Georgina M. Ellison-Hughes

Adult cardiac stem cells are multipotent and robustly myogenic: c-kit expression is necessary but not sufficient for their identification

Multipotent adult resident cardiac stem cells (CSCs) were first identified by the expression of c-kit, the stem cell factor receptor. However, in the adult myocardium c-kit alone cannot distinguish CSCs from other c-kit-expressing (c-kitpos) cells. The adult heart indeed contains a heterogeneous mixture of c-kitpos cells, mainly composed of mast and endothelial/progenitor cells. This heterogeneity of cardiac c-kitpos cells has generated confusion and controversy about the existence and role of CSCs in the adult heart. Here, to unravel CSC identity within the heterogeneous c-kit-expressing cardiac cell population, c-kitpos cardiac cells were separated through CD45-positive or -negative sorting followed by c-kitpos sorting. The blood/endothelial lineage-committed (Lineagepos) CD45posc-kitpos cardiac cells were compared to CD45neg(Lineageneg/Linneg) c-kitpos cardiac cells for stemness and myogenic properties in vitro and in vivo. The majority (~90%) of the resident c-kitpos cardiac cells are blood/endothelial lineage-committed CD45posCD31posc-kitpos cells. In contrast, the LinnegCD45negc-kitpos cardiac cell cohort, which represents ⩽10% of the total c-kitpos cells, contain all the cardiac cells with the properties of adult multipotent CSCs. These characteristics are absent from the c-kitneg and the blood/endothelial lineage-committed c-kitpos cardiac cells. Single Linnegc-kitpos cell-derived clones, which represent only 1–2% of total c-kitpos myocardial cells, when stimulated with TGF-β/Wnt molecules, acquire full transcriptome and protein expression, sarcomere organisation, spontaneous contraction and electrophysiological properties of differentiated cardiomyocytes (CMs). Genetically tagged cloned progeny of one Linnegc-kitpos cell when injected into the infarcted myocardium, results in significant regeneration of new CMs, arterioles and capillaries, derived from the injected cells. The CSC’s myogenic regenerative capacity is dependent on commitment to the CM lineage through activation of the SMAD2 pathway. Such regeneration was not apparent when blood/endothelial lineage-committed c-kitpos cardiac cells were injected. Thus, among the cardiac c-kitpos cell cohort only a very small fraction has the phenotype and the differentiation/regenerative potential characteristics of true multipotent CSCs.

Non-invasive strategies for stimulating endogenous repair and regenerative mechanisms in the damaged heart

The adult myocardium, including human, harbours a population of resident multi-potent cardiac stem cells (CSCs), which when stimulated under the right conditions can give rise to new cardiomyocytes and vasculature. Elucidation of the cellular and molecular mechanisms that govern CSC biology and their role in myocardial regeneration will allow the design and development of optimal therapeutic interventions. It is now evident that different growth factors and cytokines govern CSC survival, proliferation, migration and differentiation, as well as playing a role in activating cardiac repair mechanisms such as improving angiogenesis, cardiomyocyte survival and limiting fibrosis. This review article will summarize the evidence for a role of VEGF, NRG-1, IGF-1, HGF, EGF, FGF and TGF-β1 in modulating the repair and regeneration of cardiac tissue. It will also discuss the use of exosomes and exercise training as interventions to stimulate the endogenous repair and regenerative mechanisms in the damaged heart.

Skeletal muscle-derived interstitial progenitor cells (PICs) display stem cell properties, being clonogenic, self-renewing, and multi-potent in vitro and in vivo

BackgroundThe development of cellular therapies to treat muscle wastage with disease or age is paramount. Resident muscle satellite cells are not currently regarded as a viable cell source due to their limited migration and growth capability ex vivo. This study investigated the potential of muscle-derived PW1+/Pax7– interstitial progenitor cells (PICs) as a source of tissue-specific stem/progenitor cells with stem cell properties and multipotency.MethodsSca-1+/PW1+ PICs were identified on tissue sections from hind limb muscle of 21-day-old mice, isolated by magnetic-activated cell sorting (MACS) technology and their phenotype and characteristics assessed over time in culture. Green fluorescent protein (GFP)-labelled PICs were used to determine multipotency in vivo in a tumour formation assay.ResultsIsolated PICs expressed markers of pluripotency (Oct3/4, Sox2, and Nanog), were clonogenic, and self-renewing with >60 population doublings, and a population doubling time of 15.8u2009±u20092.9xa0h. PICs demonstrated an ability to generate both striated and smooth muscle, whilst also displaying the potential to differentiate into cell types of the three germ layers both in vitro and in vivo. Moreover, PICs did not form tumours in vivo.ConclusionThese findings open new avenues for a variety of solid tissue engineering and regeneration approaches, utilising a single multipotent stem cell type isolated from an easily accessible source such as skeletal muscle.

