Resident Stem Cell Population Research Articles

Hematopoietic, CNS And Skeletal Muscle Stem Cells As Drug Targets: Opportunities, Progress And Challenges

Lineage-committed stem and progenitor cells are currently targeted by a handful of medicines, mainly to treat conditions involving the immune and hematopoietic systems. Knowledge of new stem and progenitor cell populations in the body is accumulating at a rapid pace and a new era of targeting resident stem cell populations for therapeutic ends is coming into focus. Small-molecule regulators of body-resident stem and progenitor cell assess are now a reality both in the clinic and as promising new drugs in the development pipeline. This review will explore the current state of the art with an emphasis on emerging concepts and experimental systems in the therapeutic regulation of endogenous stem and progenitor cell populations.

Ciliary Epithelium: An Underevaluated Target for Therapeutic Regeneration

The purpose of stem cells in various organs of vertebrates is to replenish dying cells or to replace damaged tissues. However, a few organs have reasonable, while others have very limited regenerative, capacity. Until the last two decades, the organs such as brain, heart, and kidneys were known to lack regenerative capacity for lack of resident stem cell population. However, with advancement of techniques and an increase in scientific communication, new discoveries have brought novel concepts and data to discover and manipulate these valuable resources. Much focus has been devoted to understanding the regulation and maintenance of these stem cells. We discuss the preclinical data emerging from retino-vascular interactions useful in the exploitation of ciliary epithelium−derived stem cells for therapeutic regeneration.

Ciliary Epithelium: An Underevaluated Target for Therapeutic Regeneration

The purpose of stem cells in various organs of vertebrates is to replenish dying cells or to replace damaged tissues. However, a few organs have reasonable, while others have very limited regenerative, capacity. Until the last two decades, the organs such as brain, heart, and kidneys were known to lack regenerative capacity for lack of resident stem cell population. However, with advancement of techniques and an increase in scientific communication, new discoveries have brought novel concepts and data to discover and manipulate these valuable resources. Much focus has been devoted to understanding the regulation and maintenance of these stem cells. We discuss the preclinical data emerging from retino-vascular interactions useful in the exploitation of ciliary epithelium-derived stem cells for therapeutic regeneration.

Bmi1 Is Expressed in Postnatal Myogenic Satellite Cells, Controls Their Maintenance and Plays an Essential Role in Repeated Muscle Regeneration

Satellite cells are the resident stem cell population of the adult mammalian skeletal muscle and they play a crucial role in its homeostasis and in its regenerative capacity after injury. We show here that the Polycomb group (PcG) gene Bmi1 is expressed in both the Pax7 positive (+)/Myf5 negative (−) stem cell population as well as the Pax7+/Myf5+ committed myogenic progenitor population. Depletion of Pax7+/Myf5− satellite cells with reciprocal increase in Pax7+/Myf5+ as well as MyoD positive (+) cells is seen in Bmi1−/− mice leading to reduced postnatal muscle fiber size and impaired regeneration upon injury. Bmi1−/− satellite cells have a reduced proliferative capacity and fail to re-enter the cell cycle when stimulated by high serum conditions in vitro, in keeping with a cell intrinsic defect. Thus, both the in vivo and in vitro results suggest that Bmi1 plays a crucial role in the maintenance of the stem cell pool in postnatal skeletal muscle and is essential for efficient muscle regeneration after injury especially after repeated muscle injury.

Preparation of Epithelial and Mesenchymal Stem Cells from Murine Mammary Gland

The mammary gland is a complex organ consisting of multiple cell types that undergo extensive remodeling during pregnancy and involution, cyclical changes that suggest the existence of a resident stem cell population that is responsible for remarkable tissue regeneration. The basic functional unit of the mammary gland is the terminal duct lobular unit, which invades the stromal tissue (fat, connective tissue, blood vessels, etc.). Luminal epithelial cells line the ducts while outer myoepithelial cells secrete the basal lamina that separates the mammary gland parenchyma from the mesenchymal cells of the stroma. Within the epithelial cell population of the ducts resides the mammary gland stem cells and it is believed that this population is the origin of the mammary gland cancer stem cells as well. In the mouse, epithelial stem cells can be separated from mesenchymal cells on the basis of CD24, CD44, and CD49f expression. This allows for the determination of both normal and cancer stem cell potential of these two populations and permits investigation into their interaction in tumor development.

