Expansion of primitive human hematopoietic stem cells by culture in a zwitterionic hydrogel.

The ability to expand hematopoietic stem and progenitor cells (HSPCs) ex vivo is critical to fully realize the potential of HSPC-based therapies. In particular, the application of clinically effective therapies, such as cord blood transplantation, has been impeded because of limited HSPC availability. Here, using 3D culture of human HSPCs in a degradable zwitterionic hydrogel, we achieved substantial expansion of phenotypically primitive CD34+ cord blood and bone-marrow-derived HSPCs. This culture system led to a 73-fold increase in long-term hematopoietic stem cell (LT-HSC) frequency, as demonstrated by limiting dilution assays, and the expanded HSPCs were capable of hematopoietic reconstitution for at least 24 weeks in immunocompromised mice. Both the zwitterionic characteristics of the hydrogel and the 3D format were important for HSPC self-renewal. Mechanistically, the impact of 3D zwitterionic hydrogel culture on mitigating HSPC differentiation and promoting self-renewal might result from an inhibition of excessive reactive oxygen species (ROS) production via suppression of O2-related metabolism. HSPC expansion using zwitterionic hydrogels has the potential to facilitate the clinical application of hematopoietic-stem-cell therapies.

Notch-Expanded Murine Hematopoietic Stem and Progenitor Cells Mitigate Death from Lethal Radiation and Convey Immune Tolerance in Mismatched Recipients.

The hematopoietic syndrome of acute radiation syndrome (h-ARS) is characterized by severe bone marrow aplasia, resulting in a significant risk for bleeding, infections, and death. To date, clinical management of h-ARS is limited to supportive care dictated by the level of radiation exposure, with a high incidence of mortality in those exposed to high radiation doses. The ideal therapeutic agent would be an immediately available, easily distributable single-agent therapy capable of rapid in vivo hematopoietic reconstitution until recovery of autologous hematopoiesis occurs. Using a murine model of h-ARS, we herein demonstrate that infusion of ex vivo expanded murine hematopoietic stem and progenitor cells (HSPCs) into major histocompatibility complex mismatched recipient mice exposed to a lethal dose of ionizing radiation (IR) led to rapid myeloid recovery and improved survival. Survival benefit was significant in a dose-dependent manner even when infusion of the expanded cell therapy was delayed 3 days after lethal IR exposure. Most surviving mice (80%) demonstrated long-term in vivo persistence of donor T cells at low levels, and none had evidence of graft versus host disease. Furthermore, survival of donor-derived skin grafts was significantly prolonged in recipients rescued from h-ARS by infusion of the mismatched expanded cell product. These findings provide evidence that ex vivo expanded mismatched HSPCs can provide rapid, high-level hematopoietic reconstitution, mitigate IR-induced mortality, and convey donor-specific immune tolerance in a murine h-ARS model. Stem Cells Translational Medicine 2017;6:566-575.

Potential pathways to restore ß-cell mass: pluripotent stem cells, reprogramming, and endogenous regeneration.

Currently available ß-cell replacement therapies for patients with diabetes, including islet and pancreas transplantation, are largely successful in restoring normal glucose metabolism, but the scarcity of organ donors restricts their more widespread use. To solve this supply problem, several different strategies for achieving ß-cell mass restoration are being pursued. These include the generation of ß cells from stem cells and their subsequent transplantation, or regeneration-type approaches, such as stimulating endogenous regenerative mechanisms or inducing reprogramming of non-ß cells into ß cells. Because these strategies would ultimately generate allogeneic or syngeneic ß cells in humans, the control of alloimmunity and/or autoimmunity in addition to replacing lost ß cells will be of utmost importance. We briefly review the recent literature on these three promising strategies toward ß-cell replacement or restoration and point out the major issues impacting their translation to treating human diabetes.

Inhibition of fatty acid amide hydrolase suppresses referred hyperalgesia induced by bladder inflammation.

OBJECTIVE: • To determine (i) the presence of fatty acid amide hydrolase (FAAH) in the urinary bladder; (ii) whether or not endogenous fatty acid ethanolamides are synthesized by the bladder; (iii) the effects of FAAH inhibition on referred hyperalgesia associated with acute bladder inflammation in rats. MATERIALS AND METHODS: • Immunohistochemistry and immunoblotting were performed to detect FAAH in the bladder. Acrolein (1 mM, 400 µL) was instilled into bladders of female Wistar rats to induce cystitis. Referred mechanical hyperalgesia was assessed by application of Von Frey monofilaments to the hind paws. • Animals were killed 4, 24, 48 and 72 h after acrolein instillation, and the fatty acid ethanolamide content of bladders was measured using isotope-dilution liquid chromatography/mass spectrometry. • Other rats were treated with the FAAH inhibitor URB597 (0.3 mg/kg, i.p.) after the induction of cystitis, and the mechanical sensitivity of the hind paws was determined. RESULTS: • Immunohistochemistry and immunoblotting showed the presence of FAAH in the bladder, with greatest abundance in the urothelium. • Acrolein-induced cystitis increased fatty acid ethanolamide content (including anandamide) in the bladder in a time-dependent manner. Inhibition of FAAH diminished referred hyperalgesia associated with acute bladder inflammation. CONCLUSIONS: • The results obtained in the present study indicate that (i) FAAH is present in the urinary bladder; (ii) fatty acid ethanolamides are increased during bladder inflammation; (iii) inhibition of FAAH could be an effective therapeutic approach for the treatment of bladder pain. • These results raise the possibility that inhibitors of enzymes responsible for metabolism of fatty acid ethanolamides could inhibit pain associated with bladder inflammation.

