Pure Population Of Stem Cells Research Articles

In-Vitro Proliferation and Enrichment of CD34+ Autologous Haematopoietic Stem Cells, Obtained from Patients with End-Stage Left Ventricular Failure

With a rising global prevalence of end-stage heart failure and a limited availability of cardiac transplantation programmes or left ventricular assist devices in many parts of the world, there is a renewed interest in potential alternative treatment options.Along with other groups, we have previously demonstrated the beneficial effect of intracoronary autologous bone marrow derived peripheral haemopoietic stem cell transplantation, to improve myocardial contractility in end-stage heart failure.We now assess the ability of these cells to expand in numbers in-vitro, to achieve enrichment of stem cell counts, using standard cell culture methods and flow- cytometric analysis.We demonstrate that with in-vitro culturing (within 3 passages post-harvest), the CD34+ stem cell fraction increases in standard culture media, across multiple samples. However, optimal culture conditions to achieve near pure stem cell populations with rapid cell proliferation, still needs to be defined.

AB0039 ROLE OF MESENCHYMAL STEM CELLS ISOLATED FROM DENTAL PULP (DPSCS) IN IMMUNOREGULATION PROCESSES MEDIATED BY PROGRAMMED DEATH-LIGAND 1 (PD-L1)

Background:Stem cells isolated from dental pulp (DPSCs) are characterized by a high rate of proliferation, low immunogenicity and a high ability to differentiate in different lineages (i.e. osteogenic, chondrogenic, adipogenic, myogenic and neural commitment). Their multipotency can be attributed to the peculiar embryological origin from the neural crest. DPSCs represent a promising stem cell resource since they hold a low ethical impact and can be easily isolated through routine dental procedures. These cells own immuno-modulatory properties, exerted through the activation of different mechanisms, including the Fas / FasL pathway, as well as through the release of soluble factors. Currently, other molecular mechanisms are under consideration such as PD-1 / PD-L1 (Programmed Death 1 and its Ligand) which are supposed to be involved in the induction and / or maintenance of immune tolerance.Objectives:The aim of this research was to investigate whether the stimulation of PD-L1 in DPSCs can affect the immunomodulatory effects of these stem cells on peripheral blood mononuclear cells (PBMCs). Furthermore, the expression of PD-L1 was also assayed after the induction of osteogenic differentiation of DPSCs in order to evaluate a possible application of DPSCs in autoimmune inflammatory osteo-erosive diseases.Methods:Immuno-selection was performed on DPSCs, isolated from waste material, against the stemness markers c-Kit and STRO-1, to obtain a pure stem cell population. Then, STRO-1+/c-Kit+ DPSCs, were co-cultured either directly and indirectly with peripheral blood mononuclear cells (PBMCs) from healthy adult donors, previously activated by anti-CD3 and anti-CD28 antibodies. Co-cultures of PBMCs with amniotic fluid stem cells (AFSCs) and bone marrow mesenchymal stem cells (BM-MSCs) were also set up. The expression of PD-1 in PBMCs as well as of PD-L1 in DPSCs, AFSCs, BM-MSCs and PBMCs, was evaluated by Western Blot (WB) and immunofluorescence (IF) analyses, before and after osteogenic differentiation. Osteogenic differentiation of DPSCs, after 30 days of induction, was verified by IF and WB, of osteopontin, osteocalcin and RUNX2 markers. Interleukin-2 (IL-2) expression levels in PBMCs were analyzed by Real-Time PCR analysis.Results:Our data highlight that, after direct and indirect co-culture with activated PBMCs, PD-L1 expression was up-regulated not only in DPSCs, but also in BM-MSCs and AFSCs (Figure 1), thus suggesting that 1) this is a common ability of mesenchymal stem cells and 2) this event can be also mediated by soluble factors release. Moreover, when evaluating the effects of DPSCs co-culture on PBMCs an increased expression of cleaved caspase 3 was observed, together with a decreased expression of IL-2 - a growth factor essential for the proliferation and survival of T cells (Figure 2). These findings showed how DPSCs can modulate the immune system by PD-L1 up-regulation. On the other hand, it is noteworthy that, after reaching osteogenic commitment, DPSCs down-regulated the expression of PD-L1, allowing to hypothesize that PD-L1 expression is strictly related to the maintenance of stemness.Figure 1.Figure 2.Conclusion:Taken together, our findings suggest that the expression of PD-L1 in DPSCs is involved in the modulation of immune response and pave the way for further investigations on the role of PD-1/PD-L1 pathway in controlling inflammation and immune response when applied to the treatment of autoimmune inflammatory diseases.