Cardiac Stem Cells for Myocardial Regeneration: They Are Not Alone

Heart failure is the number one killer worldwide with ~50% of patients dying within 5u2009years of prognosis. The discovery of stem cells, which are capable of repairing the damaged portion of the heart, has created a field of cardiac regenerative medicine, which explores various types of stem cells, either autologous or endogenous, in the hope of finding the “holy grail” stem cell candidate to slow down and reverse the disease progression. However, there are many challenges that need to be overcome in the search of such a cell candidate. The ideal cells have to survive the harsh infarcted environment, retain their phenotype upon administration, and engraft and be activated to initiate repair and regeneration in vivo. Early bench and bedside experiments mostly focused on bone marrow-derived cells; however, heart regeneration requires multiple coordinations and interactions between various cell types and the extracellular matrix to form new cardiomyocytes and vasculature. There is an observed trend that when more than one cell is coadministered and cotransplanted into infarcted animal models the degree of regeneration is enhanced, when compared to single-cell administration. This review focuses on stem cell candidates, which have also been tested in human trials, and summarizes findings that explore the interactions between various stem cells in heart regenerative therapy.

Exploring pericyte and cardiac stem cell secretome unveils new tactics for drug discovery

ABSTRACT Ischaemic diseases remain a major cause of morbidity and mortality despite continuous advancements in medical and interventional treatments. Moreover, available drugs reduce symptoms associated with tissue ischaemia, without providing a definitive repair. Cardiovascular regenerative medicine is an expanding field of research that aims to improve the treatment of ischaemic disorders through restorative methods, such as gene therapy, stem cell therapy, and tissue engineering. Stem cell transplantation has salutary effects through direct and indirect actions, the latter being attributable to growth factors and cytokines released by stem cells and influencing the endogenous mechanisms of repair. Autologous stem cell therapies offer less scope for intellectual property coverage and have limited scalability. On the other hand, off‐the‐shelf cell products and derivatives from the stem cell secretome have a greater potential for large‐scale distribution, thus enticing commercial investors and reciprocally producing more significant medical and social benefits. This review focuses on the paracrine properties of cardiac stem cells and pericytes, two stem cell populations that are increasingly attracting the attention of regenerative medicine operators. It is likely that new cardiovascular drugs are introduced in the next future by applying different approaches based on the refinement of the stem cell secretome.

Active GSK3β and an intact β-catenin TCF complex are essential for the differentiation of human myogenic progenitor cells

Wnt-β-catenin signalling is essential for skeletal muscle myogenesis during development, but its role in adult human skeletal muscle remains unknown. Here we have used human primary CD56Pos satellite cell-derived myogenic progenitors obtained from healthy individuals to study the role of Wnt-β-catenin signalling in myogenic differentiation. We show that dephosphorylated β-catenin (active-β-catenin), the central effector of the canonical Wnt cascade, is strongly upregulated at the onset of differentiation and undergoes nuclear translocation as differentiation progresses. To establish the role of Wnt signalling in regulating the differentiation process we manipulated key nodes of this pathway through a series of β-catenin gain-of-function (GSK3 inhibition and β-catenin overexpression) or loss-of-function experiments (dominant negative TCF4). Our data showed that manipulation of these critical pathway components led to varying degrees of disruption to the normal differentiation phenotype indicating the importance of Wnt signalling in regulating this process. We reveal an independent necessity for active-β-catenin in the fusion and differentiation of human myogenic progenitors and that dominant negative inhibition of TCF4 prevents differentiation completely. Together these data add new mechanistic insights into both Wnt signalling and adult human myogenic progenitor differentiation.

