Sca-1 Cells Research Articles

Suppressing gain-of-function proteins via CRISPR/Cas9 system in SCA1 cells

SCAs are autosomal dominant neurodegenerative disorders caused by a gain-of-function protein with toxic activities, containing an expanded polyQ tract in the coding region. There are no treatments available to delay the onset, stop or slow down the progression of these pathologies. In this work we focus our attention on SCA1 which is one of the most common genotypes circulating in Italy. Here, we develop a CRISPR/Cas9-based approach to reduce both forms of the ATXN1 protein, normal and mutated with expanded polyQ. We started with the screening of 10 different sgRNAs able to target Exon 8 of the ATXN1 gene. The two most promising sgRNAs were validated in fibroblasts isolated from SCA1 patients, following the identification of the best transfection method for this type of cell. Our silencing approach significantly downregulated the expression of ataxin1, due to large deletions and the introduction of small changes in the ATXN1 gene, evidenced by NGS analysis, without major effects on cell viability. Furthermore, very few significant guide RNA-dependent off-target effects were observed. These preliminary results not only allowed us to identify the best transfection method for SCA1 fibroblasts, but strongly support CRISPR/Cas9 as a promising approach for the treatment of expanded polyQ diseases. Further investigations will be needed to verify the efficacy of our silencing system in SCA1 neurons and animal models.

Differentiation Epitopes Define Hematopoietic Stem Cells and Change with Cell Cycle Passage

Hematopoietic stem cells express differentiation markers B220 and Gr1 and are proliferative. We have shown that the expression of these entities changes with cell cycle passage. Overall, we conclude that primitive hematopoietic stem cells alter their differentiation potential with cell cycle progression.Graphical Murine derived long-term hematopoietic stem cells (LT-HSC) are cycling and thus always changing phenotype. Here we show that over one half of marrow LT-HSC are in the population expressing differentiation epitopes and that B220 and Gr-1 positive populations are replete with LT-HSC after a single FACS separation but if subjected to a second separation these cells no longer contain LT-HSC. However, with second separated cells there is a population appearing that is B220 negative and replete with cycling c-Kit, Sca-1 CD150 positive LT-HSC. There is a 3–4 h interval between the first and second B220 or GR-1 FACS separation during which the stem cells continue to cycle. Thus, the LT-HSC have lost B220 or GR-1 expression as the cells progress through cell cycle, although they have maintained the c-kit, Sca-1 and CD150 stem cells markers over this time interval. These data indicate that cycling stem cells express differentiation epitopes and alter their differentiation potential with cell cycle passage.

Adult mouse kidney stem cells orchestrate the de novo assembly of a nephron via Sirt2‐ modulated canonical Wnt/β‐catenin signaling

End‐stage renal disease affects an estimated 2.4 million people annually worldwide. With cases increasing 8% every year, regenerative strategies are needed to help alleviate, and potentially replace, the increasing demand for kidney transplants. An important question remains whether stem cells reside in the adult mammalian kidney. Here we developed a methodology for isolation of kidney stem cells from adult mouse kidney by using Sca1 and Oct4 stem cell markers. Using these cells, we generated three types of kidney‐like structures. First, we observed the assembly of self‐organizing three‐dimensional kidney organoids. These kidney organoids show characteristic of kidney structures and contain podocytes and endothelial cells that form networks of capillary loop‐like structures. We also observed the de novogeneration of a self‐organizing nephron (and its associated collecting duct) from monolayer cultures. Comma‐ and S‐shaped bodies that expressed activated Notch1 were present during this formation, suggesting that Notch signaling played a key role in driving nephrogenesis. This is consistent with our current knowledge of nephron formation in the mouse embryo. Furthermore, we observed the differentiation of monolayer cells into functionally mature tubules and self‐organizing kidney‐shaped structures that selectively endocytose dextran. Finally, we demonstrated that expression of Sirt2 was upregulated, which modulated the dynamics of canonical Wnt/β‐catenin signaling during kidney organoid development. Altogether, our observations provide the first evidence for the existence of stem cells in the adult mouse kidney. These findings offer powerful tools for future applications, including drug screening, disease modelling, cell therapy, and kidney regeneration.

Pre-Existing Anti-FVIII Immunity Alters Therapeutic Platelet-Targeted FVIII Engraftment in the System Preconditioned with Busulfan Alone through Cytotoxic CD8 T Cells

