Cardiac Side Population Cells Research Articles

Current status, challenges and perspectives of mesenchymal stem cell-based therapy for cardiac regeneration

Modern methods of treating heart failure are similar to the palliative care, since they mostly relieve the symptoms of the disease. The discovery of resident cardiac stem cells gave impetus to the development of “second generation” cell therapy, which quickly moved from animal research to clinical trials with critically ill patients. Many cardiac side population cells have been identified to have stem cells characteristics and some additional individual characteristics, both in vitro and in vivo. The results of clinical studies demonstrated that the stem cell treatment is safe, however, this type of cell-based therapy did not restore cardiac function. Its effects were limited to mildly improving left ventricular systolic pressure and reducing the scar area. Despite that, the promising nature of these therapeutic approaches for heart diseases have contributed to the development of next-generation cell therapy.

Abcg2-expressing side population cells contribute to cardiomyocyte renewal through fusion.

The adult mammalian heart has a limited regenerative capacity. Therefore, identification of endogenous cells and mechanisms that contribute to cardiac regeneration is essential for the development of targeted therapies. The side population (SP) phenotype has been used to enrich for stem cells throughout the body; however, SP cells isolated from the heart have been studied exclusively in cell culture or after transplantation, limiting our understanding of their function in vivo. We generated a new Abcg2-driven lineage-tracing mouse model with efficient labeling of SP cells. Labeled SP cells give rise to terminally differentiated cells in bone marrow and intestines. In the heart, labeled SP cells give rise to lineage-traced cardiomyocytes under homeostatic conditions with an increase in this contribution following cardiac injury. Instead of differentiating into cardiomyocytes like proposed cardiac progenitor cells, cardiac SP cells fuse with preexisting cardiomyocytes to stimulate cardiomyocyte cell cycle reentry. Our study is the first to show that fusion between cardiomyocytes and non-cardiomyocytes, identified by the SP phenotype, contribute to endogenous cardiac regeneration by triggering cardiomyocyte cell cycle reentry in the adult mammalian heart.

Abcg2-Expressing cells fuse with existing cardiomyocytes

Cardiac side population cells (cSPCs) were the first group of progenitor cells identified in the heart; however, their progenitor cell properties have only been established in cell culture and after transplantation. To determine whether cSPCs possess progenitor cell properties in vivo, we generated an Abcg2-driven, lineage-tracing mouse model. In this model, 75.8 ± 10.87% of cSPCs are labeled with GFP. Over a four-week chase period, there is a five-fold increase in cardiomyocyte-labeling from 0.17 ± 0.15% to 0.84 ± 0.24%. Surprisingly, 90.7 ± 2.66% of labeled cardiomyocytes arise from fusion events and not direct differentiation from cSPCs based on labeling in Abgc2MCM/+ R26mTmG/+ mice. Given the extensive labeling of bone marrow and endothelial cells in our model, we hypothesized that the increase in cardiomyocyte labeling over the four-week chase period arises from fusion of unlabeled cardiomyocytes with GFP-labeled bone marrow cells or endothelial cells. To test this hypothesis, we irradiated Myh7Cre/+ R26tdTomato/+ mice, transplanted them with hematopoietic stem (LSK) cells isolated from Abgc2MCM/+ R26GFP/+ and pulsed them with tamoxifen. At the end of a four-week chase period, a similar percentage of bone marrow side population cells, LSK cells and differentiated lineages were labeled with GFP. More importantly, only 0.02 ± 0.03% of cardiomyocytes were labeled with both GFP and tdTomato. No cardiomyocytes were labeled with only GFP. Next, we evaluated whether fusion with endothelial cells could account for the increase in cardiomyocyte labeling we observed. Using an endothelial-specific, lineage-tracing mouse model, Cdh5CreER/+ R26mTmG/+, we found no cardiomyocytes labeled at the end of a four-week chase period. Our results demonstrate that the primary mechanism of cardiomyocyte labeling in our Abcg2-driven, lineage-tracing mouse model arises from fusion of cardiomyocytes with lineage-traced cells, but not with bone marrow or endothelial cells. These results indicate that fusion in the heart may occur at much higher rates than previously assumed, and may play important roles in homeostatic maintenance of cardiac function.