Senescent, dysfunctional human cardiac progenitor cells (CPCs) accumulate in the aged heart and elimination of senescent cells enhances CPC activation and cardiomyocyte proliferation in aged mice

Rationale: Aging leads to increased cellular senescence and is associated with decreased potency of tissue-specific stem/progenitor cells.nObjective: To determine the impact of ageing and senescence on human cardiac stem/progenitor cell (CPC) biology and regenerative potential, and investigate whether elimination of senescent cells in aged mice enhances CPC activation and cardiomyocyte proliferation. nMethods and Results: CPCs were isolated from the right atrial appendage (~200mg) of human subjects with cardiovascular disease (n=119), aged 32-86 years, and assessed for expression of senescence-associated markers (p16INK4A, SAbetagal, DNA damage yH2AX, telomere length), Senescence-Associated Secretory Phenotype (SASP), cell growth, differentiation, and regenerative potential following transplantation into the infarcted mouse heart. Senescent cells were eliminated in aged mice (22 to 32 months) in vivo either genetically, using INK-ATTAC mice, which results in inducible elimination of p16Ink4a-expressing senescent cells upon the administration of the drug AP20187, or pharmacologically using intermittent oral administration of combined senolytics, Dasatinib (D) and Quercetin (Q). In aged subjects (>74 years old) over half of CPCs are senescent, unable to replicate, differentiate, regenerate or restore cardiac function following transplantation into the infarcted heart. Aged-senescent CPCs secrete SASP factors, which renders otherwise healthy, cycling-competent CPCs to senescence. Elimination of senescent CPCs using senolytics abrogates the SASP and its debilitative effect in vitro. Elimination of senescent cells in aged mice (INK-ATTAC or wildtype mice treated with D+Q) in vivo activates resident CPCs (0.23±0.06% vs. 0.01±0.01% vehicle; p<0.05) and increased the number of small, proliferating Ki67-, EdU-positive cardiomyocytes (0.25±0.07% vs. 0.03±0.03% vehicle; p<0.05). nConclusions: Human CPCs become senescent with age, negatively impacting their regenerative capacity. Therapeutic approaches that eliminate senescent cells may alleviate cardiac deterioration with aging and rejuvenate the regenerative capacity of the heart.Aging leads to increased cellular senescence and is associated with decreased potency of tissue-specific stem/progenitor cells. Here we have done an extensive analysis of cardiac progenitor cells (CPCs) isolated from human subjects with cardiovascular disease (n=119), aged 32-86 years. In aged subjects (>74 years old) over half of CPCs are senescent (p16INK4A, SA-β-gal, DNA damage γH2AX, telomere length, Senescence-Associated Secretory Phenotype (SASP)), unable to replicate, differentiate, regenerate or restore cardiac function following transplantation into the infarcted heart. SASP factors secreted by senescent CPCs renders otherwise healthy CPCs to senescence. Elimination of senescent CPCs using senolytics abrogates the SASP and its debilitative effect in vitro. Global elimination of senescent cells in aged mice (INK-ATTAC or wildtype mice treated with D+Q senolytics) in vivo activates resident CPCs (0.23±0.06% vs. 0.01±0.01% vehicle; p<0.05) and increased the number of small, proliferating Ki67-, EdU-positive cardiomyocytes (0.25±0.07% vs. 0.03±0.03% vehicle; p<0.05). Therapeutic approaches that eliminate senescent cells may alleviate cardiac deterioration with aging and rejuvenate the regenerative capacity of the heart.

Progenitor Cells from the Adult Heart

The adult myocardium harbours a population of resident (endogenous) multipotent cardiac stem and progenitor cells (eCSCs). Manipulation of these cells in situ and ex vivo has opened new therapeutic avenues for anatomical and functional myocardial regeneration. However, recently the ability of the c-kitpos stem and progenitor cells to transdifferentiate into new cardiomyocytes has been disputed. Within an already highly controversial research field, these publications have caused significant confusion in their interpretation. Importantly, identifying, tracing and characterising stem and progenitor cells according to expression of a single surface receptor such as c-kit do not identify eCSCs. As discussed in this chapter, eCSCs isolated from the adult heart have a specific phenotype, being negative for blood lineage markers such as CD34, CD45 and CD31, and exhibit properties of stem and progenitor cells, being clonogenic, self-renewing and multipotent. Under the appropriate conditions, eCSCs differentiate into fully functional beating cardiomyocytes and regenerate cardiomyocytes lost from damage in vivo. Finally, eCSCs are susceptible to the effects of ageing, making regulation of this parameter highly impactful in the efficacy of myocardial regenerative therapies.

Transplantation of Allogeneic PW1pos/Pax7neg Interstitial Cells Enhance Endogenous Repair of Injured Porcine Skeletal Muscle

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