Elimination of tumorigenic stem cells from differentiated progeny and selection of definitive endoderm reveals a Pdx1+ foregut endoderm stem cell lineage.

Embryonic stem cell (ESC) derivatives offer promise for generating clinically useful tissues for transplantation, yet the specter of producing tumors in patients remains a significant concern. We have developed a simple method that eliminates the tumorigenic potential from differentiated ESC cultures of murine and human origin while purifying lineage-restricted, definitive endoderm-committed cells. A three-stage scheme utilizing magnetic bead sorting and specific antibodies to remove undifferentiated ESCs and extraembryonic endoderm cells, followed by positive selection of definitive endoderm cells on the basis of epithelial cell adhesion molecule (EpCAM) expression, was used to isolate a population of EpCAM(+)SSEA1(-)SSEA3(-) cells. Sorted cells do not form teratomas after transplantation into immunodeficient mice, but display gene and protein expression profiles indicative of definitive endoderm cells. Sorted cells could be subsequently expanded in vitro and further differentiated to express key pancreas specification proteins. In vivo transplantation of sorted cells resulted in small, benign tissues that uniformly express PDX1. These studies describe a straightforward method without genetic manipulation that eliminates the risk of teratoma formation from ESC differentiated derivatives. Significantly, enriched populations isolated by this method appear to be lineage-restricted definitive endoderm cells with limited proliferation capacity.

Cannabinoid receptor 2 is increased in acutely and chronically inflamed bladder of rats.

Cannabinoid receptors 1 and 2 (CB1 and CB2) are G-protein coupled receptors that are expressed throughout the body. Cannabinoid receptors are expressed in the urinary bladder and may affect bladder function. The purpose of this study was twofold: to confirm the presence of cannabinoid receptors in the bladder, the L6/S1 spinal cord, and dorsal root ganglia (DRG), and to determine the effects of acute and chronic bladder inflammation on expression of cannabinoid receptors. Acute or chronic bladder inflammation was induced in rats by intravesical administration of acrolein. Abundance of CB1 and CB2 protein and their respective mRNA was determined using immunoblotting and quantitative real-time PCR, respectively. We confirmed the presence of CB1 and CB2 receptor protein and mRNA in bladder, L6-S spinal cord, and DRG. Acute bladder inflammation induced increased expression of CB2, but not CB1, protein in the bladder detrusor. Chronic bladder inflammation increased expression of bladder CB2 protein and mRNA but not CB1 protein or mRNA. Expression of CB1 or CB2 in spinal cord or DRG was unaffected by acute or chronic bladder inflammation. CB1 and CB2 receptors are present in the bladder and its associated innervation, and CB2 receptors are up-regulated in bladder after acute or chronic inflammation. CB2 receptors may be a viable target for pharmacological treatment of bladder inflammation and associated pain.

Lack of TRPV1 inhibits cystitis-induced increased mechanical sensitivity in mice.

Transient receptor potential vanilloid 1 (TRPV1) is highly expressed in primary afferent neurons. Tissue damage generates an array of chemical mediators that activate and sensitize afferent nerve fibers, and sensitization of afferent nerve fibers plays an important role in development of visceral pain. We investigated participation of TRPV1 in visceral pain associated with bladder inflammation induced in mice by systemic treatment with cyclophosphamide or intravesical instillation of acrolein. The effects of experimental cystitis on bladder function (an indicator of visceral pain) and the threshold of response to mechanical or thermal stimuli of the hind paws were investigated using TRPV1 knock-out (KO) and congenic wild-type (WT) mice. We found that cystitis induced bladder mechanical hyperreactivity and increased mechanical sensitivity of hind paws in WT, but not in TRPV1 KO mice. Lack of functional TRPV1 did not inhibit development of histological evidence of bladder inflammation, or increased expression of mRNAs for nerve growth factor, endothelial nitric oxide synthase, cyclooxygenase-2 and bradykinin receptors in urothelium. Cystitis did not affect the threshold of response to thermal stimuli in WT or KO mice. These results suggest that TRPV1 is essential for cystitis-induced bladder mechanical hyperreactivity. Also, TRPV1 participates in development of visceral pain, as reflected by referred increased mechanosensitivity in peripheral tissues in the presence of visceral inflammation.