Stem Cell Therapy in Cardiology

Cell therapy provides one of the most important solutions to the unmet need for new treatments in cardiovascular disease. This area of research has undergone a rapid translation into humans, with the bone marrow mononuclear cell predominating as the cell type with most clinical data. Confidence in the use of this cell type has grown over the last year with the publication of the results of Phase II/III trials in the setting of acute and chronic ischemia confirming safety and biological activity. A large pan-European outcome study is now being planned, which will definitely address the therapeutic potential of this cell type with respect to mortality. Data for the use of selected populations of cells, bioengineered cells/scaffolds and the resident stem cell population continue to grow, with some of these approaches reported in Phase I clinical trials with promising results. There is still some way to go and these more complicated cell therapy products will need to undergo the same scrutiny that has been applied to the results of the bone marrow mononuclear cell trials to date. Ultimately, these engineered biologics will have to justify the costs involved in producing them by significantly improving on results obtained by using bone marrow mononuclear cells for cardiovascular repair. The continued success of this area of translational medicine relies on the ongoing partnership between clinicians and scientists, who have thus far demonstrated a determined and pragmatic approach to solving some of the complexities of moving from bench to bedside. The next 5-years will see this partnership reach fruition as the long-awaited results of outcome studies of cell therapy in the treatment of cardiovascular disease are published.

De novo cardiomyocytes from within the activated adult heart after injury

A significant bottleneck in cardiovascular regenerative medicine is the identification of a viable source of stem/progenitor cells that could contribute new muscle after ischaemic heart disease and acute myocardial infarction. A therapeutic ideal--relative to cell transplantation--would be to stimulate a resident source, thus avoiding the caveats of limited graft survival, restricted homing to the site of injury and host immune rejection. Here we demonstrate in mice that the adult heart contains a resident stem or progenitor cell population, which has the potential to contribute bona fide terminally differentiated cardiomyocytes after myocardial infarction. We reveal a novel genetic label of the activated adult progenitors via re-expression of a key embryonic epicardial gene, Wilm's tumour 1 (Wt1), through priming by thymosin β4, a peptide previously shown to restore vascular potential to adult epicardium-derived progenitor cells with injury. Cumulative evidence indicates an epicardial origin of the progenitor population, and embryonic reprogramming results in the mobilization of this population and concomitant differentiation to give rise to de novo cardiomyocytes. Cell transplantation confirmed a progenitor source and chromosome painting of labelled donor cells revealed transdifferentiation to a myocyte fate in the absence of cell fusion. Derived cardiomyocytes are shown here to structurally and functionally integrate with resident muscle; as such, stimulation of this adult progenitor pool represents a significant step towards resident-cell-based therapy in human ischaemic heart disease.

Adult Skeletal Muscle Stem Cell Migration Is Mediated by a Blebbing/Amoeboid Mechanism

Adult skeletal muscle possesses a resident stem cell population called satellite cells, which are responsible for tissue repair following damage. Satellite cell migration is crucial in promoting rapid tissue regeneration, but it is a poorly understood process. Furthermore, the mechanisms facilitating satellite cell movement have yet to be elucidated. This study investigates the process of satellite cell migration, revealing that they undergo two distinct phases of movement, first under the basal lamina and then rapidly increasing their velocity when on the myofiber surface. Most significantly, we show that satellite cells move using a highly dynamic blebbing or amoeboid-based mechanism and not via lamellipodia-mediated propulsion. We show that nitric oxide and noncanonical Wnt signaling pathways are necessary for regulating the formation of blebs and the migration of satellite cells. In summary, we propose that the formation of blebs and their necessity for satellite cell migration has significant implications in the future development of therapeutic regimes aimed at promoting skeletal muscle regeneration.