Proliferation, differentiation and migration of SCA1−/CD31− cardiac side population cells in vitro and in vivo

BackgroundSide-population (SP) cells, identified by their capacity to efflux Hoechst dye, are highly enriched for stem/progenitor cell activity. They are found in many mammalian tissues, including mouse heart. Studies suggest that cardiac SP (CSP) cells can be divided into SCA1+/CD31−, SCA1+/CD31+ and SCA1−/CD31− CSP subpopulations. SCA1+/CD31− were shown to be cardiac and endothelial stem/progenitors while SCA1+/CD31+ CSP cells are endothelial progenitors. SCA1−/CD31− CSP cells remain to be fully characterized. In this study, we characterized SCA1−/CD31− CSP cells in the adult mouse heart, and investigated their abilities to proliferate, differentiate and migrate in vitro and in vivo. Methods and resultsUsing fluorescence-activated cell sorting, reverse transcriptase/polymerase chain reaction, assays of cell proliferation, differentiation and migration, and a murine model of myocardial infarction we show that SCA1−/CD31− CSP cells are located in the heart mesenchyme and express genes characteristic of stem cells and endothelial progenitors. These cells were capable of proliferation, differentiation, migration and vascularization in vitro and in vivo. Following experimental myocardial infarction, the SCA1−/CD31− CSP cells migrated from non-infarcted areas to the infarcted region within the myocardium where they differentiated into endothelial cells forming vascular (tube-like) structures. We further demonstrated that the SDF-1α/CXCR4 pathway may play an important role in migration of these cells after myocardial infarction. ConclusionsBased on their gene expression profile, localization and ability to proliferate, differentiate, migrate and vascularize in vitro and in vivo, we conclude that SCA1−/CD31− CSP cells may serve as endothelial progenitor cells in the adult mouse heart.

The Role of Cardiac Side Population Cells in Cardiac Regeneration

The heart has a limited ability to regenerate. It is important to identify therapeutic strategies that enhance cardiac regeneration in order to replace cardiomyocytes lost during the progression of heart failure. Cardiac progenitor cells are interesting targets for new regenerative therapies because they are self-renewing, multipotent cells located in the heart. Cardiac side population cells (cSPCs), the first cardiac progenitor cells identified in the adult heart, have the ability to differentiate into cardiomyocytes, endothelial cells, smooth muscle cells, and fibroblasts. They become activated in response to cardiac injury and transplantation of cSPCs into the injured heart improves cardiac function. In this review, we will discuss the current literature on the progenitor cell properties and therapeutic potential of cSPCs. This body of work demonstrates the great promise cSPCs hold as targets for new regenerative strategies.

Abstract 218: Cardiac Side Population Cells Give Rise to Cardiomyocytes in the Adult Heart

Therapies that enhance cardiac regeneration have the potential to improve heart failure outcomes. One strategy to enhance cardiac regeneration is by activating endogenous cardiac progenitor cells. Cardiac side population cells (cSPCs) are proposed progenitor cells that reside in the heart; however, their putative progenitor cell properties are based on cell culture data and transplantation studies performed with isolated cSPCs. To determine the endogenous regenerative potential of cSPCs in vivo , we generated a mouse model that harbors an Abcg2-driven, tamoxifen-inducible Cre recombinase and crossed it to a GFP reporter mice. One month after tamoxifen treatment, we obtained efficient GFP-labeling of side population cells with 47.0 ± 11.05% of bone marrow side population cells and 75.8 ± 10.87% of cSPCs. Importantly, during a one-month chase period, we observed a three-fold increase in GFP-labeled cardiomyocytes when compared to a 1-week chase period. We quantified the extent of GFP-labeling of cardiomyocytes using adult cardiomyocyte isolation, where we measured 0.8% GFP labeled cardiomyocytes after a one-month chase period. We also observed labeling of many other cell types in the heart, such as endothelial cells, smooth muscle cells, and pericytes. We are currently testing to what extent cardiac injury enhances cSPC derived cardiomyocyte formation. Using our mouse model that efficiently labels side population cells in vivo , we demonstrated that cSPCs contribute cardiomyocytes in the adult heart in vivo .

Oxygen-Generating Photo-Cross-Linkable Hydrogels Support Cardiac Progenitor Cell Survival by Reducing Hypoxia-Induced Necrosis.