The development of anti-FVIII inhibitory antibodies (inhibitors) is a significant problem in FVIII protein replacement therapy in hemophilia A (HA). We have developed a platelet-targeted FVIII gene therapy approach, in which human FVIII expression is driven by the platelet-specific αIIb promoter (2bF8) and demonstrated that 2bF8 gene therapy can restore hemostasis and induce FVIII-specific immune tolerance in FVIII null mice even with pre-existing anti-FVIII immunity when an effective preconditioning regimen is employed. Since busulfan, an alkylating agent with potent effects on primitive hematopoietic cells, is an important component of many hematopoietic stem cell (HSC) transplantation preparative regimens in humans, we evaluated the efficacy of busulfan conditioning regimens in 2bF8 gene therapy. We found that busulfan conditioning alone resulted in sustained therapeutic levels of platelet-FVIII expression in FVIII null mice that received 2bF8-transduced HSCs in the non-inhibitor model but not in the inhibitor model. In the current study, we explored the mechanism of platelet FVIII loss upon busulfan conditioning in the FVIII inhibitor model.FVIII null mice were immunized with recombinant human FVIII (rhF8) to induce anti-FVIII inhibitor development to establish the inhibitor model. Once the inhibitor titers were confirmed, animals received busulfan preconditioning at the dose of 50 mg/kg followed by transplantation of either whole bone marrow or Sca-1 + cells from 2bF8 transgenic (2bF8 Tg) mice. After 4 weeks of bone marrow reconstitution, platelet-FVIII expression levels in recipients transplanted with 2bF8 Tg whole bone marrow cells were 7.19±8.59 mU/10 8 platelets (n=5), which were significantly higher than those obtained from animals transplanted with 2bF8 Tg Sca-1 cells (0.55±1.02 mU/10 8 platelets [n=15]). The differences in platelet-FVIII expression between the whole bone marrow and Sca-1 groups were maintained during the study period for 6 months. When CD8 T cells were depleted in addition to busulfan preconditioning, platelet-FVIII expression was significantly enhanced in rhF8-primed recipients that received 2bF8 Tg Sca-1 cells (2.14±2.25 mU/10 8 platelets [n=8]) and sustained during the study period.We then explored which subset of cells from 2bF8 Tg mice could activate rhF8-primed CD8 T cells using the mouse IFNγ ELISpot assay. rhF8-primed CD8 T cells were stimulated with platelets, Sca-1 + cells, or megakaryocytes sorted from either 2bF8 Tg or FVIII null mice. We found that CD8 T cells from rhF8-primed FVIII null mice were efficiently activated by Sca-1 + cells from 2bF8 Tg mice and secreted IFNγ but not by platelets or megakaryocytes. These results suggest that 2bF8 Tg-Sca-1 + cells could be a potential target for rhF8-primed CD8 T cells. As a control, Sca-1 + cells from FVIII null mice did not activate rhF8-primed CD8 T cells, suggesting that IFNγ production from rhF8-primed CD8 T cells stimulated with 2bF8 Tg-Sca-1 + cells was a FVIII-specific response. To explore whether the elimination of platelet-FVIII expression in the inhibitor model relies on antibody-dependent cellular cytotoxicity (ADCC), we transplanted 2bF8 Tg-Sca-1 + cells into rhF8-primed B-cell deficient μMT mice preconditioned with busulfan. We found that no platelet-FVIII was detected in μMT recipients even though they did not produce anti-FVIII antibodies, suggesting that the loss of platelet-FVIII expression in the inhibitor model is not mediated by the ADCC pathway.In summary, our studies demonstrate that pre-existing anti-FVIII immunity can alter the engraftment of 2bF8-genetically-manipulated Sca-1 + hematopoietic stem/progenitor cells via the cytotoxic CD8 T-cell killing pathway. Sufficient eradication of FVIII-primed CD8 T cells is critical for the success of platelet-targeted gene therapy in hemophilia A with pre-existing immunity. DisclosuresNo relevant conflicts of interest to declare.

MATERNAL OBESITY NEGATIVELY IMPACT HEART DEVELOPMENT IN OFFSPRING

<h3>Background</h3> The development of the heart is a sensitive and dependent process of stem cells present during its maturation. Changes in the population of these cells through modifications in the intrauterine environment due to a metabolic program, such as during pregnancy of obese women is something of concern. It is estimated that 30% of pregnant women are obese and that 40% of women gain excessive weight during pregnancy. Thus, the objective of this work is to analyze the impacts of maternal obesity on the heart of the offspring in neonate Swiss mice. <h3>Methods</h3> After birth, litters were adjusted to nine pups and divided into overfeeding Group - nine pups until the 3rd day, when the litter was reduced to three female pups, and control Group with nine pups. Three months old females of both groups were mated to healthy males and their offspring were euthanized at the day of birth, resulting in the Offspring Control Group (OCG) and Offspring Overfed Group (OOG). Biometric parameters were analyzed, and fragments of the heart were used to analyze fibrosis by Picro-Sirius Red stanning and to analyze the population of c-kit and sca-1 cells. The results were expressed as mean ± SEM, analyzed by t-student test. <h3>Results</h3> OOG showed, at birth, 25% increased body mass compared to OCG. However, there was no difference in naso-anal length between groups, reflecting an increase of 93% in the Lee Index of OOG compared to OCG. Although the cardiac mass was 35% decreased in OOG, the analysis of collagen deposition in the heart showed an increased interstitial fibrosis (196%) in this group compared to the OCG. In addition, the quantification of cardiac stem cells demonstrated an increase of 229% in the c-kit+ cells and an increase of 218% in the sca-1+ cells when compared to the OCG. <h3>Conclusion</h3> Together, results demonstrate that maternal metabolic programming negatively impacts offspring at birth, altering cardiac development, making the heart more immature at birth, as well as increasing adverse remodeling, which may be related to the development of cardiovascular diseases in adulthood.