The mechanical coupling of adult marrow stromal stem cells during cardiac regeneration assessed in a 2-D co-culture model

Postnatal cardiomyocytes undergo terminal differentiation and a restricted number of human cardiomyocytes retain the ability to divide and regenerate in response to ischemic injury. However, whether these neo-cardiomyocytes are derived from endogenous population of resident cardiac stem cells or from the exogenous double assurance population of resident bone marrow-derived stem cells that populate the damaged myocardium is unresolved and under intense investigation. The vital challenge is to ameliorate and/or regenerate the damaged myocardium. This can be achieved by stimulating proliferation of native quiescent cardiomyocytes and/or cardiac stem cell, or by recruiting exogenous autologous or allogeneic cells such as fetal or embryonic cardiomyocyte progenitors or bone marrow-derived stromal stem cells. The prerequisites are that these neo-cardiomyocytes must have the ability to integrate well within the native myocardium and must exhibit functional synchronization. Adult bone marrow stromal cells (BMSCs) have been shown to differentiate into cardiomyocyte-like cells both in vitro and in vivo. As a result, BMSCs may potentially play an essential role in cardiac repair and regeneration, but this concept requires further validation. In this report, we have provided compelling evidence that functioning cardiac tissue can be generated by the interaction of multipotent BMSCs with embryonic cardiac myocytes (ECMs) in two-dimensional (2-D) co-cultures. The differentiating BMSCs were induced to undergo cardiomyogenic differentiation pathway and were able to express unequivocal electromechanical coupling and functional synchronization with ECMs. Our 2-D co-culture system provides a useful in vitro model to elucidate various molecular mechanisms underpinning the integration and orderly maturation and differentiation of BMSCs into neo-cardiomyocytes during myocardial repair and regeneration.

Mesenchymal stromal cells stimulate C2C12 myoblast cell proliferation: potential relevance in skeletal muscle regeneration

Skeletal muscle tissue harbors of a population of resident myoblastic stem cells which are capable of regenerating the damaged tissue. However, in case of extended injury, their myogenic potential is compromised by excessive inflammatory response and collagen deposition which hamper the environmental conducivity to muscle regeneration. Therefore, the identification of new strategies aimed at potentiating the proliferative attitude of myoblasts may have potential therapeutic application for muscle repair. In this line, we have previously demonstrated in a co-culture system, that adult mouse bone marrow-derived mesenchymal stromal/stem cells (MSCs) stimulate mouse neonatal cardiomyocyte proliferation. On these premises, in the present study, we searched whether MSCs were also able to influence C2C12 myoblast cells proliferation. We found that myoblast proliferation was significantly enhanced in co-culture as compared with the single culture, and that this response involved the activation of Notch-1 signalling in the myoblastic cells. These data were confirmed by the finding that Hes1 transcriptional regulator, the major downstream effector of Notch-1, was also upregulated in C2C12 cells cultured in the presence of MSCs or their derived conditioned medium. In particular, MSCs were able to release growth factors, including FGF and VEGF, thus underscoring potential paracrine mechanisms involved in the regulation of myoblast proliferation and Notch-1 expression by MSCs. In conclusion, the results of the present study provide strong evidence for a role of MSCs in stimulating myoblast cell proliferation and suggest that the functional interaction betweeen the two cell types may be exploited to the development of new and more efficient cell-based skeletal muscle repair strategies.

