Adoptive transfer of T regulatory cells (Treg) has been successfully exploited in the context of graft-versus-host disease, transplantation, and autoimmune disease. For the majority of applications, ...clinical administration of Treg requires laborious
expansion and typically involves open handling for culture feeds and repetitive sampling. Here we show results from our approach to translate manual Treg manufacturing to the fully closed automated CliniMACS Prodigy® system reducing contamination risk, hands-on time, and quality variation from human intervention. Polyclonal Treg were isolated from total nucleated cells obtained through leukapheresis of healthy donors by CD8
cell depletion and subsequent CD25
enrichment. Treg were expanded with the CliniMACS Prodigy® device using clinical-grade cell culture medium, rapamycin, IL-2, and αCD3/αCD28 beads for 13-14 days. We successfully integrated expansion bead removal and final formulation into the automated procedure, finalizing the process with a ready to use product for bedside transfusion. Automated Treg expansion was conducted in parallel to an established manual manufacturing process using G-Rex cell culture flasks. We could prove similar expansion kinetics leading to a cell yield of up to 2.12 × 10
cells with the CliniMACS Prodigy® and comparable product phenotype of >90% CD4
CD25
CD127
FOXP3
cells that had similar
immunosuppressive function. Efficiency of expansion bead depletion was comparable to the CliniMACS® Plus system and the final ready-to-infuse product had phenotype stability and high vitality after overnight storage. We anticipate this newly developed closed system expansion approach to be a starting point for the development of enhanced throughput clinical scale Treg manufacture, and for safe automated generation of antigen-specific Treg grafted with a chimeric antigen receptor (CAR Treg).
Understanding the physiological migration of hematopoietic progenitors is important, not only for basic stem cell research, but also in view of their therapeutic relevance. Here, we investigated the ...role of the Rho kinase pathway in the morphology and migration of hematopoietic progenitors using an ex vivo co-culture consisting of human primary CD34+ progenitors and mesenchymal stromal cells. The addition of the Rho kinase inhibitor Y-27632 led to the abolishment of the uropod and microvillar-like structures of hematopoietic progenitors, concomitant with a redistribution of proteins found therein (prominin-1 and ezrin). Y-27632-treated cells displayed a deficiency in migration. Time-lapse video microscopy revealed impairment of the rear pole retraction. Interestingly, the knockdown of ROCK I, but not ROCK II, using RNA interference (RNAi) was sufficient to cause the referred morphological and migrational changes. Unexpectedly, the addition of nocodazole to either Y-27632- or ROCK I RNAi-treated cells could restore their polarized morphology and migration suggesting an active role for the microtubule network in tail retraction. Finally, we could demonstrate using RNAi that RhoA, the upstream regulator of ROCK, is involved in these processes. Collectively, our data provide new insights regarding the role of RhoA/ROCK I and the microtubules in the migration of stem cells.
Human prominin-1/CD133 has been reported to be expressed in neural and hematopoietic stem/progenitor cells and in embryonic, but not adult, epithelia. This lack of detection of human prominin-1, as ...defined by its glycosylation-dependent AC133 epitope, is surprising given the expression of the murine ortholog in adult epithelia. Here, we demonstrate, by using a novel prominin-1 antiserum (alphahE2), that the decrease of AC133 immunoreactivity observed during differentiation of the colonic adenocarcinoma-derived Caco-2 cells is not paralleled by a down-regulation of prominin-1. We have also shown that alphahE2 immunoreactivity, but not AC133 immunoreactivity, is present in several adult human tissues, such as kidney proximal tubules and the parietal layer of Bowman's capsule of juxtamedullary nephrons, and in lactiferous ducts of the mammary gland. These observations suggest that only the AC133 epitope is down-regulated upon cell differentiation. Furthermore, alphahE2 immunoreactivity has been detected in several kidney carcinomas derived from proximal tubules, independent of their grading. Interestingly, in one particular case, the AC133 epitope, which is restricted to stem cells in normal adult tissue, was up-regulated in the vicinity of the tumor. Our data thus show that (1) in adults, the expression of human prominin-1 is not limited to stem and progenitor cells, and (2) the epitopes of prominin-1 might be useful for investigating solid cancers.
The differentiation of stem cells is a fundamental process in cell biology and understanding its mechanism might open a new avenue for therapeutic strategies. Using an ex vivo co‐culture system ...consisting of human primary haematopoietic stem and progenitor cells growing on multipotent mesenchymal stromal cells as a feeder cell layer, we describe here the exosome‐mediated release of small membrane vesicles containing the stem and cancer stem cell marker prominin‐1 (CD133) during haematopoietic cell differentiation. Surprisingly, this contrasts with the budding mechanism underlying the release of this cholesterol‐binding protein from plasma membrane protrusions of neural progenitors. Nevertheless, in both progenitor cell types, protein–lipid assemblies might be the essential structural determinant in the release process of prominin‐1. Collectively, these data support the concept that prominin‐1‐containing lipid rafts may host key determinants necessary to maintain stem cell properties and their quantitative reduction or loss may result in cellular differentiation.
In the second half of the nineteenth century, American cities began to go dark. Hulking new buildings overspread blocks, pollution obscured the skies, and glass and smog screened out the ...health-giving rays of the sun. Doctors fed anxities about these new conditions with claims about a rising tide of the "diseases of darkness, " especially rickets and tuberculosis.In American Sunshine, Daniel Freund tracks the obsession with sunlight from those bleak days into the twentieth century. Before long, social reformers, medical professionals, scientists, and a growing nudist movement proffered remedies for America's new dark age. Architects, city planners, and politicians made access to sunlight central to public housing and public health. and entrepreneurs, dairymen, and tourism boosters transformed the pursuit of sunlight and its effects into a commodity. Within this historical context, Freund sheds light on important questions about the commodification of health and nature and makes an original contribution to the histories of cities, consumerism, the environment, and medicine.