Aortic carboxypeptidase-like protein regulates vascular adventitial progenitor and fibroblast differentiation through myocardin related transcription factor A

The vascular adventitia contains numerous cell types including fibroblasts, adipocytes, inflammatory cells, and progenitors embedded within a complex extracellular matrix (ECM) network. In response to vascular injury, adventitial progenitors and fibroblasts become activated and exhibit increased proliferative capacity and differentiate into contractile cells that remodel the ECM. These processes can lead to vascular fibrosis and disease progression. Our previous work established that the ECM protein aortic carboxypeptidase-like protein (ACLP) promotes fibrotic remodeling in the lung and is activated by vascular injury. It is currently unknown what controls vascular adventitial cell differentiation and if ACLP has a role in this process. Using purified mouse aortic adventitia Sca1+ progenitors, ACLP repressed stem cell markers (CD34, KLF4) and upregulated smooth muscle actin (SMA) and collagen I expression. ACLP enhanced myocardin-related transcription factor A (MRTFA) activity in adventitial cells by promoting MRTFA nuclear translocation. Sca1 cells from MRTFA-null mice exhibited reduced SMA and collagen expression induced by ACLP, indicating Sca1 cell differentiation is regulated in part by the ACLP-MRTFA axis. We determined that ACLP induced vessel contraction and increased adventitial collagen in an explant model. Collectively these studies identified ACLP as a mediator of adventitial cellular differentiation, which may result in pathological vessel remodeling.

Protective role of ErbB3 signaling in myeloid cells during adaptation to cardiac pressure overload

BackgroundMyeloid cells play an important role in a wide variety of cardiovascular disorders, including both ischemic and non-ischemic cardiomyopathies. Neuregulin-1 (NRG-1)/ErbB signaling has recently emerged as an important factor contributing to the control of inflammatory activation of myeloid cells after an ischemic injury. However, the role of ErbB signaling in myeloid cells in non-ischemic cardiomyopathy is not fully understood. This study investigated the role of ErbB3 receptors in the regulation of early adaptive response using a mouse model of transverse aortic constriction (TAC) for non-ischemic cardiomyopathy. Methods and resultsTAC surgery was performed in groups of age- and sex-matched myeloid cell-specific ErbB3-deficient mice (ErbB3MyeKO) and control animals (ErbB3MyeWT). The number of cardiac CD45 immune cells, CD11b myeloid cells, Ly6G neutrophils, and Ly6C monocytes was determined using flow cytometric analysis. Five days after TAC, survival was dramatically reduced in male but not female ErbB3MyeKO mice or control animals. The examination of lung weight to body weight ratio suggested that acute pulmonary edema was present in ErbB3MyeKO male mice after TAC. To determine the cellular and molecular mechanisms involved in the increased mortality in ErbB3MyeKO male mice, cardiac cell populations were examined at day 3 post-TAC using flow cytometry. Myeloid cells accumulated in control but not in ErbB3MyeKO male mouse hearts. This was accompanied by increased proliferation of Sca-1 positive non-immune cells (endothelial cells and fibroblasts) in control but not ErbB3MyeKO male mice. No significant differences in intramyocardial accumulation of myeloid cells or proliferation of Sca-1 cells were found between the groups of ErbB3MyeKO and ErbB3MyeWT female mice. An antibody-based protein array analysis revealed that IGF-1 expression was significantly downregulated only in ErbB3MyeKO mice hearts compared to control animals after TAC. ConclusionOur data demonstrate the crucial role of myeloid cell-specific ErbB3 signaling in the cardiac accumulation of myeloid cells, which contributes to the activation of cardiac endothelial cells and fibroblasts and development of an early adaptive response to cardiac pressure overload in male mice.

Long-term repopulation of aged bone marrow stem cells using young Sca-1 cells promotes aged heart rejuvenation.