The Importance of Stem Cells and Bioengineering in Regenerative Medicine

The field of regenerative medicine is focused on the creation of functional human tissue to either model, repair or replace the damaged body part. This provides promise that in the future scientists will be able to bioengineer human tissue in vitro [1,2] that may allow the identification of novel medicines and has the potential for cell based therapies. If successful, such approaches could have a significant impact on the problem donor organ shortage for transplantation and improve patient treatment and medication. The liver is a fascinating organ, performing many processes necessary for life. In addition to its broad functional repertoire, the liver is the only human organ capable of large scale regeneration. Only 20-30% of healthy liver mass is required liver regeneration. The primary regenerative response of the liver is provided by its principal cell type, the hepatocyte. However when the liver is overloaded or chronically damaged, the hepatocyte response is inhibited, prompting the activation of a resident stem cell population. In both cases, acute and chronic liver injury may result in irreparable loss of human liver function marking the onset of liver disease. Presently the only real treatment strategy for critically failing liver function is organ transplantation. While highly successful, the shortage of donor organs limits its widespread implementation. The generation of liver tissue from a renewable resource therefore holds great promise for the development of extra-corporeal liver support devices and transplantation procedures.

Tissue-Resident Adult Stem Cell Populations of Rapidly Self-Renewing Organs

The epithelial lining of the intestine, stomach, and skin is continuously exposed to environmental assault, imposing a requirement for regular self-renewal. Resident adult stem cell populations drive this renewal, and much effort has been invested in revealing their identity. Reliable adult stem cell biomarkers would accelerate our understanding of stem cell roles in tissue homeostasis and cancer. Membrane-expressed markers would also facilitate isolation of these adult stem cell populations for exploitation of their regenerative potential. Here, we review recent advances in adult stem cell biology, highlighting the promise and pitfalls of the candidate biomarkers of the various stem cell populations.

Skeletal Myoblasts for Cardiac Repair

Stem cells provide an alternative curative intervention for the infarcted heart by compensating for the cardiomyocyte loss subsequent to myocardial injury. The presence of resident stem and progenitor cell populations in the heart, and nuclear reprogramming of somatic cells with genetic induction of pluripotency markers are the emerging new developments in stem cell-based regenerative medicine. However, until safety and feasibility of these cells are established by extensive experimentation in in vitro and in vivo experimental models, skeletal muscle-derived myoblasts, and bone marrow cells remain the most well-studied donor cell types for myocardial regeneration and repair. This article provides a critical review of skeletal myoblasts as donor cells for transplantation in the light of published experimental and clinical data, and indepth discussion of the advantages and disadvantages of skeletal myoblast-based therapeutic intervention for augmentation of myocardial function in the infarcted heart. Furthermore, strategies to overcome the problems of arrhythmogenicity and failure of the transplanted skeletal myoblasts to integrate with the host cardiomyocytes are discussed.

Targeting ependymal stem cells in vivo as a non-invasive therapy for spinal cord injury

Spinal cord injury (SCI) is a debilitating and devastating condition, and there are approximately 12,000 new cases in the USA each year and an estimated number of sufferers reaching 250,000–300,000 in the USA alone. Although important advances have been made in the medical treatment of SCI

Caloric restriction attenuates the age-associated increase of adipose-derived stem cells but further reduces their proliferative capacity

White adipose tissue is a promising source of mesenchymal stem cells. Currently, little is known about the effect of age and caloric restriction (CR) on adipose-derived stem cells (ASC). This is important for three reasons: firstly, age and CR cause extensive remodeling of WAT; it is currently unknown how this remodeling affects the resident stem cell population. Secondly, stem cell senescence has been theorized as one of the causes of aging and could reduce the utility of a stem cell as a reagent. Thirdly, the mechanism by which CR extends lifespan is currently not known, one theory postulates that CR maintains the resident stem cell population in youthful "fit" state. For the purpose of this study, we define ASC as lineage negative (lin(-))/CD34(+(low))/CD31(-). We show that aging increases the abundance of ASC and the expression of Cdkn2a 9.8-fold and Isl1 60.6-fold. This would suggest that aging causes an accumulation of non-replicative ASC. CR reduced the percentage of ASC in the lin(-) SVF while also reducing colony forming ability. Therefore, CR appears to have anti-proliferative effects on ASC that may be advantageous from the perspective of cancer, but our data raises the possibility that it may be disadvantageous for regenerative medicine applications.