Oxygen is essential to cell survival and tissue function. Not surprisingly, ischemia resulting from myocardial infarction induces cell death and tissue necrosis. Attempts to regenerate myocardial tissue with cell based therapies exacerbate the hypoxic stress by further increasing the metabolic burden. In consequence, implanted tissue engineered cardiac tissues suffer from hypoxia-induced cell death. Here, we report on the generation of oxygen-generating hydrogels composed of calcium peroxide (CPO) laden gelatin methacryloyl (GelMA). CPO-GelMA hydrogels released significant amounts of oxygen for over a period of 5 days under hypoxic conditions (1% O2). The released oxygen proved sufficient to relieve the metabolic stress of cardiac side population cells that were encapsulated within CPO-GelMA hydrogels. In particular, incorporation of CPO in GelMA hydrogels strongly enhanced cell viability as compared to GelMA-only hydrogels. Importantly, CPO-based oxygen generation reduced cell death by limiting hypoxia-induced necrosis. The current study demonstrates that CPO based oxygen-generating hydrogels could be used to transiently provide oxygen to cardiac cells under ischemic conditions. Therefore, oxygen generating materials such as CPO-GelMA can improve cell-based therapies aimed at treatment or regeneration of infarcted myocardial tissue.

Leukemia Inhibitory Factor Enhances Endogenous Cardiomyocyte Regeneration after Myocardial Infarction.

Cardiac stem cells or precursor cells regenerate cardiomyocytes; however, the mechanism underlying this effect remains unclear. We generated CreLacZ mice in which more than 99.9% of the cardiomyocytes in the left ventricular field were positive for 5-bromo-4-chloro-3-indolyl-β-d-galactoside (X-gal) staining immediately after tamoxifen injection. Three months after myocardial infarction (MI), the MI mice had more X-gal-negative (newly generated) cells than the control mice (3.04 ± 0.38/mm2, MI; 0.47 ± 0.16/mm2, sham; p < 0.05). The cardiac side population (CSP) cell fraction contained label-retaining cells, which differentiated into X-gal-negative cardiomyocytes after MI. We injected a leukemia inhibitory factor (LIF)-expression construct at the time of MI and identified a significant functional improvement in the LIF-treated group. At 1 month after MI, in the MI border and scar area, the LIF-injected mice had 31.41 ± 5.83 X-gal-negative cardiomyocytes/mm2, whereas the control mice had 12.34 ± 2.56 X-gal-negative cardiomyocytes/mm2 (p < 0.05). Using 5-ethynyl-2'-deoxyurinide (EdU) administration after MI, the percentages of EdU-positive CSP cells in the LIF-treated and control mice were 29.4 ± 2.7% and 10.6 ± 3.7%, respectively, which suggests that LIF influenced CSP proliferation. Moreover, LIF activated the Janus kinase (JAK)signal transducer and activator of transcription (STAT), mitogen-activated protein kinase/extracellular signal-regulated (MEK)extracellular signal-regulated kinase (ERK), and phosphatidylinositol 3-kinase (PI3K)–AKT pathways in CSPs in vivo and in vitro. The enhanced green fluorescent protein (EGFP)-bone marrow-chimeric CreLacZ mouse results indicated that LIF did not stimulate cardiogenesis via circulating bone marrow-derived cells during the 4 weeks following MI. Thus, LIF stimulates, in part, stem cell-derived cardiomyocyte regeneration by activating cardiac stem or precursor cells. This approach may represent a novel therapeutic strategy for cardiogenesis.

A role for matrix stiffness in the regulation of cardiac side population cell function.

The mechanical properties of the local microenvironment may have important influence on the fate and function of adult tissue progenitor cells, altering the regenerative process. This is particularly critical following a myocardial infarction, in which the normal, compliant myocardial tissue is replaced with fibrotic, stiff scar tissue. In this study, we examined the effects of matrix stiffness on adult cardiac side population (CSP) progenitor cell behavior. Ovine and murine CSP cells were isolated and cultured on polydimethylsiloxane substrates, replicating the elastic moduli of normal and fibrotic myocardium. Proliferation capacity and cell cycling were increased in CSP cells cultured on the stiff substrate with an associated reduction in cardiomyogeneic differentiation and accelerated cell ageing. In addition, culture on stiff substrate stimulated upregulation of extracellular matrix and adhesion proteins gene expression in CSP cells. Collectively, we demonstrate that microenvironment properties, including matrix stiffness, play a critical role in regulating progenitor cell functions of endogenous resident CSP cells. Understanding the effects of the tissue microenvironment on resident cardiac progenitor cells is a critical step toward achieving functional cardiac regeneration.

