initial observations that endothelial progenitor cells and mesenchymal stem cells can differentiate in vitro to cells expressing lung epithelial markers and contribute to mature functional bioengineered tissues.
Throughout the pulmonary tract there exist many different niche environments containing distinct epithelial cell types that contribute to the complexity of the lung. Identification of a true endogenous stem cell population that is responsible for maintaining lung tissue under steady state and injury has been challenging and has been a source of controversy.75 Evidence from rodent models and human lungs suggests that the adult endogenous airway, alveolar epithelial cells, lung stroma, and pulmonary vasculature all contain putative stem cell populations that can repair damaged tissue.76,77 These studies suggest the lung has a regional hierarchy of stem and progenitor cells that are specific for proximal versus distal airways as well as alveoli.
Identifying endogenous lung stem cells is complex because many different subpopulations of basal epithelial cells exhibit restricted patterns or roles in self-renewal for steady-state maintenance or after injury.78,79 In the distal airway, putative progenitor cells have been identified in the neuroepithelial body,80 bronchoalveolar duct junction,81 by specific markers of self-renewing lung epithelial cells,82,83 and by function as bronchiolar alveolar stem cells (BASCs). This is in contrast to alveolar epithelial repair thought to be regulated by type 2 alveolar epithelial cells (ATII) because they have been shown to be precursors to type 1 (ATI) cells.84,85 Identification of regionally specific stem cell populations is further complicated by the demonstration that isolated distal airway progenitors (BASCs, CK5+/p63+) can differentiate into ATII and ATI cells.86,87 Regardless, all of these cells show unique functions in repair after injury, reside in different locations in the distal airway and alveolar epithelium, and play different roles as endogenous lung epithelial progenitors.
Many preclinical studies have shown that EPCs can increase function in pulmonary lung injury models.88,89,90 This improvement in function could be because of contributions to structure, paracrine effects, modulation of immune responses, or a combination of these.90 EPCs have also been demonstrated to preferentially home to sites of injury in the lung after systemic administration91; consequently, autologous EPCs have been used clinically in pulmonary hypertension patients and showed improved cardiopulmonary outcomes.92,93
Marrow MSCs are known for their immunomodulatory effects in a wide range of diseases.94,95 The beneficial effect of MSCs results from secretion of soluble mediators and microsomal particles that influence lung progenitor cells directly or indirectly through mediation of inflammatory cells that subsequently promote repair.96,97 Both preclinical and clinical studies have shown efficacy in either systemic or intratracheal administration of MSCs in acute lung injury models, asthma, COPD, and a host of other inflammation-related lung injuries or diseases.75,98 Although different studies have shown varying degrees of efficacy, there are still significant gaps in our understanding of the mechanisms of MSC action on ameliorating disease symptoms and of the specific subtype of MSCs used. This is important, as studies have demonstrated that certain MSCs can have negative effects in some lung disease models, such as pulmonary fibrosis.99,100
Although there has been significant preclinical investigation into using EPC and MSC therapy approaches to lung repair, clinical studies have been slow to develop. However, there are a growing number of clinical trials in development focused on using MSCs for chronic lung diseases where preclinical data show the most promise. The most recent is the PROCHYMAL phase II trial looking at systemic administration of marrow MSCs for moderate to severe COPD,101 which showed the safety of using MSCs and also preliminary evidence for decrease in markers of inflammation.
BRAIN AND SPINAL CORD REPAIR
Stem cell-based therapy is rapidly developing as a way to improve outcomes following brain and spinal cord injury or disease. The human CNS is composed of more than 100 billion nerve cells connected in a complex network that must work seamlessly throughout our lives. Conditions affecting the CNS—such as stroke, brain, spinal cord injury, and neurodegenerative diseases—affect millions of people worldwide. Challenges for therapeutic intervention include the complex pathology of these conditions, as well as the specialized anatomical structures of the CNS that prevent easy access from systemic administration of therapies (e.g., the blood–brain barrier).
As with other areas of regenerative medicine, much effort has been spent on the identification of cell types with the best potential for CNS repair. Although it was initially thought that the adult CNS did not contain progenitor cells for repair, it is now recognized that the human CNS does retain an endogenous neural stem cell (NSC) population that retains some capacity for repair, although only in select regions and of a limited nature. Isolated adult and fetal NSCs can be expanded and differentiated into neurons, astrocytes, and oligodendrocytes, the three main CNS cell types. Adult NSCs are retained throughout life, and are found in the striatal subventricular zone and the dentate gyrus of the hippocampus. In preclinical studies, endogenous NSCs have shown to provide the most significant improvement in functional recovery in rodent stroke models.102 Isolated human fetal NSCs (CD133+),103 are currently being investigated in a number of clinical studies.104,105 In addition, multiple NSC-based cell therapy trials are being conducted to determine the safety and efficacy in patients with amyotrophic lateral sclerosis (ALS, sometimes called Lou Gehrig disease).106,107 Although all trials to date have confirmed the safety of using these cells, any benefit from them has yet to be reported.