Germline self-renewal requires cyst stem cells and stat regulates niche adhesion in Drosophila testes

Adults maintain tissue-specific stem cells through niche signals. A model for niche function is the Drosophila melanogaster testis, where a small cluster of cells called the hub produce locally available signals that allow only adjacent cells to self-renew. We show here that the principal signalling pathway implicated in this niche, chemokine activation of STAT, does not primarily regulate self-renewal of germline stem cells (GSCs), but rather governs GSC adhesion to hub cells. In fact, GSC renewal does not require hub cell contact, as GSCs can be renewed solely by contact with the second resident stem cell population, somatic cyst stem cells (CySCs), and this involves BMP signalling. These data suggest a modified paradigm whereby the hub cells function as architects of the stem cell environment, drawing into proximity cellular components necessary for niche function. Self-renewal functions are shared by the hub cells and the CySCs. This work also reconciles key differences in GSC renewal between Drosophila testis and ovary niches, and highlights how a niche can coordinate the production of distinct lineages by having one stem cell type rely on a second.

Potential and pitfalls of stem cell therapy in old age

Our increasing understanding of resident stem cell populations in various tissues of the adult body provides promise for the development of cell-based therapies to treat trauma and disease. With the sharp rise in the aging population, the need for effective regenerative medicine strategies for the aged is more important then ever. Yet, the vast majority of research fuelling our understanding of the mechanisms that control stem cell behaviour, and their role in tissue regeneration, is conducted in young animals. Evidence collected in the last several years indicates that, although stem cells remain active into old age, changes in the stem cells and their microenvironments inhibit their regenerative potential. An understanding of both the cell-intrinsic stem cell changes, as well as concomitant changes to the stem cell niche and the systemic environment, are crucial for the development of regenerative medicine strategies that might be successful in aged patients.

The production of fluorescent transgenic trout to study in vitro myogenic cell differentiation

BackgroundFish skeletal muscle growth involves the activation of a resident myogenic stem cell population, referred to as satellite cells, that can fuse with pre-existing muscle fibers or among themselves to generate a new fiber. In order to monitor the regulation of myogenic cell differentiation and fusion by various extrinsic factors, we generated transgenic trout (Oncorhynchus mykiss) carrying a construct containing the green fluorescent protein reporter gene driven by a fast myosin light chain 2 (MlC2f) promoter, and cultivated genetically modified myogenic cells derived from these fish.ResultsIn transgenic trout, green fluorescence appeared in fast muscle fibers as early as the somitogenesis stage and persisted throughout life. Using an in vitro myogenesis system we observed that satellite cells isolated from the myotomal muscle of transgenic trout expressed GFP about 5 days post-plating as they started to fuse. GFP fluorescence persisted subsequently in myosatellite cell-derived myotubes. Using this in vitro myogenesis system, we showed that the rate of muscle cell differentiation was strongly dependent on temperature, one of the most important environmental factors in the muscle growth of poikilotherms.ConclusionsWe produced MLC2f-gfp transgenic trout that exhibited fluorescence in their fast muscle fibers. The culture of muscle cells extracted from these trout enabled the real-time monitoring of myogenic differentiation. This in vitro myogenesis system could have numerous applications in fish physiology to evaluate the myogenic activity of circulating growth factors, to test interfering RNA and to assess the myogenic potential of fish mesenchymal stem cells. In ecotoxicology, this system could be useful to assess the impact of environmental factors and marine pollutants on fish muscle growth.