Reduced quantity and quality of stem cells in aged individuals hinders cardiac repair and regeneration after injury. We used young bone marrow (BM) stem cell antigen 1 (Sca‐1) cells to reconstitute aged BM and rejuvenate the aged heart, and examined the underlying molecular mechanisms. BM Sca‐1+ or Sca‐1− cells from young (2–3 months) or aged (18–19 months) GFP transgenic mice were transplanted into lethally irradiated aged mice to generate 4 groups of chimeras: young Sca‐1+, young Sca‐1−, old Sca‐1+, and old Sca‐1−. Four months later, expression of rejuvenation‐related genes (Bmi1, Cbx8, PNUTS, Sirt1, Sirt2, Sirt6) and proteins (CDK2, CDK4) was increased along with telomerase activity and telomerase‐related protein (DNA‐PKcs, TRF‐2) expression, whereas expression of senescence‐related genes (p16INK4a, P19ARF, p27Kip1) and proteins (p16INK4a, p27Kip1) was decreased in Sca‐1+ chimeric hearts, especially in the young group. Host cardiac endothelial cells (GFP−CD31+) but not cardiomyocytes were the primary cell type rejuvenated by young Sca‐1+ cells as shown by improved proliferation, migration, and tubular formation abilities. C‐X‐C chemokine CXCL12 was the factor most highly expressed in homed donor BM (GFP+) cells isolated from young Sca‐1+ chimeric hearts. Protein expression of Cxcr4, phospho‐Akt, and phospho‐FoxO3a in endothelial cells derived from the aged chimeric heart was increased, especially in the young Sca‐1+ group. Reconstitution of aged BM with young Sca‐1+ cells resulted in effective homing of functional stem cells in the aged heart. These young, regenerative stem cells promoted aged heart rejuvenation through activation of the Cxcl12/Cxcr4 pathway of cardiac endothelial cells.

SAT-184 Diabetic Neuropathy as a Hematopoietic Stem Cell Disease in the Bone Marrow

Diabetic neuropathy, a major complication of diabetes mellitus, is induced at a relatively early stage after the development of diabetes, and progressively worsens throughout life. A number of pathological mechanisms for the development of neuropathy have been proposed, including ischemia, up-regulation of inflammatory cytokines, and the deficiency of nerve growth factor in peripheral nerve tissues. However, therapeutic approaches targeting these proposed mechanisms have yielded little success. We previously identified a mechanism by which aberrant bone marrow-derived cells pathologically expressing proinsulin/TNF-alpha fuse with residential neurons to impair neuronal function. Here we show that hematopoietic stem cells (HSCs) in c-kit (+), Sca-1 (+) and Lineage (-) cells, are the culprits that underlie the pathogenesis of diabetic neuropathy in both streptozotocin-induced type 1 diabetic mice and high fat diet-induced type 2 diabetic mice. Furthermore, the important role for these cells is supported by the fact that transplantation of HSCs from diabetic mice to non-diabetic mice produces diabetic neuronal dysfunction in the recipient mice via cell fusion without hyperglycemia. On the other hand, transplantation of HSCs from non-diabetic mice to non-diabetic mice produces no dysfunction and no cell fusion. In conclusion, we have identified hyperglycemia-induced aberrant HSCs underlie the development of diabetic neuropathy, which may constitute a novel therapeutic target for the treatment of diabetic neuropathy.

Young Bone Marrow Sca-1 Cells Rejuvenate the Aged Heart by Promoting Epithelial-to-Mesenchymal Transition.

Background: To improve the regenerative capacity of aged individuals, we reconstituted bone marrow (BM) of aged mice with young Sca-1 cells, which repopulated cardiac progenitors and prevented cardiac dysfunction after a myocardial infarction (MI). However, the mechanisms involved were incompletely elucidated. This study aimed to investigate whether young, highly regenerative BM Sca-1 cells exert their cardio-protective effects on the aged heart through reactivation of the epithelial-to-mesenchymal transition (EMT) process.Methods: In vitro, BM Sca-1 cells were co-cultured with epicardial-derived cells (EPDCs) under hypoxia condition; mRNA and protein levels of EMT genes were measured along with cellular proliferation and migration. In vivo, BM Sca-1+ or Sca-1- cells from young mice (2-3 months) were transplanted into lethally-irradiated old mice (20-22 months) to generate chimeras. In addition, Sca-1 knockout (KO) mice were reconstituted with wild type (WT) BM Sca-1+ cells. The effects of BM Sca-1 cell on EMT reactivation and improvement of cardiac function after MI were evaluated.Results: In vitro, BM Sca-1+ cells increased EPDC proliferation, migration, and EMT relative to Sca-1- cells and these effects were inhibited by a TGF-β blocker. In vivo, more young BM Sca-1+ than Sca-1- cells homed to the epicardium and induced greater host EPDC proliferation, migration, and EMT after MI. Furthermore, reconstitution of Sca-1 KO mice with WT Sca-1+ cells was associated with the reactivation of EMT and improved cardiac function after MI.Conclusions: Young BM Sca-1+ cells improved cardiac regeneration through promoting EPDC proliferation, migration and reactivation of EMT via the TGF-β signaling pathway.

Flow Cytometry (FCM) Analysis and Fluorescence-Activated Cell Sorting (FACS) of Erythroid Cells.