Urotensin II inhibited the proliferation of cardiac side population cells in mice during pressure overload by JNK ‐ LRP 6 signalling

Cardiac side population cells (CSPs) are promising cell resource for the regeneration in diseased heart as intrinsic cardiac stem cells. However, the relative low ratio of CSPs in the heart limited the ability of CSPs to repair heart and improve cardiac function effectively under pathophysiological condition. Which factors limiting the proliferation of CSPs in diseased heart are unclear. Here, we show that urotensin II (UII) regulates the proliferation of CSPs by c-Jun N-terminal kinase (JNK) and low density lipoprotein receptor-related protein 6 (LRP6) signalling during pressure overload. Pressure overload greatly upregulated UII level in plasma, UII receptor (UT) antagonist, urantide, promoted CSPs proliferation and improved cardiac dysfunction during chronic pressure overload. In cultured CSPs subjected to mechanical stretch (MS), UII significantly inhibited the proliferation by UT. Nanofluidic proteomic immunoassay showed that it is the JNK activation, but not the extracellular signal-regulated kinase signalling, that involved in the UII-inhibited- proliferation of CSPs during pressure overload. Further analysis in vitro indicated UII-induced-phospho-JNK regulates phosphorylation of LRP6 in cultured CSPs after MS, which is important in the inhibitory effect of UII on the CSPs during pressure overload. In conclusion, UII inhibited the proliferation of CSPs by JNK/LRP6 signalling during pressure overload. Pharmacological inhibition of UII promotes CSPs proliferation in mice, offering a possible therapeutic approach for cardiac failure induced by pressure overload.

Microfluidics-Assisted Fabrication of Gelatin-Silica Core–Shell Microgels for Injectable Tissue Constructs

Microfabrication technology provides a highly versatile platform for engineering hydrogels used in biomedical applications with high-resolution control and injectability. Herein, we present a strategy of microfluidics-assisted fabrication photo-cross-linkable gelatin microgels, coupled with providing protective silica hydrogel layer on the microgel surface to ultimately generate gelatin-silica core–shell microgels for applications as in vitro cell culture platform and injectable tissue constructs. A microfluidic device having flow-focusing channel geometry was utilized to generate droplets containing methacrylated gelatin (GelMA), followed by a photo-cross-linking step to synthesize GelMA microgels. The size of the microgels could easily be controlled by varying the ratio of flow rates of aqueous and oil phases. Then, the GelMA microgels were used as in vitro cell culture platform to grow cardiac side population cells on the microgel surface. The cells readily adhered on the microgel surface and proliferated over time while maintaining high viability (∼90%). The cells on the microgels were also able to migrate to their surrounding area. In addition, the microgels eventually degraded over time. These results demonstrate that cell-seeded GelMA microgels have a great potential as injectable tissue constructs. Furthermore, we demonstrated that coating the cells on GelMA microgels with biocompatible and biodegradable silica hydrogels via sol–gel method provided significant protection against oxidative stress which is often encountered during and after injection into host tissues, and detrimental to the cells. Overall, the microfluidic approach to generate cell-adhesive microgel core, coupled with silica hydrogels as a protective shell, will be highly useful as a cell culture platform to generate a wide range of injectable tissue constructs.

GW24-e1861 Urotensin II inhibited the proliferation of cardiac side population cells in mice during pressure overload by JNK-LRP6 signalling

ObjectivesCardiac side population cells (CSPs) are promising cell resource for the regeneration in diseased heart as intrinsic cardiac stem cells. However the relative low ratio of CSPs in the heart...

Optimised Protocols for the Identification of the Murine Cardiac Side Population

Cardiac side population (CSP) cells, defined by their ability to efflux the vital dye Hoechst 33342, have been identified as putative cardiac stem cells based on their potential to give rise to both cardiomyocytes and endothelial cells. The CSP phenotype relies on an active metabolic pathway and cell viability to identify a rare population of cells and therefore technical differences in the CSP staining protocol can lead to inconsistent results and discrepancies between studies. Here we describe an established protocol for CSP identification and have optimised a protocol for CSP analysis utilizing an automated cardiac digestion procedure using gentleMACs dissociation and Hoechst 33342 staining followed by dual wavelength flow cytometric analysis.