Recent studies suggest that both facial and bodily dominance promote high status positions and predict status-seeking behaviors such as aggression and social dominance. An evolutionarily relevant ...context in which associations between these dominance signals and status outcomes may be prevalent are face-to-face status contests. The present study examined whether facial and bodily dominance predicted success in dyadic competitions (one physical discipline, arm wrestling, and three nonphysical disciplines) in men (N = 125) in a controlled laboratory setting. Men’s bodies and faces were independently rated for physical dominance, and associations of these ratings with contest outcomes as well as mediating and moderating variables (such as physical strength, body height, trait dominance, baseline and reactive testosterone) were examined. Both facial and bodily dominance positively predicted success in the physical discipline, mediated by physical strength, but not in the three nonphysical disciplines. Our findings demonstrate that facial and bodily physical dominance may be honest signals for men’s formidability and hence status potential, at least in a physically competitive context.
During ontogenesis and the entire adult life hematopoietic stem and progenitor cells have the capability to migrate. In comparison to the process of peripheral leukocyte migration in inflammatory ...responses, the molecular and cellular mechanisms governing the migration of these cells remain poorly understood. A common feature of migrating cells is that they need to become polarized before they migrate. Here we have investigated the issue of cell polarity of hematopoietic stem/progenitor cells in detail. We found that human CD34+ hematopoietic cells (1) acquire a polarized cell shape upon cultivation, with the formation of a leading edge at the front pole and a uropod at the rear pole; (2) exhibit an amoeboid movement, which is similar to the one described for migrating peripheral leukocytes; and (3) redistribute several lipid raft markers including cholesterol-binding protein prominin-1 (CD133) in specialized plasma membrane domains. Furthermore, polarization of CD34+ cells is stimulated by early acting cytokines and requires the activity of phosphoinositol-3-kinase as previously reported for peripheral leukocyte polarization. Together, our data reveal a strong correlation between polarization and migration of peripheral leukocytes and hematopoietic stem/progenitor cells and suggest that they are governed by similar mechanisms.
Human prominin-1 (CD133) is expressed by various stem and progenitor cells originating from diverse sources. In addition to stem cells, its mouse ortholog is expressed in a broad range of adult ...epithelial cells, where it is selectively concentrated in their apical domain. The lack of detection of prominin-1 in adult human epithelia might be explained, at least in part, by the specificity of the widely used AC133 antibody, which recognizes an epitope that seems dependent on glycosylation. Here we decided to re-examine its expression in adult human tissues, particularly in glandular epithelia, using a novel monoclonal antibody (80B258) generated against the human prominin-1 polypeptide. In examined tissues, we observed 80B258 immunoreactivity at the apical or apicolateral membranes of polarized cells. For instance, we found expression in secretory serous and mucous cells as well as intercalated ducts of the large salivary and lacrimal glands. In sweat glands including the gland of Moll, 80B258 immunoreactivity was found in the secretory (eccrine and apocrine glands) and duct (eccrine glands) portion. In the liver, 80B258 immunoreactivity was identified in the canals of Hering, bile ductules, and small interlobular bile ducts. In the uterus, we detected 80B258 immunoreactivity in endometrial and cervical glands. Together these data show that the overall expression of human prominin-1 is beyond the rare primitive cells, and it seems to be a general marker of apical or apicolateral membrane of glandular epithelia. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.
The optimal management of shared vehicle systems, such as bike-, scooter-, car-, or ride-sharing, is more challenging compared with traditional resource allocation settings because of the presence of ...spatial externalities—changes in the demand/supply at any location affect future supply throughout the system within short timescales. These externalities are well captured by steady-state Markovian models, which are therefore widely used to analyze such systems. However, using Markovian models to design pricing and other control policies is computationally difficult because the resulting optimization problems are high dimensional and nonconvex. In our work, we design a framework that provides near-optimal policies, for a range of possible controls, that are based on applying the possible controls to achieve spatial balance on average. The optimality gap of these policies improves as the ratio between supply and the number of locations increases and asymptotically goes to zero.
Optimizing shared vehicle systems (bike-/scooter-/car-/ride-sharing) are more challenging compared with traditional resource allocation settings because of the presence of
complex network externalities
—changes in the demand/supply at any location affect future supply throughout the system within short timescales. These externalities are well captured by steady-state Markovian models, which are therefore widely used to analyze such systems. However, using such models to design pricing and other control policies is computationally difficult because the resulting optimization problems are high dimensional and nonconvex. To this end, we develop a
rigorous approximation framework
for shared vehicle systems, providing a unified approach for a wide range of controls (pricing, matching, rebalancing), objective functions (throughput, revenue, welfare), and system constraints (travel times, welfare benchmarks, posted-price constraints). Our approach is based on the analysis of natural convex relaxations and obtains as special cases existing approximate optimal policies for limited settings, asymptotic optimality results, and heuristic policies. The resulting guarantees are nonasymptotic and parametric and provide operational insights into the design of real-world systems. In particular, for any shared vehicle system with
n
stations and
m
vehicles, our framework obtains an approximation ratio of
1
+
(
n
−
1
)
/
m
, which is particularly meaningful when
m
/
n
, the average number of vehicles per station, is large, as is often the case in practice.
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