To study the process of erythropoiesis, it is important to be able to isolate erythroid progenitors and erythroblasts at distinct stages of development. During the past decade, considerable progress has been made on the development of flow cytometry (FCM) and fluorescence-activated cell sorting (FACS) methods for the analysis and isolation of both murine and human erythroid cells at distinct stages of erythropoiesis, based on changes in the expression of cell surface markers. A method for the identification of murine BFU-E and CFU-E cells was reported by Flygare et al., by negative selection for Ter119, B220, Mac-1, CD3, Gr1, Sca-1, CD16/CD32, CD41, and CD34 cells, followed by separation based on the expression levels of CD71. We developed an alternative method in which Ter119 is used as an erythroid lineage marker, and in conjunction with CD44 and cell size as differentiation markers, it is possible to unambiguously distinguish erythroblasts at each developmental stage during murine terminal erythroid differentiation. We also developed methods for the analysis and isolation of human erythroid cells at all developmental stages. BFU-E and CFU-E are characterized by CD45+GPA-IL-3R-CD34+CD36-CD71low and CD45+GPA-IL-3R-CD34-CD36+CD71high phenotypes, respectively; the combination of GPA, band 3 and α4-integrin are used to isolate erythroid cells at all of the terminal stages of human erythropoiesis, including proerythroblasts, early basophilic, late basophilic, polychromatic and orthochromatic erythroblasts.

* Calvarial Bone Regeneration Is Enhanced by Sequential Delivery of FGF-2 and BMP-2 from Layer-by-Layer Coatings with a Biomimetic Calcium Phosphate Barrier Layer.

A drug delivery coating for synthetic bone grafts has been developed to provide sequential delivery of multiple osteoinductive factors to better mimic aspects of the natural regenerative process. The coating is composed of a biomimetic calcium phosphate (bCaP) layer that is applied to a synthetic bone graft and then covered with a poly-l-Lysine/poly-l-Glutamic acid polyelectrolyte multilayer (PEM) film. Bone morphogenetic protein-2 (BMP-2) was applied before the coating process directly on the synthetic bone graft and then, bCaP-PEM was deposited followed by adsorption of fibroblast growth factor-2 (FGF-2) into the PEM layer. Cells access the FGF-2 immediately, while the bCaP-PEM temporally delays the cell access to BMP-2. In vitro studies with cells derived from mouse calvarial bones demonstrated that Sca-1 and CD-166 positive osteoblast progenitor cells proliferated in response to media dosing with FGF-2. Coated scaffolds with BMP-2 and FGF-2 were implanted in mouse calvarial bone defects and harvested at 1 and 3 weeks. After 1 week in vivo, proliferation of cells, including Sca-1+ progenitors, was observed with low dose FGF-2 and BMP-2 compared to BMP-2 alone, indicating that in vivo delivery of FGF-2 activated a similar population of cells as shown by in vitro testing. At 3 weeks, FGF-2 and BMP-2 delivery increased bone formation more than BMP-2 alone, particularly in the center of the defect, confirming that the proliferation of the Sca-1 positive osteoprogenitors by FGF-2 was associated with increased bone healing. Areas of bone mineralization were positive for double fluorochrome labeling of calcium and alkaline phosphatase staining of osteoblasts, along with increased TRAP+ osteoclasts, demonstrating active bone formation distinct from the bone-like collagen/hydroxyapatite scaffold. In conclusion, the addition of a bCaP layer to PEM delayed access to BMP-2 and allowed the FGF-2 stimulated progenitors to populate the scaffold before differentiating in response to BMP-2, leading to improved bone defect healing.

Dual roles for bone marrow-derived Sca-1 cells in cardiac function.

Recruitment of stem cells from the bone marrow (BM) is an important aspect of cardiac healing that becomes inefficient with age. We investigated the role of young stem cell antigen 1 (Sca-1)-positive BM cells on the aged heart by microarray analysis after BM reconstitution. Sca-1+ and Sca-1- BM cells from young green fluorescent protein (GFP)-positive mice were used to reconstitute the BM of aged mice. Myocardial infarction (MI) was induced 3 mo later. GFP+ cells were more abundant in the BM, blood, and heart of Sca-1+ mice, which corresponded to preserved cardiac function after MI. At baseline, Sca-1+ BM reconstitution increased cardiac expression of serum response factor, vascular endothelial growth factor A, and myogenic genes, but reduced the expression of Il-1β. After MI, inflammation was identified as a key difference between Sca-1- and Sca-1+ groups, as cytokine expression and cell surface markers associated with inflammatory cells were up-regulated with Sca-1+ reconstitution. Mac-3 and F4/80 staining showed that the postinfarction heart was composed of a mixture of GFP+ (donor) macrophages, GFP- (host) macrophages, and GFP+ cells that did not contribute to the macrophage population. This study demonstrates that Sca-1+ BM cells regulate cardiac healing though an acute inflammatory response and also before injury by stimulating formation of a beneficial cardiac niche.-Tobin, S. W., Li, S.-H., Li, J., Wu, J., Yeganeh, A., Yu, P., Weisel, R. D., Li, R.-K. Dual roles for bone marrow-derived Sca-1 cells in cardiac function.