Although other adult stem cell types (endothelial progenitor cells, umbilical cord blood cells [UBCs], dental pulp stem cells [DPSCs]) have been investigated preclinically, only MSCs have been shown to have the same level of efficacy as NSCs. Marrow-derived MSCs have been the primary focus of preclinical and clinical studies because of their relative abundance and potential for autologous cell transplantation.108,109 Administration of MSCs, regardless of route, have been shown to improve outcome measures in rodent models of injury and disease.106,110 Based on these findings, there have been a number of early phase clinical trials initiated to study the effects of MSC transplantation following CNS injury111,112 or disease.113,114 In both cases, MSCs have been shown to be both safe and a feasible approach. Although not designed to test efficacy, many trials have observed improvements in functional outcomes.115
From studies of the efficacy of adult stem cells for therapy in preclinical models of CNS injury or disease over the past 20 years, there is strong evidence that transplanted adult stem cells can migrate to the site of injury and promote functional improvement. The mechanism of action, however, remains controversial.116 There is speculation that the benefits of cell-based therapy arise from multiple factors. From the host of preclinical studies on MSCs for treatment of stroke, improvements in function have been attributed to increased angiogenesis, neurogenesis, prosurvival signals, and mitigation of immune responses.117,118 These mechanisms are potentially mediated by various soluble factors that act through a paracrine mechanism secreted by transplanted stem cells that benefit the local environment. Many of these secreted factors have begun to be identified and are well-known mediators of neurogenesis (bone-derived neurotrophic factor [BDNF]), angiogenesis (vascular endothelial growth factor [VEGF]), and immune regulation (transforming growth factor-β1[TGF-β1]).117,119
The use of pluripotent cell types (ESCs, iPSCs) holds significant potential as a therapeutic approach for CNS repair. Clinical application of ESCs/iPSCs is limited because of the inability to isolate pure differentiated populations of neuronal cell types.120,121 Despite this limitation, considerable progress has been made in preclinical studies for remyelination following spinal cord injury.122,123 These studies and others have demonstrated that ESCs/iPSCs can be differentiated to oligodendrocyte progenitor cells (OPCs), migrate within the spinal cord, and produce myelin. Again, the efficacy and mechanism of recovery remain controversial.124,125,126,127 In 2010, the Geron Company began recruitment of patients for a phase I clinical trial for treatment of spinal cord injury with ESC-derived OPCs. Despite significant enthusiasm from the patient population, and report of no adverse events on a small cohort of treated patients (n = 4), significant methodologic128 and economic obstacles forced the early discontinuation of the trial.
LIVER AND PANCREAS REPAIR
The liver is an essential organ that coordinates glycogen storage, drug detoxification, production of various serum proteins, and secretion of bile, which plays a critical role in food digestion and metabolism. It is interspersed with small microscopic canals known as canaliculi through which the bile drains to the gall bladder. The numerous bile canaliculi join together into many larger bile ducts, which join to become a branched structure that forms the common hepatic duct. The part of the common hepatic duct that is outside the liver is called the extra hepatic bile duct, which joins the cystic duct from the gall bladder to form the common bile duct and connect to the exocrine pancreas. This whole branched structure forms the biliary tree.129 Liver, biliary tree, and pancreas originate from the anterior definitive endoderm and have been found to share stem cell populations during the early stages of development.130
The normal liver has extraordinary potential to regenerate following partial removal of the liver or liver injury. The hepatocytes and cholangiocytes (biliary epithelial cells) are normally quiescent, but in response to liver injury, these cells proliferate and contribute to regeneration.131 Average liver turnover is maintained by differentiation and proliferation of parenchymal or nonparenchymal cells. However, in chronic liver disease, when the liver cannot be repaired by self-duplication of existing hepatocytes, small bipotential progenitor cells are activated that have the ability to differentiate into either hepatocytes or cholangiocytes.132 Severe injury in alcoholic liver disease has been found to stimulate increased proliferation of hepatic stem/progenitor cells in humans133 and oval cells in rodents.134 Some experiments have suggested that stem progenitor cells are not only activated in the injured liver, but that the normal adult human liver contains a large number of liver progenitor cells that may contribute to liver homeostasis.
Liver transplantation is currently the only option in acute liver failure. Two small clinical trials conducted with hepatocyte transplantation resulted in limited restored enzyme function in one patient, but it was not enough for survival and the patient eventually needed a liver transplant.135 It remains unknown if hepatocytes can contribute to long-term rescue in patients. Additionally, hepatocytes are difficult to produce in clinically sufficient numbers, as they lose their viability and function when cultured in vitro.
