Correcting ß-thalassemia by combined therapies that restrict iron and modulate erythropoietin activity.

ß-Thalassemia intermedia is a disorder characterized by ineffective erythropoiesis (IE), anemia, splenomegaly, and systemic iron overload. Novel approaches are being explored based on the modulation of pathways that reduce iron absorption (ie, using hepcidin activators like Tmprss6-antisense oligonucleotides [ASOs]) or increase erythropoiesis (by erythropoietin [EPO] administration or modulating the ability of transferrin receptor 2 [Tfr2] to control red blood cell [RBC] synthesis). Targeting Tmprss6 messenger RNA by Tmprss6-ASO was proven to be effective in improving IE and splenomegaly by inducing iron restriction. However, we postulated that combinatorial strategies might be superior to single therapies. Here, we combined Tmprss6-ASO with EPO administration or removal of a single Tfr2 allele in the bone marrow of animals affected by ß-thalassemia intermedia (Hbbth3/+). EPO administration alone or removal of a single Tfr2 allele increased hemoglobin levels and RBCs. However, EPO or Tfr2 single-allele deletion alone, respectively, exacerbated or did not improve splenomegaly in ß-thalassemic mice. To overcome this issue, we postulated that some level of iron restriction (by targeting Tmprss6) would improve splenomegaly while preserving the beneficial effects on RBC production mediated by EPO or Tfr2 deletion. While administration of Tmprss6-ASO alone improved the anemia, the combination of Tmprss6-ASO + EPO or Tmprss6-ASO + Tfr2 single-allele deletion produced significantly higher hemoglobin levels and reduced splenomegaly. In conclusion, our results clearly indicate that these combinatorial approaches are superior to single treatments in ameliorating IE and anemia in ß-thalassemia and could provide guidance to translate some of these approaches into viable therapies.

Multipotent vasculogenic pericytes from human pluripotent stem cells promote recovery of murine ischemic limb.

BACKGROUND: Pericytes represent a unique subtype of microvessel-residing perivascular cells with diverse angiogenic functions and multilineage developmental features of mesenchymal stem cells. Although various protocols for derivation of endothelial and/or smooth muscle cells from human pluripotent stem cells (hPSC, either embryonic or induced) have been described, the emergence of pericytes in the course of hPSC maturation has not yet been elucidated. METHODS AND RESULTS: We found that during hPSC development, spontaneously differentiating embryoid bodies give rise to CD105(+)CD90(+)CD73(+)CD31(-) multipotent clonogenic mesodermal precursors, which can be isolated and efficiently expanded. Isolated and propagated cells expressed characteristic pericytic markers, including CD146, NG2, and platelet-derived growth factor receptor ß, but not the smooth muscle cell marker α-smooth muscle actin. Coimplantation of hPSC-derived endothelial cells with pericytes resulted in functional and rapid anastomosis to the murine vasculature. Administration of pericytes into immunodeficient mice with limb ischemia promoted significant vascular and muscle regeneration. At day 21 after transplantation, recruited hPSC pericytes were found incorporated into recovered muscle and vasculature. CONCLUSIONS: Derivation of vasculogenic and multipotent pericytes from hPSC can be used for the development of vasculogenic models using multiple vasculogenic cell types for basic research and drug screening and can contribute to angiogenic regenerative medicine.

Immunoevasive pericytes from human pluripotent stem cells preferentially modulate induction of allogeneic regulatory T cells.