Young Bone-Marrow Sca-1+ Stem Cells Rejuvenate the Aged Heart and Improve Function after Injury through PDGFR\u03b2-Akt pathway

Bone marrow (BM) reconstitution with young BM cells in aged recipients restores the functionality of cardiac resident BM-derived progenitors. This study investigated the cell type primarily responsible for this effect. We reconstituted old mice with BM cells from young or old mice and found that the number of stem cell antigen 1 (Sca-1) cells homing to the heart was significantly greater in young than old chimeras. We then reconstituted old mice with young BM Sca-1+ or Sca-1− cells. We found that Sca-1 cells repopulated the recipient BM and homed to the heart. The number of BM-derived cells in the aged myocardium co-expressing PDGFRβ was 3 times greater in Sca-1+ than Sca-1− chimeric mice. Sca-1+ chimeras had more active cell proliferation in the infarcted heart and improved ventricular function after MI. The improved regeneration involved activation of the PDGFRβ/Akt/p27Kip1 pathway. Sca-1+ stem cells rejuvenated cardiac tissue in aged mice. Restoration of the Sca-1+ subset of stem cells by BM reconstitution improved cardiac tissue regeneration after injury in aged mice.

Prostaglandin E2 Promotes Early Bone Marrow Reconstitution for Survival after Lethal Irradiation

Introduction: High dose total body irradiation (TBI) causes severe damage to the hematopoietic system, resulting in ablation of both red and white blood cells and a high probability of infection, hemorrhage, and death. This is known as the hematopoietic syndrome of the acute radiation syndrome (HS-ARS). TBI is used therapeutically, and incidental exposure through nuclear accidents or radical terrorism is a current threat for which preparation is critical. Safe, effective, and pragmatic agents against HS-ARS are needed. We have identified prostaglandin E2 (PGE2) as a promising medical countermeasure to promote hematopoietic recovery and increase survival from lethal irradiation. Here, using the PGE2 analog 16,16-dimethyl PGE2 (dmPGE2), pre-irradiation dosing strategies were assessed in survival studies with an established mouse model of HS-ARS, and time course analyses were performed to elucidate the early cellular events associated with PGE2 radioprotection.Methods: C57BL/6 mice (n = 20/group) were exposed to LD70/30 (872 cGy) or LD90/30 (904 cGy) and treated with one of the following four dmPGE2 dosing strategies: a single dose of 35 μg at time -30 min, -1 hr, or -3 hr, or a double-dose of 20 μg each at time -45 min and -15 min pre-irradiation. Thirty-day survival was evaluated. The double-dose regimen was further utilized to assess cellular effects over time following LD70/30. Peripheral blood (PB) and bone marrow (BM) cells were harvested on days 1, 2, 4, 7, and 10 post-irradiation (n = 3/group/day). BM was analyzed by flow cytometry with immunostaining for surface markers (Sca-1, c-Kit, CD150, CD48 and blood cell lineage markers), cell cycle (Hoechst/PyroninY), and apoptosis (AnnexinV/7-AAD). PB differentials were assessed by a veterinary hematology analyzer.Results: All dmPGE2 regimens conferred 100% survival to mice receiving LD70/30, and all regimens provided a distinct survival advantage to mice receiving LD90/30 with the double-dose strategy conferring 100% survival at LD90/30. The double-dose was then chosen to assess cellular effects of dmPGE2 throughout the critical 10-day time period following LD70/30. Within the BM, marker-defined HSC numbers were significantly preserved by day 1 post-irradiation compared to vehicle controls, with a drop to vehicle levels observed on day 2 only. Cell cycling frequency among primitive hematopoietic cells was also conserved on day 1, decreased on day 2, then restored to non-irradiated control levels by days 7-10, while that of the unprotected mice remained significantly lower. Differentiated BM cells (lineage marker-positive) were substantially less apoptotic by day 2 and, in significant contrast to the untreated mice, returned to control levels of basal apoptosis by days 7-10. Overall BM cell populations were found to be largely replenished within one week as compared to the vehicle-treated mice, which remained significantly depressed. Further, 73-88% of detectable BM cells from the untreated mice were non-viable by day 4 post-irradiation based on sub-diploid DNA content and did not recover through day 10; dmPGE2-treated mice maintained significantly greater BM cell viability at each time point, returning to only 17-26% sub-diploid by day 7 (non-irradiated controls demonstrated ~5% sub-diploid DNA content). A corresponding effect on PB resulted in significant rescue from neutropenia, anemia, and thrombocytopenia by or before day 10.Conclusions: A single treatment with PGE2 prior to lethal irradiation can facilitate virtually complete survival, though a double treatment regimen may be most effective; further work will test whether this may be primarily due to the combined higher dose or to the altered timing. This PGE2-mediated survival involves early protection of both primitive and differentiated hematopoietic BM cells, affecting cell number, cell cycling, and apoptosis. Early hematopoietic BM recovery subsequently rescues PB components, facilitating ultimate survival. With further study, PGE2 may serve to provide an effective radioprotectant with the potential to safeguard patients undergoing TBI therapy, as well as military personnel and first responders, from the risk of radiation-associated mortality. DisclosuresNo relevant conflicts of interest to declare.