Transplanted human ESC/iPSC-derived mature hepatocytes can express liver-specific enzymes such as albumin, antitrypsin, and cytochrome P450, which can improve liver function.136,137 Furthermore, a functional liver organ bud generated from iPSCs was found to be engrafted and integrated within the host organism, even including development of blood vessels.138 When iPSCs are injected into the blastocysts of fumarylacetoacetate hydrolase (FAH)-deficient mice, iPSC-derived hepatocytes can repopulate the damaged liver efficiently and restore liver function.139
The liver and pancreas both harbor a niche of stem cells, collectively known as the hepatic stem/progenitor cells.140,141 The fetal biliary tree, arising from the ductal plate cells during development and consisting of the intrahepatic and extrahepatic ducts, has been shown to harbor a rich source of stem/progenitor cells.142 These stem/progenitor cells are quite distinctive from hepatoblasts, which contribute toward the hepatocytes or cholangiocytes during development.143 These stem/progenitor cells present in the biliary tree differ from hepatoblasts by their ability to self-renew and differentiate into hepatocytes, cholangiocytes, or pancreatic islets, depending on the microenvironment. Sox9 expression is detected throughout the pancreatic ducts, intra- and extrahepatic ducts, and in the intestinal crypt connected through the major duodenal papilla, forming a contiguous Sox9+ zone.144,145 Remarkably, when an adenovirus contacting three pancreatic transcription factors (Pdx1, Ngn3, and MafA) was delivered into the liver, it was able to reprogram the Sox9+ population of cells within the bile ducts into functional insulin-secreting, beta-like cells.25 When both Ad-PNM and a peroxisome proliferator-activated receptor (PPAR) agonist, WY14643, were given in animals, it resulted in injury to the liver, led to an increase in the cell division rate of Sox9+ cells lining bile ducts, and contributed toward making more insulin-positive beta cells.146 In an injured liver, the Wnt signaling pathway is activated, which stimulates discreet subsets of progenitor cell populations, which, in turn, engage in liver regeneration.147 Isolation of stem/progenitor cell populations based on their surface markers like Lgr5 or EpCAM has identified the possibility of differentiating them toward a hepatocyte- or beta-like cell fate.145,148
The pancreas plays an important role in digestion, as well as in maintaining blood glucose homeostasis. It is composed of ducts, with the main duct (pancreatic duct) running the length of the pancreas. The pancreatic duct merges with the bile duct to form the major duodenal ampulla, which drains the pancreatic fluid into the first portion of the small intestine, the duodenum. The pancreas is composed of exocrine and endocrine parts. The exocrine component plays an integral part in digestion. The endocrine component contains the islets of Langerhans that produce and secrete hormones into the bloodstream. The pancreatic hormones, insulin and glucagon, work together to maintain proper sugar levels in the blood.
Diabetes mellitus is a metabolic syndrome caused by having an insufficient number of insulin-producing beta cells. In type 1 diabetes, beta cells are destroyed by the body’s own immune system. In type 2 diabetes, although the pancreas has functioning beta cells, insulin resistance causes the liver to release too much sugar into the bloodstream, and the beta cells cannot secrete enough insulin to maintain normal glucose homeostasis. The American Diabetes Association estimated in 2012 that approximately 9.3 percent of the United States population is living with diabetes.149 Beta-cell therapy holds promise for treating type 1 diabetes by replenishing beta cells in the body; however, donor pancreases are in short supply and the demand for transplantable beta cells cannot be met.
In addition, beta-like cells have been successfully generated from hESCs and iPSCs using overexpression of transcription factors, chemicals, or growth factors.150 Current protocols for differentiation of pluripotent stem cells to beta cells follow a five-stage procedure that recapitulates the embryonic stages of development.151 Only the first four stages have been carried out successfully in vitro. The fifth stage—which involves maturation to glucose-responsive, insulin-secreting beta cells and other islet cells—until recently could only be carried out by implantation in vivo.152 A long-awaited directed differentiation of insulin-producing cells from hESCs has been accomplished; this fully defined ex vivo technology is immediately relevant.153
Also nonendocrine cells within the pancreas have been found to transdifferentiate or reprogram to a beta cell fate24 upon their being induced with the three-pancreatic-gene cocktail (Pdx1, Ngn3, and MafA). Another source for adult cell reprogramming to beta cells has been described from glucagon-producing alpha cells. A study showed that overexpression of Pax4 (a gene responsible for specifying endocrine fate) in the alpha cells was able to force them to become beta-like cells.154 In another study, it was observed that near-complete ablation of beta cells forced regeneration of beta cells from former alpha cells.155 However such in vivo studies have not been established in humans or other primates.