Stem Cells and Tissue Regeneration

Helen M. Blau, Ph.D.[1]

Stem cells have great potential to increase the function of defective or aged tissues through replacement and rejuvenation. Aging is often depicted as in the painting by Gustav Klimt “The three Ages of Woman” – as a progression from a wondrous child to a mature radiant woman to a stooped elderly individual – decrepit and aged. Although longevity, or lifespan, is increasing for both men and women, quality of life, or healthspan, is not. As posed in Nature (Bellantuono, 2018), increased longevity means “more years of what?” Indeed, longer lifespan is associated with more years with chronic disease. Regenerative medicine aims to change that and to increase healthspan, the quality of life, so that one can enjoy life – jog, ski, dance and do all the things one likes to do, even when aged.

The overarching goal of stem cell biology and tissue engineering is to replace or regenerate damaged or diseased tissues, for instance to treat Parkinson’s, myocardial infarction (heart attack) or stroke (Blau and Daley, 2019). Additionally, stem cells can be used to deliver genes that are missing in inherited diseases, like Duchenne muscular dystrophy or hemophilia. Finally, stem cells afford a unique opportunity to improve our understanding of early human development by modeling it in tissue culture. The ability to model human disease processes in culture facilitates screens for drugs in an unprecedented manner. There are three fundamental types of stem cells (Figure 1 and Table 1): embryonic stem (ES) cells, induced pluripotent stem cells (iPSC), and adult tissue-specific stem cells, each with its relative advantages and disadvantages.

Embryonic stem cells

Stem cell biology began with embryonic stem cells. As is well known, the cells of the embryo are pluripotent, capable of specializing into the diverse range of cell types that comprise the human body. Once the oocyte is fertilized, it divides multiple times, giving rise to a blastula with an inner cell mass. These cells, if isolated and cultured, give rise to embryonic stem cells. Embryonic stem cells are pluripotent – they can be directed to become any cell type in the body – for example skeletal muscle, cardiomyocytes, neurons or epidermal cells.

ES cells have distinct advantages (Blau and Daley, 2019) (Figure 1 and Table 1). They divide extensively, provide an almost unlimited cell source, and can be differentiated into almost any given cell type. The challenges of using ES cells are that they are obtained by dissociating the inner cell mass which involves destruction of the embryo created by in vitro fertilization. Accordingly, there are ethical concerns regarding exploitation of egg donors. Additionally, there are challenges to their use, as directing ES cells to become mature cell types is not readily achieved. Indeed, cells differentiated from ES cells usually express fetal genes, not adult genes, and there is an inherent risk of tumorgenicity, as the cells are often karyotypically unstable (Figure2).

Plasticity of the differentiated state

I entered the field with a quest to discover if the differentiated state of a cell could be changed. In the 1980s, cells specialized for specific functions were thought to be stably and irreversibly committed. The dogma at the time was that once a cell became a liver cell or a skin cell, that was its destiny. A cell’s fate was thought to be fixed and set for life. I decided to challenge this premise and probe if specialized cells could be induced to change and adopt properties of other cell types. Specifically, I asked the question: is the destiny of a skin or liver cell terminal or is it reversible? Can it be changed? To address this, I devised a cell fusion system called a heterokaryon wherein multiple mouse muscle cells were fused with human liver cells (hepatocytes) to form a mixed species non-dividing syncytial cell (Figure 3). In this microenvironment, there was no loss of chromosomes and all genetic material remained stably intact. Such heterokaryons are advantageous over hybrid cells, dividing interspecies cell fusion products, in which genetic instability and chromosome loss are pervasive. The use of hybrids allowed genes to be mapped to chromosomes, but precluded conclusions as to whether de novo gene expression in mammalian cells resulted from the activity of activators or the loss of repressors, a question of major interest at the time. Using heterokaryons, Cecelia Webster, Choy Pik Chiu and Grace Pavlath showed that hepatocytes could be induced to express muscle genes that they would never normally express. Further, we found that this held true for a number of human differentiated cell types representative of all three embryonic lineages. We were able to show definitively that this occurred by transactivation, because all chromosomes remained stably intact and the human and mouse muscle gene products could be distinguished. Our experiments provided evidence that reprogramming of the nuclei of highly specialized human cells was possible and resulted in the activation of previously silent genes in specialized mammalian cells (Blau et al., 1983, 1985; Chiu and Blau, 1984). Our discovery provided early evidence that John Gurdon’s elegant Nobel Prize winning nuclear reprogramming experiments in frogs had relevance to mammals. This demonstration of the “Plasticity of the Differentiated State” was highlighted in Science in the Frontiers in Biology issue for the year 1985 (Blau et al., 1985).