Abstract 3357: Isolation of tumor cell subpopulations using semi-automated tissue dissociation and magnetic cell separation

Abstract The analysis of tumor resident cell and stem cell populations requires proper methods for tissue dissociation and cell purification. Over the past few years, there has been a particular focus on the study of tumor infiltrating lymphocytes (TIL) and cancer stem cells (CSC). TIL can be isolated and characterized using conventional lymphocyte markers whereas CSC-specific cell surface markers are currently established (1). Two important markers, namely CD24 and CD44, have been shown to define CSC in various neoplasms such as breast cancer (2) or colon cancer (3). We have established methods for the enzymatic and mechanical dissociation of solid tumors and optimized them according to the specific needs of a given tissue or cell type. Solid tumors are built up of a mixture of cell types, which are interconnected to each other and surrounded by an extracellular matrix composed of a variety of proteins and polysaccharides. The major goal was to disrupt the extracellular matrix and cell adhesion components without harming the integrity of the cell membrane. This was achieved by a combination of varying enzyme mixes, mechanical forces, incubation periods, and temperatures. The automation of all mechanical steps (gentleMACS™ dissociator) led to reproducible and moreover user independent results with reduced overall processing times. Viable tumor infiltrating lymphocytes (TIL) as well as tumor cells were obtained from melanoma metastases in high numbers. The isolated TIL expanded significantly faster than those from manually dissociated tumors and showed functionality in vitro and in vivo. As for cancer stem cells, we present novel tools for the immunomagnetic isolation of CD44+ and CD44+/CD24- cells. Using magnetic cell sorting (MACS®), a subpopulation of human teratocarcinoma cells could be isolated to a purity of 92% based on their expression of CD44. Furthermore, a combined depletion and positive selection approach allowed for the enrichment of a CD44+/CD24- human CML subpopulation from a starting frequency of 34% to final purity of 95%. The separation of pure tumor cell populations will be necessary for their further characterization and also targeted drug screening approaches. The methods presented here are broadly applicable as they can be easily adapted to other cancer tissues and cell types and should help to optimize and standardize the future basic and clinical cancer research. 1. Trumpp, A. and Wiestler, O. D. (2008); Nature Clin. Prac. Oncology.; Vol. 5 No. 6 2. Al-Hajj, M. et al. (2003); Proc. Natl. Acad. Sci. USA; Vol. 100 No. 7 3. Du, L. et al. (2008); Clin. Cancer Res.; 6751 14 (21) Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3357.

Seizure induces activation of multiple subtypes of neural progenitors and growth factors in hippocampus with neuronal maturation confined to dentate gyrus

Adult hippocampal neurogenesis is altered in response to different physiological and pathological stimuli. GFAP +ve/nestin +ve radial glial like Type-1 progenitors are considered to be the resident stem cell population in adult hippocampus. During neurogenesis these Type-1 progenitors matures to GFAP −ve/nestin +ve Type-2 progenitors and then to Type-3 neuroblasts and finally differentiates into granule cell neurons. In our study, using pilocarpine-induced seizure model, we showed that seizure initiated activation of multiple progenitors in the entire hippocampal area such as DG, CA1 and CA3. Seizure induction resulted in activation of two subtypes of Type-1 progenitors, Type-1a (GFAP +ve/nestin +ve/BrdU +ve) and Type-1b (GFAP +ve/nestin +ve/BrdU −ve). We showed that majority of Type-1b progenitors were undergoing only a transition from a state of dormancy to activated form immediately after seizures rather than proliferating, whereas Type-1a showed maximum proliferation by 3 days post-seizure induction. Type-2 (GFAP −ve/nestin +ve/BrdU +ve) progenitors were few compared to Type-1. Type-3 (DCX +ve) progenitors showed increased expression of immature neurons only in DG region by 3 days after seizure induction indicating maturation of progenitors happens only in microenvironment of DG even though progenitors are activated in CA1 and CA3 regions of hippocampus. Also parallel increase in growth factors expression after seizure induction suggests that microenvironmental niche has a profound effect on stimulation of adult neural progenitors.