Mitos y realidades del uso de las células troncales en la terapia oftalmológica

During the last 20 years, stem cells became a central issue for many research groups, and the ophthalmologic field is not the exception. Under such scenario, the huge amount of available information together with contradictory results may lead to confusion; making the continuous updating by health professionals difficult and challenging. Here, we describe some of the essential properties of stem cells, as well as the intricacies associated with their use in ophthalmic therapy. It is clear that despite stem cells may be a very useful tool in the treatment of several diseases, we still ignore many of their properties as well as the mechanisms that regulate them. Also, it is significant the lack of procedures that could allow their isolation as a pure stem cell population and their subsequent controlled transplantation into patients as completely differentiated cells; facts that imply a real risk for the candidate to this kind of treatment. At present, therapies that involve the use of stem cells have focused on treatment of wet or dry AMD, as well as LSCD. However, considering the current difficulties, one can conclude that maybe it is time to pause possible additional therapeutic uses and gain a better understanding of stem cells before we continue treating ophthalmologic disease.

In vitro cementoblast-like differentiation of postmigratory neural crest-derived p75+ stem cells with dental follicle cell conditioned medium

Cranial neural crest-derived cells (CNCCs) play important role in epithelial–mesenchymal interactions during tooth morphogenesis. However, the heterogeneity of CNCCs and their tendency to spontaneously differentiate along smooth muscle or osteoblast lineages in vitro limit further understanding of their biological properties. We studied the differentiation properties of isolated rat embryonic postmigratory CNCCs, expressing p75 neurotrophin receptor (p75NTR). These p75NTR positive (p75+) CNCCs, isolated using fluorescence activated cell sorter, exhibited fibroblast-like morphology and characteristics of mesenchymal stem cells. Incubation of p75+ CNCCs in dental follicle cell conditioned medium (DFCCM) combined with dentin non-collagenous proteins (dNCPs), altered their morphological features to cementoblast-like appearance. These cells also showed low proliferative activity, high ALP activity and significantly increased calcified nodule formation. Markers related to mineralization or specific to cementoblast lineage were highly expressed in dNCPs/DFCCM-treated p75+ cells, suggesting their differentiation along cementoblast-like lineage. p75+ stem cells selected from postmigratory CNCCs represent a pure stem cell population and could be used as a stem cell model for in vitro studies due to their intrinsic ability to differentiate to neuronal cells and transform from neuroectoderm to ectomesenchyme. They can provide a potential stem cell resource for tooth engineering studies and help to further investigate mechanisms of epithelial–mesenchymal interactions in tooth morphogenesis.

Human epidermal neural crest stem cells as candidates for cell-based therapies, disease modeling, and drug discovery.

In this review article I explore the suitability of human epidermal neural crest stem cells (hEPI-NCSC) for translational medicine. hEPI-NCSC are multipotent somatic stem cells that are derived from the embryonic neural crest. hEPI-NCSC are located in the bulge of hair follicles where they persist postnatally and into adulthood. Because of their location in the hairy skin and their migratory behavior, hEPI-NCSC can be easily isolated as a highly pure population of stem cells without the need for purification. Furthermore they can be expanded ex vivo into millions of stem cells, they do not form tumors in vivo, and they can undergo directed differentiation into crest and noncrest-derived cell types of clinical relevance. Taken together, these characteristics make hEPI-NCSC attractive candidates for cell-based therapies, drug discovery, and disease modeling.

184 ISOLATION AND CHARACTERIZATION OF BOVINE ENDOMETRIAL STEM CELLS

Adult mesenchymal stem cells had been isolated from various tissues of different species, including endometrial tissue of humans, mice, and pigs, but not from cattle. The aim of our work was to identify such cells in the bovine endometrium and to establish a model system in which to test inducers of differentiation and recruiters of stem cell niches, for potential therapeutic use in this and other species, such as horses. We searched for endometrial stem cells in healthy cycling cows and in cattle with clinical (C) or subclinical (SC) endometritis. For this, the uterine tracts of slaughtered cows were collected at early (Days 2 to 5; ELF) and late luteal phases (Days 11 to 15; LLF) of the oestrus cycle of healthy cows. For endometritis-diseased cattle, uterine biopsies were taken in live animals. In all cases, markers of stemness, inflammation, uterine function, and housekeeping were studied both at mRNA and protein level, by RT-qPCR and Western blot/immunohistochemistry respectively. In addition, cell primary cultures were established in vitro from all the animals (n = 4 for ELF, n = 4 for LLF; n = 4 for C and n = 4 for SC). We found that the endometrium of the majority of studied animals expressed embryonic stem cell markers, OCT4 and SOX2, but not or little NANOG, as well as CD44, c-Kit, and STAT3, all markers of mesenchymal stem cells. The expression profile of these markers was not related to the stage of the oestrus cycle; however there was a statistically significant reduction in the expression of embryonic stem cell markers in ill animals, being the lowest in clinically ill and intermediate in subclinical endometritis, (P < 0.05 and Pearson correlation coefficient 0.92). For markers of multipotency (mesenchymal), the expression was lower in clinical endometritis (P < 0.05). In conclusion, the expression profile of stem cell markers is indicative of the presence of stem cells in the bovine endometrium. At the protein level, we verified our findings for OCT4, SOX2, and CD44 using Western blot and immunohistochemistry. In general, there was a concordance between mRNA and protein profiles. Inflammatory markers showed a pattern characteristic for each of the studied stages. In order to have an ultimate criterion of the presence of stem cells, we tested the differentiation potential of the isolated cell lines, upon induction towards chondrogenic, osteogenic, and adipogenic lineages. We found that all the cell lines tested (n = 8) displayed mesenchymal differentiation potential as demonstrated by specific staining and gene expression markers. At present, work is in progress to isolate pure stem cell populations from these primary cultures to further characterise these cells. Conclusion: we showed for the first time the presence and differentiation potential of endometrial stem cells in cattle. This can have an effect on the development of new therapeutic approaches to combat uterine diseases, such as endometritis or endometriosis (in horses). This work was supported by grant FONDECYT REGULAR 1110642, from the Government of Chile.