Isolated microvessel-residing pericytes and pericytes from human pluripotent stem cells (hPSCs) exhibit mesenchymal stem cell-like characteristics and therapeutic properties. Despite growing interest in pericyte-based stem cell therapy, their immunogenicity and immunomodulatory effects on nonactivated T cells are still poorly defined, in particular those of vasculogenic hPSC pericytes. We found that tissue-embedded and unstimulated cultured hPSC- or tissue-derived pericytes constitutively expressed major histocompatibility complex (MHC) class I and the inhibitory programmed cell death-ligand 1/2 (PD-L1/2) molecules but not MHC class II or CD80/CD86 costimulatory molecules. Pretreatment with inflammatory mediators failed to induce an antigen-presenting cell-like phenotype in stimulated pericytes. CD146+ pericytes from hPSCs did not induce activation and proliferation of allogeneic resting T cells independent of interferon (IFN)-γ prestimulation, similarly to pericytes from human brain or placenta. Instead, pericytes mediated a significant increase in the frequency of allogeneic CD25highFoxP3+ regulatory T cells when cocultured with nonactivated peripheral blood T cells. Furthermore, when peripheral blood CD25high regulatory T cells (Tregs) were depleted from isolated CD3+ T cells, pericytes preferentially induced de novo formation of CD4+CD25highFoxP3+CD127-, suppressive regulatory T cells. Constitutive expression of PD-L1/2 and secretion of transforming growth factor-ß by hPSC pericytes directly regulated generation of pericyte-induced Tregs. Pericytes cotransplanted into immunodeficient mice with allogeneic CD25- T cells maintained a nonimmunogenic phenotype and mediated the development of functional regulatory T cells. Together, these findings reveal a novel feature of pericyte-mediated immunomodulation distinguished from immunosuppression, shared by native tissue pericytes and hPSC pericytes, and support the notion that pericytes can be applied for allogeneic cell therapy.

ADAR1 is involved in the regulation of reprogramming human fibroblasts to induced pluripotent stem cells.

Adenosine-to-inosine (A-to-I) RNA editing is a post-transcriptional, site-specific modification process that is catalyzed by Adenosine Deaminase Acting on RNA (ADAR) gene family members. Since ADARs act on double-stranded RNA, most A-to-I editing occurs within repetitive elements, particularly Alu elements, as the result of the inherent property of these sequences to fold and form double strands. ADAR1-mediated A-to-I RNA editing was recently implicated in the regulation of human embryonic stem cells (hESCs). Spontaneous and neuronal differentiation of hESC was shown to result in a decrease in A-to-I editing levels. Knockdown of ADAR1 in hESCs results in an elevation of the expression of differentiation-related genes. In addition, we found that hESCs over-expressing ADAR1 could not be generated. The current study shows that the editing levels of induced pluripotent stem cells (iPSCs) change throughout reprogramming, from a source cell level to a level similar to that of hESCs. Up- or down-regulation of the ADAR1 level in human foreskin fibroblast (HFF) cells before induction of reprogramming results in varied reprogramming efficiencies. Furthermore, HFF-iPSC early clones derived from source cells in which the ADAR1 level was down-regulated lose their iPSC properties shortly after iPSC colony formation and instead exhibit characteristics of cancer cells. Taken together, our results imply a role for ADAR1 in the regulation of pluripotency induction as well as in the maintenance of early iPSC properties.

Efficient engineering of vascularized ectopic bone from human embryonic stem cell-derived mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) can be derived from various adult and fetal tissues. However, the quality of tissues for the isolation of adult and fetal hMSCs is donor dependent with a nonreproducible yield. In addition, tissue engineering and cell therapy require large-scale production of a pure population of lineage-restricted stem cells that can be easily induced to differentiate into a specific cell type. Therefore, human embryonic stem cells (hESCs) can provide an alternative, plentiful source for generation of reproducible hMSCs. We have developed efficient differentiation protocols for derivation of hMSCs from hESCs, including coculture with murine OP9 stromal cells and feeder layer-free system. Our protocols have resulted in the generation of up to 49% of hMSCs, which expressed CD105, CD90, CD29, and CD44. The hMSCs exhibited high adipogenic, chondrocytic, and osteogenic differentiation in vitro. The latter correlated with osteocalcin secretion and vascular endothelial growth factor (VEGF) production by the differentiating hMSCs. hMSC-derived osteoblasts further differentiated and formed ectopic bone in vivo, and induced the formation of blood vessels in Matrigel implants. Our protocol enables generation of a purified population of hESC-derived MSCs, with the potential of differentiating into several mesodermal lineages, and particularly into vasculogenesis-inducing osteoblasts, which can contribute to the development of bone repair protocols.

Enhanced reprogramming and cardiac differentiation of human keratinocytes derived from plucked hair follicles, using a single excisable lentivirus.