Other scientists were also addressing this question using different experimental paradigms. A particularly creative approach was developed by Nicole Le Douarin in Paris, reviewed in (Le Douarin and Dupin, 2016). She showed the plasticity of cells derived from the brain using a very elegant method for following and monitoring cell fate, wherein nuclei of quail cells transplanted into chickens could be distinguished microscopically. Her experiments revealed that neural crest cells are multipotent and change in response to alterations in their microenvironment in the developing avian embryo.

Induced pluripotent stem cells

A major breakthrough in the stem cell field was achieved by Shinya Yamanaka in 2006, which led to the development of an artificial pluripotent cell type, known as “induced pluripotent stem cell” (iPSC), an alternative to embryonic stem cells (Takahashi and Yamanaka, 2006). This discovery was transformative, as it enabled pluripotent cells to be obtained without sacrificing embryos. It also revolutionized the field by creating cells that could be envisioned, not only as sources for cell therapy, but also for studying the development of human disorders in vitro. iPSCs can be derived from a patient’s readily accessible tissues: blood, urine, or skin. Yamanaka identified four transcription factors in a clever screen, that if overexpressed in such cells, converted them into pluripotent cells with features similar to ES cells and the potential to differentiate into essentially any differentiated cell type, as shown in Figure 1 and Table 1 and reviewed in (Blau and Daley, 2019). iPSCs are advantageous, as they comprise an unlimited cell source and are, in effect, immortal. iPSCs can be converted into diverse cell types such as beating cardiomyocytes or electrically coupled neurons, and if derived from a patient with a heritable disorder, will manifest features of the disease in culture. They enable experiments in which CRISPR-corrected controls have an identical genetic background and rescued mutation. iPSC, like ES cells, constitute an unlimited cell source for in vivo tissue replacement. In contrast to ES cells, if donor-derived, they can be immunologically matched to the patient, thereby avoiding immune rejection or lifelong immunosuppression. Obtaining the cells entails a simple basic procedure – obtaining blood, urine or a piece of skin. iPSCs afford an unprecedented opportunity to study human disease mechanisms in tissue culture and perform screens for drugs with therapeutic potential.

The challenges of using iPSC clinically are that, like embryonic cells, they are difficult to differentiate beyond the fetal stage of gene expression (Figure 2 and Table 1). They also have a tendency to be tumorigenic, as they characteristically express oncogenes, harbor an unstable chromosome composition, and have elongated telomeres. Thus, safety is a major barrier to their use in cell therapy. Additionally, it is difficult to scale up production, as rendering the quality control of the cells at a reasonable cost is an immense challenge. As a result, autologous cells, which would overcome immune mismatches, are currently impractical and the current focus of research is on generating non-autologous cell banks lacking major histocompatibility antigens in the hope that they will evade immune clearance.

As an example of “disease in a dish” using iPSC, I will draw on work from my own laboratory where we have been modeling heart failure due to Duchenne Muscular Dystrophy (DMD). DMD affects 1 in 3,600 boys and is due to a mutation in the gene encoding dystrophin, a large structural protein that connects the cytoskeleton with the extracellular matrix. We obtained blood cells from DMD patients, converted them to iPSC by overexpressing the four Yamanaka transcription factors with the help of collaborators at Stanford, and then differentiated them into contractile cardiomyocytes. A talented mechanical engineer, Gaspard Pardon, in my laboratory generated hydrogel patterns with an aspect ratio of 1:7, the typical dimensions of a cardiomyocyte in vivo. This causes the cardiomyocytes to align and form well-organized sarcomeres that aid in their maturation and contractile function. Additionally, using traction force microscopy, Gaspard was able to determine the contractile strength, based on displacement of fluorescent beads within the hydrogel (Figure 4). Video-microscopy and an analysis of Fourier transforms revealed contractile defects due to a single mutation in a gene encoding the contractile protein, dystrophin, on gels of a stiffness comparable to that of a fibrotic heart.