Developmental origin and lineage plasticity of endogenous cardiac stem cells.

Over the past two decades, several populations of cardiac stem cells have been described in the adult mammalian heart. For the most part, however, their lineage origins and in vivo functions remain largely unexplored. This Review summarizes what is known about different populations of embryonic and adult cardiac stem cells, including KIT(+), PDGFRα(+), ISL1(+)and SCA1(+)cells, side population cells, cardiospheres and epicardial cells. We discuss their developmental origins and defining characteristics, and consider their possible contribution to heart organogenesis and regeneration. We also summarize the origin and plasticity of cardiac fibroblasts and circulating endothelial progenitor cells, and consider what role these cells have in contributing to cardiac repair.

Preclinical Development and Validation of a Hematopoietic Stem Cell-Based Lentivector Treatment for Hemophilia a

Hemophilia A is a monogenic bleeding disorder currently treated by lifelong infusions of factor VIII (fVIII). The development of gene-based treatments for hemophilia A has been hampered by several biological hurdles including the low level expression of fVIII from genetically engineered target cells, the large transgene size, and transgene product immunogenicity. We now show that these major hurdles are overcome by i) using a bioengineered FVIII transgene (ET3) that expresses at 10 - 100-fold higher levels compared to other human FVIII sequences, ii) targeting hematopoietic stem cells (HSC) ex vivo using a self-inactivating (SIN) lentiviral vector (LV) encoding ET3, and iii) using a non-myeloablative conditioning regimen prior to HSC transplantation that results in transgene product associated immune tolerance. The clinical trial design and preclinical data were favorably reviewed by the RAC, Institutional Biosafety Committee, and FDA. However upon full-scale clinical production of the SIN-ET3-LV, an additional hurdle was discovered that resulted in low LV manufacturing titer, which we showed is due to promoter competition between the CMV/HIV-LTR and the internal promoter driving ET3 production (eg. EF1a). We show that high-titer recombinant LV encoding ET-3 can be achieved by replacing the EF1a internal promoter with one that has low activity in the 293T producer cells. CD68 is a monocyte-restricted promoter that is not active in 293T producer cell lines and thus minimizes promoter interference. Using a SIN-CD68-ET3-LV expression vector, high titer virus was generated using a laboratory scale production strategy as well as a 6 L scalable clinical LV production platform. Our scalable manufacturing process generated titers >2 x 109/ml, which is sufficient to support an ex vivo HSC transduction/transplantation pilot/phase I clinical trial. Late stage preclinical testing using clinically processed vector showed that the high titer virus efficiently transduces hematopoietic cell lines, as determined by quantitative PCR, and Southern analysis, which confirmed integrated transgene stability over 8 weeks. Efficient and stable gene transfer into murine Sca-1+ cells harvested from hemophilia A mice was also achieved. A dose response of LV addition to Sca-1 cells and FVIII expression was observed in transplanted mice. Expression of >40% normal FVIII levels was achieved at multiplicity of infections (MOIs) as low 20, and long term FVIII expression was observed post HSCT. No difference in the engraftment of genetically modified cells as compared to mock-modified cells was observed. In addition, efficient, stable and dose responsive gene marking (up to 0.6 VCN/cell) of human mobilized peripheral blood(mPB)-derived CD34+ cells was reproducibly achieved. Transduction of mPB-CD34+ cells at MOIs ranging from 1 to 125 had no effect on methylcellulose colony forming unit growth when comparing SIN-CD68-ET3-LV to non-modified cells. In addition, there was no effect on mPB-CD34+ cell engraftment of NSG mice when comparing modified and mock modified cells. Furthermore, next generation sequencing-based insertion site analysis showed integration of SIN-CD68-ET3-LV to be similar, with respect to integration events near oncogenes, to that of other vectors in current clinical trials. Collectively, this comprehensive, preclinical data set provides support for regulatory submissions to conduct a pilot/phase I clinical trial of a SIN-CD68-ET3-LV transduced-HSC transplantation product for the treatment of hemophilia A. DisclosuresDoering:Bayer Healthcare: Consultancy, Honoraria, Research Funding; Expression Therapeutics: Equity Ownership. Denning:Expression Therapeutics: Employment. Spencer:Expression Therapeutics: Equity Ownership.

MSCs relieve lung injury of COPD mice through promoting proliferation of endogenous lung stem cells.