Induced pluripotent stem cells (iPSCs) represent an ideal cell source for future cell therapy and regenerative medicine. However, most iPSC lines described to date have been isolated from skin fibroblasts or other cell types that require harvesting by surgical intervention. Because it is desirable to avoid such intervention, an alternative cell source that can be readily and noninvasively isolated from patients and efficiently reprogrammed, is required. Here we describe a detailed and reproducible method to derive iPSCs from plucked human hair follicle keratinocytes (HFKTs). HFKTs were isolated from single plucked hair, then expanded and reprogrammed by a single polycistronic excisable lentiviral vector. The reprogrammed HFKTs were found to be very sensitive to human embryonic stem cell (hESC) growth conditions, generating a built-in selection with easily obtainable and very stable iPSCs. All emerging colonies were true iPSCs, with characteristics typical of human embryonic stem cells, differentiated into derivatives of all three germ layers in vitro and in vivo. Spontenaeouly differentiating functional cardiomyocytes (CMs) were successfully derived and characterized from these HFKT-iPSCs. The contracting CMs exhibited well-coordinated intracellular Ca²+ transients and contractions that were readily responsive to ß-adrenergic stimulation with isoproterenol. The introduction of Cre-recombinase to HFKT-iPSC clones was able to successfully excise the integrated vector and generate transgene-free HFKT-iPSC clone that could be better differentiated into contracting CMs, thereby revealing the desired cells for modeling human diseases. Thus, HFKTs are easily obtainable, and highly reprogrammed human cell source for all iPSC applications.

Immunomodulatory therapeutic effect of glatiramer acetate on several murine models of inflammatory bowel disease.

Inflammatory bowel disease (IBD) is characterized by detrimental immune reactivity in the gut and imbalance between proinflammatory and anti-inflammatory reactivity. In an attempt to down-regulate colitis, we investigated the effect of the immunomodulator glatiramer acetate (GA, Copaxone, copolymer 1) on two murine models of IBD, chemically induced and spontaneous. Acute experimental colitis of different levels of severity was induced in C57BL/6 mice by dextran sulfate sodium (DSS) administered orally at different concentrations and frequencies. It was manifested in weight loss, intestinal bleeding, and diarrhea, as well as by macroscopic and microscopic colon damage. GA treatment led to amelioration of all of these pathological manifestations, resulting in improved long-term survival. Moreover, even when colitis was induced by three cycles of DSS in this highly susceptible mouse strain, as well as in BALB/c mice that exhibit a chronic disease pattern, a substantial reduction in disease activity and mortality was obtained. GA treatment induced a beneficial effect also in a spontaneous model of colitis developed in the C3H/HeJBir IL-10-deficient mice. The detrimental proinflammatory response manifested by proliferation, tumor necrosis factor-alpha, and interferon-gamma expression was modulated by GA, whereas the regulatory anti-inflammatory transforming growth factor-beta and IL-10 cytokines response was elevated. This was demonstrated on the level of protein secretion in splenocytes and local mesenteric lymphocytes in response to syngeneic colon extract and in the overall response to anti-CD3, as well as on the level of mRNA expression in the colon.

The immunomodulator glatiramer acetate augments the expression of neurotrophic factors in brains of experimental autoimmune encephalomyelitis mice.

Neurotrophins (NTs) such as BDNF, NT-3, and NT-4 are important modulators of neuronal function and survival. Their expression in the CNS after various insults is thus of major therapeutic consequence. Glatiramer acetate [(GA) Copaxone], an approved drug for the treatment of multiple sclerosis, has been shown to induce Th2/3 cells that accumulate in the CNS, expressing in situ antiinflammatory cytokines and BDNF. In the present study, we investigated whether s.c. injections of GA, applied at various stages of experimental autoimmune encephalomyelitis, affect the expression of NTs, particularly BDNF, in the brain. In untreated experimental autoimmune encephalomyelitis mice, the expression of NTs was elevated shortly after disease appearance but subsequently declined below that of naive mice. In contrast, GA treatment led to sustained augmentation in the expression of BDNF, NT-3, and NT-4 in various brain regions as demonstrated by histological analysis of immunostained brain sections. GA treatment, even when started 45 days after disease induction, restored the impaired level of NTs to that of healthy mice. BDNF elevation after GA treatment was demonstrated on both protein and mRNA levels. Prominent staining was manifested not only by infiltrating GA-induced T cells, but also by CNS resident cells (neurons and astrocytes), indicative of a bystander therapeutic effect. Of importance, in GA-treated mice, intense BDNF expression was manifested by neuronal progenitors that migrated into lesions in injured regions. These results indicate that the immunomodulator GA exerts not only an antiinflammatory effect, but also enhances neuroprotection and regeneration of neural elements in the diseased brain.