In 2013, Alessandra Sacco, Faye Mourkioti and Alex Chang in my laboratory made the unexpected discovery that telomeres are 50% shorter in the cardiomyocytes of DMD patient hearts relative to other cell types in the same tissue, such as smooth muscle cells, which do not normally express dystrophin and are not affected by its absence. Telomeres are protective caps at the ends of chromosomes that shorten with aging – usually in the course of cell division. The mdx mouse, which lacks dystrophin like DMD patients, does not manifest the cardiomyopathy from which patients succumb. We reasoned that mice might be protected from cardiac failure by the length of their telomeres. When we bred the mice with mice lacking the RNA component of telomerase, they exhibited all of the features of DMD dilated cardiomyopathy, including early mortality due to heart failure (Chang et al., 2016; Mourkioti et al., 2013; Sacco et al., 2010). We can recapitulate the telomere shortening seen in the cardiomyocytes of mouse and in DMD cardiac tissues in the cardiomyocytes differentiated from DMD patient-derived iPSC. The telomeres or iPSC are uniformly long, whether derived from DMD patients or normal controls. Upon differentiation, the telomeres progressively shorten (Chang et al., 2018). We have now determined that a similar telomere attrition is characteristic of both tissue cardiomyocytes and iPSC-derived cardiomyocytes from patients who succumbed from dilated cardiomyopathy due, not only to dystrophin deficiency, but also to troponinT or titin deficiencies(Chang et al., 2018). Remarkably, this telomere shortening occurs independent of cell division in differentiated cardiomyocytes, recapitulating 30 years of life in 30 days in culture. This modeling of “disease in a dish” now enables elucidation of the cause and effect and the underlying molecular mechanisms by which replication-independent telomere attrition occurs, in a manner not previously possible. To date we have found that a DNA damage response is induced by contraction in the absence of a crucial contractile protein, which in turn leads to increased reactive oxygen species (ROS) and mitochondrial failure, culminating in cardiomyocyte death. Tests of interventions that could arrest telomere shortening and serve as a therapeutic intervention are now currently underway in my laboratory. This example highlights the potential for the human iPSC model system to provide fundamental insights into human diseases and serve as a potent platform for drug discovery.

Applications of ES cells and iPSC to the treatment of disease

A major cause of blindness with aging is age-related macular degeneration (AMD), of which the dry form comprises 90% of cases and is associated with loss of vision (Figure 5). ES or iPSC are being used to regenerate the lost retinal pigment epithelial cells (RPE). ES- or iPSC are differentiated to RPE and transplanted as a suspension or sheet just below the photoreceptor layer of the eye, which they support. Although not as advanced as corneal transplants, several studies suggest substantial promise for restoring sight to individuals with AMD (Stern et al., 2018) (da Cruz et al., 2018)(Davis et al., 2017)(Song et al., 2015) (Mandai et al., 2017) There are still challenges to overcome, as one clinical trial was halted in Japan due to the acquisition by iPSC of potentially oncogenic mutations (Garber, 2015; Merkle et al., 2017).

Applications of tissue-specific stem cells in the treatment of disease

Significant therapeutic advances have been achieved using adult tissue-specific stem cells – the third stem cell type (Figure 1). These are stem cells that reside in tissues like muscle, skin or blood and are dedicated to replacing those tissues throughout life. These stem cells spring into action upon injury, proliferate and regenerate the damaged tissue. They constitute a cell source for isolation, cultivation, and transplantation, serving as a cell therapeutic. Additionally, a major focus is to identify drugs that stimulate and rejuvenate the function of the endogenous tissue-resident stem cells, enlisting them in tissue replenishment, for example in aging.

A striking therapeutic success entails regeneration of the cornea following physical damage or chemical burn. The injured cornea becomes opaque, leading to loss of vision. When limbal cells, the stem cells that give rise to the cornea, are isolated from a 1-2 mm biopsy taken from the unaffected cornea of the other eye, minced and directly injected or cultured on a hydrogel contact lens and implanted into the damaged eye, sight is restored (Hirsch et al., 2017) (Hayashi et al., 2016)(Shukla et al., 2019)(Rama et al., 2010) (Figure 6).