Hematopoietic Stem Cell Niche Interactions In The Embryo Can Modulate The Size Of The Stem Cell Pool Into Adulthood

Hematopoietic stem cells (HSC) reside in the bone marrow niche and sustain the production of blood throughout life. The entire pool of these rare and important cells is generated during a brief window of embryonic development. HSC are produced by the hemogenic endothelium of the dorsal aorta, migrate to and expand in the fetal liver, and then migrate again to seed the bone marrow. The zebrafish is a highly conserved and well-established model for HSC development. Similar to mammals, HSC emerge from the dorsal aorta, but then colonize a vascular plexus in the tail of the embryo—the caudal hematopoietic tissue (CHT). It is difficult to directly observe the interactions between an endogenous HSC and its niche, so we have developed the CHT as a model for HSC-niche interactions. To track HSC in vivo we have generated a transgenic reporter using the previously described mouse Runx1 +23 kb intronic enhancer. The purity of the stem cell pool marked by this reporter was determined. Using adult-to-adult limiting dilution transplantation with as few as one Runx1+23 positive cell, we have estimated the HSC purity to be approximately 1/35 (without immune matching), or similar to Kit+Sca1+Lin- (KSL) in mouse. This is the most pure stem cell population defined in the zebrafish system. Using embryo-to-embryo transplantation, a technique that is unique to zebrafish, we sorted Runx1+23 positive cells from one group of embryos and transplanted them to another by injection directly into circulation. Embryos are then grown to adulthood and marrow is tested for long-term engraftment between 3 and 5 months. This transplantation technique precedes formation of the thymus, thereby removing any chance of immune rejection. Highly stringent dilution of HSC in our embryo-to-embryo transplants has estimated a stem cell purity of one in two cells. Next, we applied our highly specific reporter to visualize HSC migration to the CHT niche. After arrival of the HSC, we have described 5 distinct steps during colonization: 1) adherence; 2) extravasation; 3) abluminal migration; 4) endothelial niche formation (“cuddling”); and 5) cell fate decisions. Live imaging analysis of HSC together with endothelial and stromal transgenic reporters has allowed us to quantify the relationship between different cell types within the CHT. For example, we observe preferential localization of HSC in close proximity to cxcl12a positive stromal cells. Lastly, we have sought to identify the molecular mechanisms involved in interactions between HSC and their niche. A chemical genetic screen identified the natural product lycorine as a small molecule that increases hematopoiesis in the CHT and promotes HSC-endothelial cell interactions. Combined chemical treatment and live imaging revealed that lycorine significantly increased the residence time of HSC in the niche. To test if treatment during the window of CHT colonization (2-3 days post fertilization) had long-term effects on HSC and the stem cell pool, the compound was washed off at 3 days and the Runx1+23 positive population was quantified by FACS. At 7 days post fertilization, after colonization of the marrow, there was a sustained and significant increase in Runx1+23 positive HSC. Strikingly, after 3 months, when treated embryos were raised to adulthood, we discovered that the increased HSC-endothelial cell interactions we observed in the CHT niche had in fact had an impact on the number of HSC in the adult. Our studies establish that the Runx1+23 transgenic is a highly specific reporter of HSC both in the embryo and adult, and that we can use this reporter for in vivo observation of an endogenous HSC niche. Furthermore, we show that the size of the adult stem cell pool can be altered by a transient signal during development. Disclosures:Tamplin:Boston Children's Hospital: Patents & Royalties. Zon:FATE Therapeutics, Inc: Consultancy, Equity Ownership, Founder Other, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Stemgent, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Stocks, Stocks Other; Scholar Rock: Consultancy, Equity Ownership, Founder, Founder Other, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