ASSA13-03-51 Urotensin II Inhibited the Proliferation of Cardiac Side Population Cells in Mice During Pressure Overload by JNK-LRP6 Signalling

ASSA13-03-51 Urotensin II Inhibited the Proliferation of Cardiac Side Population Cells in Mice During Pressure Overload by JNK-LRP6 Signalling

Abstract 18573: Unmasking Phenotypic Micro-heterogeneities in Adult Cardiac Progenitor Cells: Clonal Analysis, Single Cell QRT-PCR, and Fate Mapping

Characterization of adult cardiac progenitor cells (CPC) is incompletely resolved and their potential heterogeneity needs further investigation. Here, we assess the molecular signature, heterogeneity, and origin of adult heart-derived Lin-/Sca-1+ mouse CPCs by clonal analysis, single cell QRT-PCR and fate-mapping. Clonal growth was predicted by the side population (SP) phenotype. Cloned cardiac Lin-/Sca-1+/SP+ CPCs (cSPC) were propagated for &gt;10 months without senescence or loss of the Sca-1/SP phenotype. QRT-PCR of 43 genes in &gt;30 clones showed a consensus signature defined by stem cell-associated markers and key cardiac transcription factors, but lacking markers of pluripotency, primitive or pre-cardiac mesoderm, hematopoiesis, and cardiomyocytes. Clonal analysis revealed mutually exclusive patterns of three core factors (Gata4, Mef2c, Tbx5), masked in the mixed population. Cloned cSPCs showed cardiac (cTnI, MLC2v, sarc MyHC, sarc α-actin), endothelial (vWF) and smooth muscle (SM-MyHC) differentiation after grafting to the heart. Fresh (non-propagated) cSPCs were profiled with microarrays, compared to 3 clones at passage 40, heart, and ESCs: clones largely resembled fresh cSPCs, excepting an increase in proliferation-associated GO categories. Single cell QRT-PCR (Fluidigm) on &gt;40 single fresh cSPCs confirmed both the transcriptional signature of the clones and heterogeneous expression of core cardiac factors. Thus, cSPCs resemble an incomplete but stable form of heart-forming mesoderm. By Cre/lox fate-mapping, we traced cSPCs’ embryological origin. Cardiac SP cells are derived from pre-cardiac mesoderm (98% labelled in Mesp1-Cre/R26R-tdTomato mice) and are not “lapsed” myocytes, neural crest, or hematopoietic in origin (0.2%, 1.2%, 0.2% with Myh6-, Wnt1-, or Vav-Cre). Half are derived from cells formerly expressing Nkx2.5 or Isl1 (52%, 57%), though these are rarely detected in fresh adult single cells (&lt;2%). Thus, our analyses unmask micro-heterogeneities in adult cSPCs, which may represent different states of differentiation or lineage. Expression of key cardiogenic transcription factors in mutually exclusive patterns may represent a mechanism to maintain the stem cell pool and prevent precocious differentiation.

ATP-Binding Cassette G-Subfamily Transporter 2 Regulates Cell Cycle Progression and Asymmetric Division in Mouse Cardiac Side Population Progenitor Cells

After cardiac injury, cardiac progenitor cells are acutely reduced and are replenished in part by regulated self-renewal and proliferation, which occurs through symmetric and asymmetric cellular division. Understanding the molecular cues controlling progenitor cell self-renewal and lineage commitment is critical for harnessing these cells for therapeutic regeneration. We previously have found that the cell surface ATP-binding cassette G-subfamily transporter 2 (Abcg2) influences the proliferation of cardiac side population (CSP) progenitor cells, but through unclear mechanisms. To determine the role of Abcg2 on cell cycle progression and mode of division in mouse CSP cells. Herein, using CSP cells isolated from wild-type and Abcg2 knockout mice, we found that Abcg2 regulates G1-S cell cycle transition by fluorescence ubiquitination cell cycle indicators, cell cycle-focused gene expression arrays, and confocal live-cell fluorescent microscopy. Moreover, we found that modulation of cell cycle results in transition from symmetric to asymmetric cellular division in CSP cells lacking Abcg2. Abcg2 modulates CSP cell cycle progression and asymmetric cell division, establishing a mechanistic link between this surface transporter and cardiac progenitor cell function. Greater understanding of progenitor cell biology and, in particular, the regulation of resident progenitor cell homeostasis is vital for guiding the future development of cell-based therapies for cardiac regeneration.