Bone marrow mesenchymal stem cells (MSCs) transplantation could repair injury tissue, but no study confirms whether MSCs can promote the proliferation of endogenous lung stem cells to repair alveolar epithelial cells of mice with chronic obstructive pulmonary disease (COPD). This study was designed to investigate the effect of MSCs on the proliferation of endogenous lung stem cells in COPD mice to confirm the repair mechanism of MSCs. The mice were divided into control group, COPD group, and COPD+MSCs group. The following indexes were detected: HE staining of lung tissue, the mean linear intercept (MLI) and alveolar destructive index (DI), the total cell number in bronchoalveolar lavage fluid (BALF), pulmonary function, alveolar wall apoptosis index (AI) and proliferation index (PI), the number of CD45(-)/CD31(-)/Sca-1(+) cells by flow cytometry (FCM), and the number of bronchoalveolar stem cells (BASCs) in bronchoalveolar duct junction (BADJ) by immunofluorescence. As compared with control group, the number of inflammatory cells in lung tissue was increased, alveolar septa was destroyed and the emphysema-like changes were seen, and the changes of lung function were in line with COPD in COPD group; AI of alveolar wall was significantly increased and PI significantly decreased in COPD group. There was no significant difference in the number of CD45(-)/CD31(-)/Sca-1(+) cells and BASCs between control group and COPD group. As compared with COPD group, the number of inflammatory cells in BALF was decreased, the number of CD45(-)/CD31(-)/Sca-1(+) cells and BASCs was increased, AI of alveolar wall was decreased and PI was increased, and emphysema-like changes were relieved in COPD+MSCs group. These findings suggested that MSCs transplantation can relieve lung injury by promoting proliferation of endogenous lung stem cells in the cigarette smoke-induced COPD mice.

Scaffold-free and scaffold-assisted 3D culture enhances differentiation of bone marrow stromal cells.

3D cultures of stem cells can preserve differentiation potential or increase the efficiency of methods that induce differentiation. Mouse bone marrow-derived stromal cells (BMSCs) were cultured in 3D as scaffold-free spheroids or "mesoid bodies" (MBs) and as aggregates on poly(lactic) acid microspheres (MB/MS). 3D cultures demonstrated viable cells, interaction on multiple planes, altered cell morphology, and the formation of structures similar to epithelial cell bridges. Cell proliferation was limited in suspension cultures of MB and MB/MS; however, cells regained proliferative capacity when transferred to flat substrates of tissue culture plates (TCPs). Expanded as monolayer, cells retained expression of Sca-1 and CD44 stem cell markers. 3D cultures demonstrated enhanced potential for adipogenic and osteogenic differentiation showing higher triglyceride accumulation and robust mineralization in comparison with TCP cultures. Enhanced and efficient adipogenesis was also observed in 3D cultures generated in a rotating cell culture system. Preservation of multilineage potential of BMSC was demonstrated in 5-azacytidine treatment of 3D cultures and TCP by expression of cardiac markers GATA4 and ACTA1 although functioning cardiomyocytes were not derived.

ORIGINS OF ADVENTITIAL SCA1 PROGENITOR CELLS

Adventitial Sca1 progenitor cells are abundant vascular smooth muscle progenitor cells that play a role in atherosclerosis and in the neointima during vascular injury. Currently, the origins of adventitial Sca1 progenitor cells are unknown and shown not to be neural crest or bone marrow derived. The purpose of this study was to identify a novel stem cell present in the adult murine aorta that gives rise to vascular smooth muscle cells and to identify the origins of adventitial Sca1 progenitor cells. Using a Myf5-Cre animal crossed to a floxed tdTomato reporter, which labels the somitic mesoderm, we were able to culture stem cells derived from the 8-12 week old mouse aorta in defined serum-free stem/progenitor cell medium. These stem cells displayed multilineage potential and could differentiate into Calponin+ vascular smooth muscle cells and S100β+ glial cells in vitro. In accord with their multilineage potential, these stem cells were positive for the stem cell marker Sox2 by indirect immunofluorescence. Intriguingly, a significant proportion of Sca1 progenitor cells were lineage marked in vivo in the Myf5-Cre animal suggesting a somitic origin for Sca1 progenitor cells in the aorta. In order to determine at which stage Sca1 progenitor cells were specified in vivo, we acquired a tamoxifen inducible Myf5-Cre ER mouse. We demonstrate that adventitial Sca1+ progenitors are specified during early somitogenesis starting at E8.5 which labeled 39±17% of Sca1 cells in the aorta of adult animals. Since the stem cells cultured in vitro also expressed Sox2, we wanted to determine if adult Sox2 stem cells in the adult aorta could also give rise to Sca1 adventitial progenitor cells. Using a Sox2-Cre ERT2 mouse crossed to a floxed tdTomato reporter, we demonstrated that approximately 34±5% of adventitial Sca1 progenitor cells were also derived from Sox2 stem cells in the aortas of adult animals. When we histologically examined the aorta for Sox2 expression, we observed that the majority of Sox2 staining was observed in the medial layer of the aorta. Thus, we suggest that somite-derived Sox2 stem cells reside in the medial layer where they give rise to transit amplifying Sca1 progenitor cells that reside in the adventitia. In the adult mouse, Sca1 adventitial progenitor cells are also replenished de novo by somite-derived Sox2 stem cells that reside in the medial layer of the aorta. Further studies will be performed to determine the physiological significance of Sox2 stem cells in disease.