The CD44+ALDH+ Population of Human Keratinocytes Is Enriched for Epidermal Stem Cells with Long-Term Repopulating Ability

Like for other somatic tissues, isolation of a pure population of stem cells has been a primary goal in epidermal biology. We isolated discrete populations of freshly obtained human neonatal keratinocytes (HNKs) using previously untested candidate stem cell markers aldehyde dehydrogenase (ALDH) and CD44 as well as the previously studied combination of integrin α6 and CD71. An in vivo transplantation assay combined with limiting dilution analysis was used to quantify enrichment for long-term repopulating cells in the isolated populations. The ALDH(+) CD44(+) population was enriched 12.6-fold for long-term repopulating epidermal stem cells (EpiSCs) and the integrin α6(hi) CD71(lo) population was enriched 5.6-fold, over unfractionated cells. In addition to long-term repopulation, CD44(+) ALDH(+) keratinocytes exhibited other stem cell properties. CD44(+) ALDH(+) keratinocytes had self-renewal ability, demonstrated by increased numbers of cells expressing nuclear Bmi-1, serial transplantation of CD44(+) ALDH(+) cells, and holoclone formation in vitro. CD44(+) ALDH(+) cells were multipotent, producing greater numbers of hair follicle-like structures than CD44(-) ALDH(-) cells. Furthermore, 58% ± 7% of CD44(+) ALDH(+) cells exhibited label-retention. In vitro, CD44(+) ALDH(+) cells showed enhanced colony formation, in both keratinocyte and embryonic stem cell growth media. In summary, the CD44(+) ALDH(+) population exhibits stem cell properties including long-term epidermal regeneration, multipotency, label retention, and holoclone formation. This study shows that it is possible to quantify the relative number of EpiSCs in human keratinocyte populations using long-term repopulation as a functional test of stem cell nature. Future studies will combine isolation strategies as dictated by the results of quantitative transplantation assays, in order to achieve a nearly pure population of EpiSCs.

CD133 Is a Marker for Long-Term Repopulating Murine Epidermal Stem Cells

Maintenance, repair and renewal of the epidermis are thought to depend on a pool of dedicated epidermal stem cells. Like for many somatic tissues, isolation of a nearly pure population of stem cells is a primary goal in cutaneous biology. We used a quantitative transplantation assay, using injection of keratinocytes into subcutis combined with limiting dilution analysis, to assess the long-term repopulating ability of putative murine epidermal stem populations. Putative epidermal stem cell populations were isolated by FACS sorting. The CD133+ population and the subpopulation of CD133+ cells that exhibits high mitochondrial membrane potential (DΨmhi), were enriched for long-term repopulating epidermal stem cells vs. unfractionated cells (3.9 and 5.2-fold, respectively). Evidence for self-renewal capacity was obtained by serial transplantation of long-term epidermal repopulating units derived from CD133+ and CD133+ΔΨmhi keratinocytes. CD133+ keratinocytes were multipotent and produced significantly more hair follicles than CD133− cells. CD133+ cells were a subset of the previously described integrin α6+CD34+ bulge cell population and 28.9±8.6% were label retaining cells. Thus, murine keratinocytes within the CD133+ and CD133+ΔΨmhi populations contain epidermal stem cells that regenerate epidermis for the long-term, are self-renewing, multipotent, and label-retaining cells.

GROWTH AND DEVELOPMENT SYMPOSIUM: Stem cell therapy in equine tendon injury1

Tendon injuries affect all levels of athletic horses and represent a significant loss to the equine industry. Accumulation of microdamage within the tendon architecture leads to formation of core lesions. Traditional approaches to tendon repair are based on an initial period of rest to limit the inflammatory process followed by a controlled reloading program designed to promote the maturation and linear arrangement of scar tissue within the lesion. However, these treatment protocols are inefficient, resulting in prolonged recovery periods and frequent recurrence. Current alternative therapies include the use of bone marrow-derived mesenchymal stem cells (BMSC) and a population of nucleated cells from adipose containing adipose-derived mesenchymal stem cells (AdMSC). Umbilical cord blood-derived stem cells (UCB) have recently received attention for their increased plasticity in vitro and potential as a therapeutic aid. Both BMSC and AdMSC require expansion in culture before implantation to obtain a pure stem cell population, limiting the time frame for implantation. Collected at parturition, UCB can be cryopreserved for future use. Furthermore, the low immunogenicity of the UCB population allows for allogeneic implantation. Current research indicates that BMSC, AdMSC, and UCB can differentiate into tenocyte-like cells in vitro, increasing expression of scleraxis, tenascin c, and extracellular matrix proteins. When implanted, BMSC and AdMSC engraft into the tendon and improve tendon architecture. However, treatment with these stem cells does not decrease recovery period. Furthermore, the resulting regeneration is not optimal, as the resulting tissue is still inferior to native tendon. Umbilical cord blood-derived stem cells may provide an alternate source of stem cells that promote improved regeneration of tendon tissue. A more naïve cell population, these cells may have a greater rate of engraftment as well as an increased ability to secrete bioactive factors and recruit additional reparative cells. Further work should clarify the role of distinct stem cell sources in the regenerating tendon and the need for a naïve or differentiated cell type for implantation.

