Erin A. Kimbrel

Current status of pluripotent stem cells: moving the first therapies to the clinic

Pluripotent stem cells (PSCs) hold great promise for drug discovery and regenerative medicine owing to their ability to differentiate into any cell type in the body. After more than three decades of research, including delays due to the potential tumorigenicity of PSCs and inefficiencies in differentiation methods, the field is at a turning point, with a number of clinical trials across the globe now testing PSC-derived products in humans. Ocular diseases dominate these first-in-man trials, and Phase l/ll results are showing promising safety data as well as possible efficacy. In addition, the advent of induced PSC (iPSC) technology is enabling the development of a wide range of cell-based disease models from genetically predisposed patients, thereby facilitating drug discovery. In this Review, we discuss the recent progress and remaining challenges for the use of PSCs in regenerative medicine and drug development.

Mesenchymal Stem Cell Population Derived from Human Pluripotent Stem Cells Displays Potent Immunomodulatory and Therapeutic Properties

Mesenchymal stem cells (MSCs) are being tested in a wide range of human diseases; however, loss of potency and inconsistent quality severely limit their use. To overcome these issues, we have utilized a developmental precursor called the hemangioblast as an intermediate cell type in the derivation of a highly potent and replenishable population of MSCs from human embryonic stem cells (hESCs). This method circumvents the need for labor-intensive hand-picking, scraping, and sorting that other hESC-MSC derivation methods require. Moreover, unlike previous reports on hESC-MSCs, we have systematically evaluated their immunomodulatory properties and in vivo potency. As expected, they dynamically secrete a range of bioactive factors, display enzymatic activity, and suppress T-cell proliferation that is induced by either allogeneic cells or mitogenic stimuli. However, they also display unique immunophenotypic properties, as well as a smaller size and >30,000-fold proliferative capacity than bone marrow-derived MSCs. In addition, this is the first report which demonstrates that hESC-MSCs can inhibit CD83 up-regulation and IL-12p70 secretion from dendritic cells and enhance regulatory T-cell populations induced by interleukin 2 (IL-2). This is also the first report which shows that hESC-MSCs have therapeutic efficacy in two different autoimmune disorder models, including a marked increase in survival of lupus-prone mice and a reduction of symptoms in an autoimmune model of uveitis. Our data suggest that this novel and therapeutically active population of MSCs could overcome many of the obstacles that plague the use of MSCs in regenerative medicine and serve as a scalable alternative to current MSC sources.

Systematic in vivo structure-function analysis of p300 in hematopoiesis

Cyclic adenosine monophosphate response element binding (CREB)-binding protein (CBP) and p300 are multidomain transcriptional coactivators that help assemble large regulatory complexes at sites of active transcription. Nullizygosity of CBP or p300 results in pervasive defects in hematopoiesis. To systematically assess the structural domains of p300 required for normal hematopoiesis, we used recombinase-mediated cassette exchange to create an allelic series of coisogenic embryonic stem cells, each expressing a different mutant of p300 from the endogenous locus. We found that deletion of either the KIX or CH1 domain caused profound and pervasive defects in hematopoiesis, whereas the loss of most other domains had only lineage-restricted effects. When expressed from the p300 locus, an extra copy of CBP largely compensated for a lack of p300. Surprisingly, mutation of the p300 histone acetyltransferase (HAT) domain had minimal effects on hematopoiesis, and actually increased progenitor and stem cell numbers and proliferative potential. Our results suggest that, in distinct contrast to other organ systems, HAT activity does not provide a critical function for hematopoietic development and emphasizes the importance of enzyme-independent functions of p300.