MTERT EXPRESSION IS INCREASED IN THE MDX MODEL OF CARDIOMYOPATHY

Recent studies have suggested the mammalian heart has innate regenerative potential, but evidence for this remains limited and incomplete. Previously, using the murine telomerase reverse transcriptase (mTert) reporter and fate...

Abstract 267: The Impact of Modulating Afterload on Cardiac Progenitor Cells

Background: Increased hemodynamic stress induces left ventricular hypertrophy followed by heart failure. Accumulating evidence suggests that the adult mammalian heart possess regeneration ability through the action of resident stem/progenitor cell. However, the effects of hemodynamic fluctuation on cardiac progenitor cell proliferation and cardiac repair remain unknown. Results: Proliferation of Cardiac Side Population (CSP) cells and cardiomyocytes was evaluated in experimental models of Ascending Aortic Constriction (AAC)-induced left ventricular hypertrophy (LVH) in mice, over a period of seven weeks. Continuous AAC caused LVH while increasing the number of BrdU + CSP cells (one week post-AAC) and the amount of BrdU + cardiomyocytes (seven weeks post-AAC). To evaluate the effects of pressure fluctuation on CSP cell and cardiomyocyte proliferation we generated another group (de-AAC) in which the constriction was removed one week post-AAC. Interestingly, restoration of cardiac pressure further increased the proliferative capacity of CSP cells and cardiomyocytes. Conclusion: Our data show that restoration of hemodynamic stress confers favorable effects on cardiac repair through increase proliferation of CSP cells and cardiomyocytes. Thus, the hemodynamic state of the heart might be related to CSP cell proliferation and cardiomyogenesis.

Spot Identification and Quality Control in Cell-Based Microarrays

Cell-based microarrays are being increasingly used as a tool for combinatorial and high throughput screening of cellular microenvironments. Analysis of microarrays requires several steps, including microarray imaging, identification of cell spots, quality control, and data exploration. While high content image analysis, cell counting, and cell pattern recognition methods are established, there is a need for new postprocessing and quality control methods for cell-based microarrays used to investigate combinatorial microenvironments. Previously, microarrayed cell spot identification and quality control were performed manually, leading to excessive processing time and potentially resulting in human bias. This work introduces an automated approach to identify cell-based microarray spots and spot quality control. The approach was used to analyze the adhesion of murine cardiac side population cells on combinatorial arrays of extracellular matrix proteins. Microarrays were imaged by automated fluorescence microscopy and cells were identified using open-source image analysis software (CellProfiler). From these images, clusters of cells making up single cell spots were reliably identified by analyzing the distances between cells using a density-based clustering algorithm (OPTICS). Naïve Bayesian classifiers trained on manually scored training sets identified good and poor quality spots using spot size, number of cells per spot, and cell location as quality control criteria. Combined, the approach identified 78% of high quality spots and 87% of poor quality spots. Full factorial analysis of the resulting microarray data revealed that collagen IV exhibited the highest positive effect on cell attachment. This data processing approach allows for fast and unbiased analysis of cell-based microarray data.

Mitochondrial DNA deletion mutations in adult mouse cardiac side population cells

We investigated the presence and potential role of mitochondrial DNA (mtDNA) deletion mutations in adult cardiac stem cells. Cardiac side population (SP) cells were isolated from 12-week-old mice. Standard polymerase chain reaction (PCR) was used to screen for the presence of mtDNA deletion mutations in (a) freshly isolated SP cells and (b) SP cells cultured to passage 10. When present, the abundance of mtDNA deletion mutation was analyzed in single cell colonies. The effect of different levels of deletion mutations on SP cell growth and differentiation was determined. MtDNA deletion mutations were found in both freshly isolated and cultured cells from 12-week-old mice. While there was no significant difference in the number of single cell colonies with mtDNA deletion mutations from any of the groups mentioned above, the abundance of mtDNA deletion mutations was significantly higher in the cultured cells, as determined by quantitative PCR. Within a single clonal cell population, the detectable mtDNA deletion mutations were the same in all cells and unique when compared to deletions of other colonies. We also found that cells harboring high levels of mtDNA deletion mutations (i.e. where deleted mtDNA comprised more than 60% of total mtDNA) had slower proliferation rates and decreased differentiation capacities. Screening cultured adult stem cells for mtDNA deletion mutations as a routine assessment will benefit the biomedical application of adult stem cells.