Human Epidermal Neural Crest Stem Cells (hEPI-NCSC)—Characterization and Directed Differentiation into Osteocytes and Melanocytes

Here we describe the isolation, characterisation and ex-vivo expansion of human epidermal neural crest stem cells (hEPI-NCSC) and we provide protocols for their directed differentiation into osteocytes and melanocytes. hEPI-NCSC are neural crest-derived multipotent stem cells that persist into adulthood in the bulge of hair follicles. Multipotency and self-renewal were determined by in vitro clonal analyses. hEPI-NCSC generate all major neural crest derivatives, including bone/cartilage cells, neurons, Schwann cells, myofibroblasts and melanocytes. Furthermore, hEPI-NCSC express additional neural crest stem cell markers and global stem cell genes. To variable degrees and in a donor-dependent manner, hEPI-NCSC express the six essential pluripotency genes C-MYC, KLF4, SOX2, LIN28, OCT-4/POU5F1 and NANOG. hEPI-NCSC can be expanded ex vivo into millions of stem cells that remain mulitpotent and continue to express stem cell genes. The novelty of hEPI-NCSC lies in the combination of their highly desirable traits. hEPI-NCSC are embryonic remnants in a postnatal location, the bulge of hair follicles. Therefore they are readily accessible in the hairy skin by minimal invasive procedure. hEPI-NCSC are multipotent somatic stem cells that can be isolated reproducibly and with high yield. By taking advantage of their migratory ability, hEPI-NCSC can be isolated as a highly pure population of stem cells. hEPI-NCSC can undergo robust ex vivo expansion and directed differentiation. As somatic stem cells, hEPI-NCSC are conducive to autologous transplantation, which avoids graft rejection. Together, these traits make hEPI-NCSC novel and attractive candidates for future cell-based therapies and regenerative medicine.Electronic supplementary materialThe online version of this article (doi:10.1007/s12015-011-9255-5) contains supplementary material, which is available to authorized users.

Taking embryonic stem cell potential to the bank: vitrification of the ICM

As much as our fascination with the potential of stem cells in the future of biomedicine has captured the fancy of scientists and non-scientists alike, the analysis of the ontogeny of this remarkable cell remains within the purview two well-defined research camps. On the one hand are the embryologists and developmental biologists who continue to wrestle with the problem of lineage specification. Their challenge has been one of a kind: how does a single fertilized ovum give rise to the progenitors of the placenta or the fetus? At the other end of the spectrum are those who deal with the isolation and characterization of ES cells, usually originating from blastocysts, which can ultimately be propagated, expanded, and manipulated into embryonic germ layer phenotypes capable of deploying their differentiation programs to completion. Here, the challenges have been manifold. Optimizing conditions for inner cell mass isolation and survival in primary culture, sorting out ES cells from the various other cell types that result from blastocyst outgrowths, and maintaining derivative lines under conditions that allow for adequate proliferation and totipotency-each of these representing years of dedicated efforts draw in large measure from pioneering studies made using the mouse model. Our lead article this month takes a giant step towards harnessing the remarkable birthright of ES cells. Desai and collaborators report on the use of vitrification to store inner cell masses from mouse embryos for future derivation of stem cells. Their findings yet again illustrate that departure from traditional methodological approaches can open new doors for improving the efficiency and utilization of ES cells for research and therapeutic applications and, if successful in human embryos, would provide direction for ethically appropriate use of these cells. As discussed in previous issues of JARG, the day to day repertoire used for isolating and establishing human ES cells takes place on the battlefield of the culture dish. War is waged in due course between inner cell mass and trophectoderm cells, amongst others, competing for survival and dominance in the earliest days of derivation. And whether initiated with frozen blastocysts (most commonly) or deposited into cryo storage after several passages, the human ES cell production line as we know it today is one that typically draws upon the use of slow freeze protocols. What Desai and colleagues alert us to is the potential of vitirification. Vitrification has emerged as an alternative method for embryo storage, and has been adopted for a number of human ART applications that include gonadal tissue and oocyte cryopreservation. It has yet to have gone mainstream in the field of stem cell biology. Given cost effectiveness, improved cell survival, and day to day applicability in the laboratory setting it seems likely that vitrification will find utility for cryopreservation of many cell types of interest in the stem cell field including those produced by induced pluripotent stem cell technology or derived from sources such as cord blood and Wharton’s jelly (mesenchymal stem cells). That it has now been demonstrated as a means for storage of the inner cell mass in mouse embryos raises a number of additional opportunities for human materials ranging from derivation efficiency to the prospect of banking the stem cell potential of supernumerary embryos. The paper by Desai and colleagues from the Cleveland Clinic explores the problem of the culture dish battlefield by testing the effects of various methods for inner cell mass isolation that in and of itself hampers isolation of pure stem cell populations due to the intrinsic heterogeneity occurring during intact blastocyst outgrowths. They report good survival of ICM following vitrification using either of two methods and most importantly demonstrate successful passaging of cells that retain major stem cell markers. Many challenges remain including validation of stem cell potential using traditional transplantion approaches to monitor expression of all embryonic germ layer cell types. Moreover, the notorious proclivity that ES cell lines show towards genetic instability upon further passaging will require further investigation using both mouse and human blastocysts. And most importantly, the utility of this approach for human materials must be demonstrated. If vitrification of isolated human ICMs can be accomplished and used to isolate totipotential ES cell lines, then banking of raw materials will become a reality for dealing with the many stored embryos that have clinical or research utility. Moreover, the investigative framework revealed by this study will offer new insights and a nexus for scientists who seek a deeper understanding of the birthright of these remarkable cells or a level of proficiency consonant with the therapeutic management of human disease.