In Vivo Pharmacodynamic Imaging of Proteasome Inhibition

Inhibiting the proteolytic activity of the 26S proteasome has been shown to have selective apoptotic effects on cancer cells and to be clinically efficacious in certain malignancies. There is an unmet medical need for additional proteasome inhibitors, and their development will be facilitated by surrogate markers of proteasome function. Toward this end, ectopic fusion of the destruction domain from ornithine decarboxylase (ODC) to reporter proteins is often used for assessing proteasome function. For luciferase-based reporters, we hypothesized that the oxygen-dependent destruction domain (ODD) from hypoxia-inducible factor 1α (HIF-1α) may provide improved sensitivity over luciferase-ODC, owing to its extremely rapid turnover by the proteasome (HIF-1α has a half-life of less than 5 minutes). In the current study, we show that ODD-luciferase affords a greater dynamic range and faster kinetics than luciferase-ODC in sensing proteasome inhibition in vitro. Importantly, ODD-luciferase also serves as an effective in vivo marker of proteasome function in xenograft tumor models, with inhibition being detected by noninvasive imaging within 3 hours of bortezomib administration. These data establish ODD-luciferase as a surrogate marker of proteasome function that can be used both in vitro and in vivo for the development of novel proteasome inhibitors.

The F-box protein beta-TrCp1/Fbw1a interacts with p300 to enhance beta-catenin transcriptional activity.

Hyperactivated β-catenin is a commonly found molecular abnormality in colon cancer, and its nuclear accumulation is thought to promote the expression of genes associated with cellular proliferation and transformation. The p300 transcriptional co-activator binds to β-catenin and facilitates transcription by recruiting chromatin remodeling complexes and general transcriptional apparatus. We have found that β-TrCp1/Fbw1a, a member of the Skp1/Cullin/Rbx1/F-box E3 ubiquitin ligase complex, binds directly to p300 and co-localizes with it to β-catenin target gene promoters. Our data show that Fbw1a, which normally targets β-catenin for degradation, works together with p300 to enhance the transcriptional activity of β-catenin, whereas other F-box/WD40 proteins do not. Fbw1a also cooperates with p300 to co-activate transcription by SMAD3, another Fbw1a ubiquitylation target, but not p53 or HIF-1α, which are substrates for other ubiquitin ligase complexes. These results suggest that, although Fbw1a is part of a negative feedback loop for controlling β-catenin levels in normal cells, its overexpression and binding to p300 may contribute to hyperactivated β-catenin transcriptional activity in colon cancer cells.

Stomping out barriers for hRBC mouse models

In this issue of Blood , Hu et al show that depletion of mouse macrophages enables the development and persistence of human red blood cells (hRBCs) in immunodeficient mouse models.[1][1] ![Figure][2] Suppression of mouse macrophages by clodronate liposomes (CLO-LIP) enables hRBC survival in

Cell Therapy for Blood Substitutes

The ability of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) to divide indefinitely without losing pluripotency and to theoretically differentiate into any cell type in the body makes them highly attractive cell sources for regenerative medicine purposes. Limitations in the supply of transfusable red blood cells and/or platelets can have potentially life-threatening consequences for patients with massive blood loss, or who suffer from diseases that cause severe anemia. During the past decade, many researchers have already been able to differentiate hESCs and/or iPSCs into specific mature blood cell types. For example, hESC-derived red blood cells and platelets are functional in tasks such as oxygen delivery and blood clotting, respectively and may be able to serve as substitutes for their donor-derived counterparts in emergencies. The ability to create banks of hESC lines with matched or reduced incompatibility could potentially reduce or eliminate the need for immunosuppressive drugs and/or immunomodulatory protocols altogether, for example, (O) RhD–lines for generation of universal red blood cells. Inasmuch as human iPSCs could potentially be produced from a patients own cells, all types of blood cells derived from such iPSC lines would be histocompatible with the patient. For transfusion-dependent patients with unusual or rare blood types, particularly those who are alloimmunized, cells generated in this manner could be an invaluable source of transfusable cells. In this chapter, we highlight biological functions of mature cells of the blood, clinical conditions requiring the transfusion or stimulation of these cells, and the potential for hESC/iPSC-derivatives to serve as functional replacements. However, in vitro differentiation systems used to generate these cells will need further optimization before hESC/iPSC-derived blood components can be used clinically.