Another landmark example of a successful stem cell therapy was achieved for a disorder of skin, entailing the bodywide replacement of 80% of the epidermal layer of a 7-year old boy. de Luca and colleagues in Modena, Italy, identified the quintessential stem cell for the epidermis, the holoclone, and showed that it was capable of longterm tissue reconstitution, enabling a boy with a severe rare genetic skin blistering disease, Epidermolysis Bullosa (EBD) to assume a normal life. EBD is due to the absence of a crucial extracellular matrix protein encoded by one of 18 genes, a laminin or collagen, that is integral to skin function and if missing results in chronic skin erosions and ulcers (Figure 7). Affected children typically live in the hospital and die at a young age, as minor injuries or friction yields blisters that readily become infected. Holoclones were isolated from an unaffected region of the boy’s body and engineered to express the gene encoding the missing protein, a laminin, and then transplanted to cover all affected skin regions (Hirsch et al., 2017). Three years later, this child is playing soccer. During this period, it is estimated that the epidermis underwent approximately 30 cycles of self-renewal. This success highlights the need to characterize the true tissue-specific stem cell with longterm self-renewal and differentiation potential, a major challenge in the adult stem cell field. Although this is now known to be a holoclone in the case of skin and cornea, the identification of specific cell surface markers enabling prospective isolation of the stem cells by fluorescence activated cell sorting has yet to be achieved. Notably, in some cases, such as the heart, the very existence of a tissue-specific stem cell remains in question, despite claims to the contrary.

In my laboratory, we focus on skeletal muscle stem cells (Figure 8). 40% of the body mass is muscle and with age muscles atrophy and become weaker, compromising quality of life, leading to an increasing incidence of falls, loss of mobility, and an associated increase in morbidity. The muscle stem cells, known as satellite cells, reside in a membrane-enclosed niche juxtaposed to the myofiber. They express the hallmark transcription factor, Pax7. In response to injury, these cells become activated, divide and become committed progenitors that fuse into the muscle fibers and restore muscle strength. Alessandra Sacco in my laboratory defined cell surface markers that enable the prospective isolation of muscle stem cells (MuSCs) capable of self-renewal, engraftment and differentiation, as we showed by using single-cell transplants monitored by dynamic bioluminescence imaging (Sacco et al., 2010). We encountered a problem in their further study, as the stem cell properties were rapidly lost when isolated MuSCs were grown on tissue culture plastic. By fabricating hydrogels with an elasticity mimicking the muscle tissue, Penney Gilbert and Karen Havenstrite were able to overcome this limitation and maintain the stem cell state of MuSCs (Gilbert et al., 2010), enabling experiments not previously possible. We identified intrinsic regulators that become dysfunctional with aging, including the acquisition of phosphorylated p38 MAP kinase. Ben Cosgrove showed that by inhibiting this kinase with a small molecule drug in aged MuSCs grown on elastic hydrogels, we could rejuvenate the function of the aged MuSC population and restore strength upon transplantation into injured muscles (Cosgrove et al., 2014). We sought to discover an extrinsic regulator of MuSC function that could target and augment the function of the stem cells in situ. Since the first response to wound healing is an inflammatory response, Andrew Ho and Adelaida Palla performed an in silico screen, a bioinformatics analysis, of the transcriptome of activated MuSCs and identified the EP4 receptor on the surface of the stem cell. EP4 is the receptor for prostaglandin E2. We found that a transient 24-hour exposure to PGE2 led to a robust increase in muscle stem cell numbers a week later (Ho et al., 2017). A timelapse microscopy analysis revealed that PGE2 acts as a potent mitogen and pro-survival metabolite. This signaling pathway is essential to the function of MuSCs, as if the EP4 receptor is genetically ablated on MuSCs in mice, they lose strength post-injury. In addition, if the natural synthesis of PGE2 is blocked by giving mice a non-steroidal anti-inflammatory agent, like ibuprofen, strength drops by 50% post-injury. A major focus in my laboratory now is to capitalize on this natural wound healing regenerative response to determine if we can rejuvenate muscle stem cell function, counter muscle atrophy, increase strength, and increase quality of life of the elderly.