Gpx3 Determines Competitiveness of Normal and Leukemic Stem Cells.

AbstractAbstract 1587Although important efforts have been invested in the discovery of genes that regulate normal or leukemic hematopoietic stem cells (HSC) self-renewal, the number of validated candidates remains low, due largely to the unavailability of functionally pure stem cell populations. Moreover, it is often difficult to identify the normal counterpart cell from which leukemia originated, further complicating studies based on comparative gene expression. In this study, we used a series of recently characterized Hoxa9 + Meis1 acute myeloid leukemias (AML) derived from fetal liver (FL) cells (Wilhelm BT et al., submitted). These leukemias are remarkably similar in several aspects including their L-HSC frequency (between ∼1 in 100 to 350) except for one leukemia (FLA2) in which 70% of the cells show repopulation ability (i.e., L-HSC).We reasoned that comparative mRNA profiling of FLA2 to the phenotypically similar FLB1 (0.3% L-HSC) might identify genes uniquely associated with L-HSC self-renewal. We observed a 2–3-fold upregulation of Gpx3 in FLA2, which was confirmed by qRT-PCR. In accordance with this, all 14 of the tested Gpx3 promoter region CpG sequences were methylated in FLB1 and hypomethylated in FLA2 cells. The higher expression of GPx3 in FLA2 was confirmed at the protein level and reflected in elevated glutathione peroxidase activity in comparison to FLB1. Importantly, we also observed in FLA2 a relative reduction in reactive oxygen species (ROS) level (DCFDA) and a concomitant decrease in p38 MAPK activation (western blot and mass spectrometry). The correlation of Gpx3 levels with L-HSC frequency could be reflective of their functional dependence on this enzyme. FLA2 cells being difficult to manipulate ex vivo, to address this we utilized retroviruses encoding shRNAs and a GFP reporter to explore the in vivo function of FLA2 cells with downregulated Gpx3. The decrease in percentage of GFP+ donor cells when leukemia became apparent (∼19 days) from that of populations initially transplanted, was 4-fold higher following Gpx3 knockdown in comparison to shLuciferase transduction. Moreover, those shGpx3 infected FLA2 remaining at day 19 displayed a 3-fold decrease in GFP mean fluorescence intensity relative to their control counterparts. These results show that GFPhigh cells were selectively depleted, and suggest that Gpx3 is critical for the competitiveness of L-HSCs.Because redox metabolism has been implicated in HSC self-renewal, we also analyzed its expression and function in normal HSC to gain further insight into the role of GPx3 in stem cell activity. Interestingly, compared to FL-HSCs, isolated 3 and 4 week bone marrow (BM), HSCs exhibited a 39- and 6-fold decrease in Gpx3 expression, respectively. A correlation of Gpx3 levels with enhanced self-renewal was also observed in vitro as overexpression of several nuclear determinants of HSC expansion such as Hoxb4, NA10HD, Klf10 and Prdm16 promoted Gpx3 expression by 3.2 to 19.2-fold. We next infected BM cells enriched for HSCs with retroviruses carrying shRNAs to Gpx3. shRNA targeting of Gpx3 dramatically inhibited hematopoietic reconstitution. Transplantations of sublethally irradiated recipients indicated that Gpx3 knockdown significantly impaired both early and late donor-derived hematopoiesis. These results suggest that GPx3 is critical for repopulation mediated by both short and long-term repopulating cells. In reciprocal gain-of-function experiments, Lin-CD150+CD48- cells engineered to overexpress Gpx3, showed a marked competitive advantage over controls when transplanted following a 7-day ex vivo culture step. Insertional mutagenesis was ruled out as proviral integration analyses of six recipients confirmed polyclonal hematopoiesis. Moreover, some mice were in part reconstituted by the same clones, indicating that self-renewal occurred in vitro prior to transplantation. Phenotypic analysis of late-transplant hematopoietic tissues showed that Gpx3-transduced cells contributed to lymphoid and myeloid repopulation, confirming their multipotentiality. Together, these results indicate that Gpx3 enhances HSC expansion ex vivo possibly through modulation of self-renewal activity.In conclusion, a unique model of primary L-HSC was exploited to identify Gpx3 as a critical determinant for the competitiveness of L-HSCs and complementary experiments demonstrated a key role for this gene in normal HSC self-renewal. Disclosures:No relevant conflicts of interest to declare.

A novel method to derive amniotic fluid stem cells for therapeutic purposes

BackgroundHuman amniotic fluid stem (hAFS) cells have become an attractive stem cell source for medical therapy due to both their ability to propagate as stem cells and the lack of ethical debate that comes with the use of embryonic stem cells. Although techniques to derive stem cells from amniotic fluid are available, the techniques have limitations for clinical uses, including a requirement of long periods of time for stem cell production, population heterogeneity and xeno-contamination from using animal antibody-coated magnetic beads. Herein we describe a novel isolation method that fits for hAFS derivation for cell-based therapy.Methods and ResultsWith our method, single hAFS cells generate colonies in a primary culture of amniotic fluid cells. Individual hAFS colonies are then expanded by subculturing in order to make a clonal hAFS cell line. This method allows derivation of a substantial amount of a pure stem cell population within a short period of time. Indeed, 108 cells from a clonal hAFS line can be derived in two weeks using our method, while previous techniques require two months. The resultant hAFS cells show a 2-5 times greater proliferative ability than with previous techniques and a population doubling time of 0.8 days. The hAFS cells exhibit typical hAFS cell characteristics including the ability to differentiate into adipogenic-, osteogenic- and neurogenic lineages, expression of specific stem cell markers including Oct4, SSEA4, CD29, CD44, CD73, CD90, CD105 and CD133, and maintenance of a normal karyotype over long culture periods.ConclusionsWe have created a novel hAFS cell derivation method that can produce a vast amount of high quality stem cells within a short period of time. Our technique makes possibility for providing autogenic fetal stem cells and allogeneic cells for future cell-based therapy.

“The Good into the Pot, the Bad into the Crop!”—A New Technology to Free Stem Cells from Feeder Cells

A variety of embryonic and adult stem cell lines require an intial co-culturing with feeder cells for non-differentiated growth, self renewal and maintenance of pluripotency. However for many downstream ES cell applications the feeder cells have to be considered contaminations that might interfere not just with the analysis of experimental data but also with clinical application and tissue engineering approaches. Here we introduce a novel technique that allows for the selection of pure feeder-freed stem cells, following stem cell proliferation on feeder cell layers. Complete and reproducible separation of feeder and embryonic stem cells was accomplished by adaptation of an automated cell selection system that resulted in the aspiration of distinct cell colonies or fraction of colonies according to predefined physical parameters. Analyzing neuronal differentiation we demonstrated feeder-freed stem cells to exhibit differentiation potentials comparable to embryonic stem cells differentiated under standard conditions. However, embryoid body growth as well as differentiation of stem cells into cardiomyocytes was significantly enhanced in feeder-freed cells, indicating a feeder cell dependent modulation of lineage differentiation during early embryoid body development. These findings underline the necessity to separate stem and feeder cells before the initiation of in vitro differentiation. The complete separation of stem and feeder cells by this new technology results in pure stem cell populations for translational approaches. Furthermore, a more detailed analysis of the effect of feeder cells on stem cell differentiation is now possible, that might facilitate the identification and development of new optimized human or genetically modified feeder cell lines.