Although challenges remain, the promise of stem cells for regenerative medicine is beginning to be realized (Blau and Daley, 2019). Stem cell therapeutics are now in clinical trials for two major disorders of vision, age-related macular degeneration and corneal damage. In addition, the epidermal layer of the skin can be effectively regenerated longterm. Efforts to identify the stem cell that regenerates the dermal skin layer are underway and essential for the treatment of burn victims, as both the epidermis and dermis are damaged. Treatment of Type I diabetes is in clinical trials using ES- or iPSC-derived pancreatic beta-cell progenitors, cleverly encapsulated to evade the immune response, while allowing diffusion of glucose into and secretion of insulin out of the capsule. Advances in the differentiation of dopaminergic cells and their integration into the host striatum of the brain has reduced symptoms of Parkinson’s disease in primates. The derivation of engraftable hematopoietic stem cells represents an as yet unattained holy grail for addressing the immense need for blood transfusion due to accidents or disease, as allogeneic bone marrow transplants are associated with a high risk of death. The loss of heart muscle, most commonly through myocardial infarction is life-threatening, and constitutes perhaps the most daunting challenge of electrical and mechanical integration. Nonetheless, the hurdles facing stem cells and their use in the treatment of disease are likely to be surmounted with regenerative medicine assuming an ever-increasing role in the therapeutic armamentarium.

Figure Legends:

Figure 1: There are three main sources of human stem cells: adult tissue-specific stem cells, embryonic stem cells (ES), and induced pluripotent stem cells (iPSCs). Each source has distinct advantages and disadvantages.

Figure 2: Challenges in the clinical use of stem cells.

Figure 3: Plasticity of the differentiated state. Schematic of an experiment showing heterokaryons: stable fusion products of multinucleate mouse muscle cells and human hepatocytes with no chromosome loss or rearrangements. Heterokaryon experiments provided novel evidence that the reprogramming and activation of silent mammalian genes is possible and that the human differentiated state is not fixed and irreversible.

Figure 4: IPSC-derived cardiomyocytes grown on patterned hydrogels exhibit well-organized sarcomeres that generate force that can be measured by traction force microscopy. Images of a cardiomyocyte embedded in a hydrogel are shown. Fluorescent beads (top, cyan) enable visualization of the hydrogel and facilitate monitoring and quantification of cardiomyocyte contraction. The sarcomere, the contractile apparatus of the cell, is labeled red and the cell’s nucleus is blue (middle). Brightfield image of the experimental setup (bottom). Contributed by Gaspard Pardon.

Figure 5: Regeneration of the retinal pigment epithelium (RPE) that supports the photoreceptors of the eye and is lost due to age-related macular degeneration (AMD) is possible with ES or IPSC-derived RPE.

Figure 6: Replacement of corneal tissue subjected to physical or chemical damage can be achieved by transplantation of limbal tissue-specific stem cells from the unaffected eye.

Figure 7: 80% of the epidermis of a boy with a rare heritable highly disabling blistering disorder, Epidermolysis Bullosa, was replaced longterm by transplanting the boy’s genetically corrected tissue-specific stem cells (holoclones), allowing the boy to assume a normal life.

Figure 8: Muscle stem cells (satellite cells) that reside in niches along the length of the myofiber, can be envisioned as a cell therapeutic to treat a wide variety of muscle-related disorders, such as muscular dystrophies. An alternative strategy entails identification of drugs to activate and rejuvenate endogenous tissue-resident stem cells to treat disorders such as sarcopenia, progressive muscle wasting with aging.

Acknowledgements

I wish to thank the many talented young scientists who worked in my laboratory and helped to develop this exciting field. I have been lucky to have an exceptionally creative and dynamic group of international colleagues throughout my career. In particular, I thank Andrew Ho for his stimulating discussions and beautiful artwork that comprises most of the figures in this chapter.

 

References:

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[1] Donald E. and Delia B. Baxter Professor; Director, Baxter Foundation Laboratory for Stem Cell Biology; Stanford University, CA 94305.

 

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