The Inhibitory Role of Stromal Cell Mesenchyme on Human Embryonic Stem Cell Hepatocyte Differentiation is Overcome by Wnt3a Treatment

Pluripotent stem cells are derived from the inner cell mass of preimplantation embryos, and display the ability of the embryonic founder cells by forming all three germ lineages in vitro. It is well established that the cellular niche plays an important role in stem cell maintenance and differentiation. Stem cells generally have limited function without the specialized microenvironment of the niche that provides key cell-cell contact, soluble mediators, and extracellular matrices. We were interested in the role that Wnt signaling, in particular Wnt3a, played in human embryonic stem cell (hESC) differentiation to hepatic endoderm in vitro. hESC differentiation to hepatic endoderm was efficient in pure stem cell populations. However, in younger hESC lines, generating stromal cell mesenchyme, our model was very inefficient. The negative effect of stroma could be reversed by pretreating hESCs with Wnt3a prior to the onset of hepatocyte differentiation. Wnt3a pretreatment reinstated efficient hESC differentiation to hepatic endoderm. These studies represent an important step in understanding hepatocyte differentiation from hESCs and the role played by the cellular niche in vitro.

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

ABCG2 Expression as Prospective Marker for Hematopoeitic Stem Cell Purification.

ABCG2 (BCRP/MXR/ABCP) is an ABC transporter that effluxes Hoechst 33342 and is required for the side population (SP) phenotype of hematopoietic stem cells (HSCs). Although SP cells are enriched for HSC activity, they are not a pure stem cell population and contain a significant number of differentiated and committed cells. We hypothesized that expression of ABCG2 would provide a more specific marker for HSCs, given our prior results showing that other ABC transporters were expressed in committed SP cells. To test this hypothesis, we have generated a mouse strain in which an IRES-GFP element was inserted immediately after the stop codon of Abcg2, so that GFP and the endogenous Abcg2 gene were co-expressed from the same transcriptional regulatory elements. Immunohistochemical analysis using an anti-GFP antibody showed that Abcg2/GFP allele was strongly expressed in proximal tubule cells of kidney, in the epithelial cells of small intestine, and in vascular endothelium, recapitulating the normal expression pattern of Abcg2. Using flow cytometry to analyze GFP expression in bone marrow cells, we found that 48% of SP cells expressed GFP. In the distal tip of the SP region, previously shown to have the highest concentration of HSCs, 93% of cells expressed GFP. In gated Lin- GFP+ cells within the whole bone marrow population, 55 % of the cells expressed c-Kit and Sca1, a previously identified phenotype for HSCs. We sorted Lin- cells into GFP+ and GFP− subpopulations and determined repopulation frequencies in a limiting dilution assay. Fifteen percent of recipient mice were reconstituted with 10 Lin- GFP+ cells, and 50% mice with 60 Lin- GFP+ cells. In groups transplanted with 125 to 1000 Lin- GFP+ cells, 100% of the mice were reconstituted. In contrast, no reconstitution was seen in any mice transplanted with 60,000 Lin− GFP− cells, indicating that most if not all HSCs expressed the Abcg2/GFP allele. Secondary transplant experiments and individual lineage analysis of peripheral blood cells confirmed that the Lin- GFP+ cells were long-term HSCs. These results demonstrate that expression of Abcg2 in Lin- cells defines a highly enriched population of HSCs, and is a more specific HSC marker than the SP phenotype. We are now increasing the stringency of the GFP sorting gate and transplanting mice with single cells to determine if a pure HSC population can be isolated with this simple 2 marker system.

The Continuing Saga of Epidermal Stem Cells

Ever sinceTill and McCulloch (1961) identified cells with the ability to recapitulate the hematopoietic tissue after radiation damage, isolating a pure population of stem cells from somatic tissues has been a primary goal. However, achieving success has been limited. Even today, four decades later, a unique characteristic that can be used to isolate a pure population of hematopoietic stem cells is lacking. The same can be said for epidermal stem cells.

Isolating a pure population of epidermal stem cells for use in tissue engineering.

Continuously renewing tissues, such as the epidermis, are maintained by stem cells that slowly proliferate and remain in the tissue for life. Although it has been known for decades that epithelial stem cells can be identified as label-retaining cells (LRCs) by long term retention of a nuclear label, isolating a pure population of stem cells has been problematic. Using a Hoechst and propidium iodide dye combination and specifically defined gating, we sorted mouse epidermal basal cells into three fractions, which we have now identified as stem, transient amplifying (TA), and non-proliferative basal cells. More than 90% of freshly isolated stem cells showed a G0/G1 cell cycle profile, while greater than 20% of the TA cells were actively dividing. Both stem and TA cells retained proliferative capacity, but the stem cells formed larger, more expandable colonies in culture. Both populations could be transduced with a retroviral vector and used to bioengineer an epidermis. However, only the epidermis from the stem cell population continued to grow and express the reporter gene for 6 months in organotypic culture. The epidermis from the transient amplifying cell fraction completely differentiated by 2 months. This novel sorting method yields pure viable epithelial stem cells that can be used to bioengineer a tissue and to test permanent recombinant gene expression.