Prolongevity hormone FGF21 protects against immune senescence by delaying age-related thymic involution

Age-related thymic degeneration is associated with loss of naïve T cells, restriction of peripheral T-cell diversity, and reduced healthspan due to lower immune competence. The mechanistic basis of age-related thymic demise is unclear, but prior evidence suggests that caloric restriction (CR) can slow thymic aging by maintaining thymic epithelial cell integrity and reducing the generation of intrathymic lipid. Here we show that the prolongevity ketogenic hormone fibroblast growth factor 21 (FGF21), a member of the endocrine FGF subfamily, is expressed in thymic stromal cells along with FGF receptors and its obligate coreceptor, βKlotho. We found that FGF21 expression in thymus declines with age and is induced by CR. Genetic gain of FGF21 function in mice protects against age-related thymic involution with an increase in earliest thymocyte progenitors and cortical thymic epithelial cells. Importantly, FGF21 overexpression reduced intrathymic lipid, increased perithymic brown adipose tissue, and elevated thymic T-cell export and naïve T-cell frequencies in old mice. Conversely, loss of FGF21 function in middle-aged mice accelerated thymic aging, increased lethality, and delayed T-cell reconstitution postirradiation and hematopoietic stem cell transplantation (HSCT). Collectively, FGF21 integrates metabolic and immune systems to prevent thymic injury and may aid in the reestablishment of a diverse T-cell repertoire in cancer patients following HSCT.The degenerative changes in thymus precede age-related loss of function in other organs (1–4). As human lifespan continues to increase, it has been hypothesized that the ability to retain a functional level of thymic lymphopoiesis beyond the time limit set by evolutionary pressures may be an important strategy to extend healthspan (3, 4). Therefore, the ability to enhance thymic lymphopoiesis is thought to be central to the rejuvenation of T-cell–mediated immune surveillance in the elderly (1–7). Aging is associated with marked perturbations in the stromal cell microenvironment of the thymus (8, 9). This includes a reduction in thymopoiesis-supporting thymic epithelial cells (TECs) (10), an increase in fibroblasts (11, 12), and emergence of adipocytes (4, 13) of unknown origin and function. Accordingly, recent efforts have focused on targeting TECs for the rejuvenation of the aging thymus (12, 14). Emerging evidence indicates that immune–metabolic interactions control several aspects of the thymic involution process and age-related inflammation (13). We have shown that byproducts of thymic fatty acids and lipids result in accumulation of “lipotoxic DAMPs” (damage-associated molecular patterns), which triggers innate immune-sensing mechanisms such as inflammasome activation that link aging to thymic demise (15). Immune–metabolic interactions within the aging thymus produce a local proinflammatory state that directly compromises the thymic stromal microenvironment, thymic lymphopoiesis, and serves as a precursor of systemic immune dysregulation in the elderly (5, 8). Despite progress in the field, the thymic growth factors that regulate thymic involution are incompletely understood.The fibroblast growth factors (FGFs) constitute a family of 22 proteins that regulate diverse biological processes such as growth, development, differentiation, and wound repair (16). Prior studies showed that FGF7/keratinocyte growth factor (KGF) administration in aged mice partially reversed thymic involution (17–19). Notably, unlike most FGFs, FGF21 lacks affinity for heparan sulfate in the extracellular matrix and thus can be secreted to act in an endocrine fashion (20). FGF21 is predominantly secreted from liver but is also expressed in thymus (21). FGF21 is a prolongevity hormone that elicits it biological effects by binding to βKlotho in complex with FGF receptor (FGFR) 1c, 2c, or 3c, but not FGFR4 (16, 22, 23). FGF21 supports host survival during states of energy deficit by increasing ketogenesis and fuel utilization through mitochondrial fatty acid oxidation (16, 23, 24). Interestingly, energy deficit induced by the prolongevity intervention of caloric restriction (CR) reduces ectopic thymic lipid and maintains thymopoiesis in aged mice (13). This raises the question of whether signals that stimulate mobilization of ectopic lipid mediate the salutary effects of CR on immune function. Here we present evidence that FGF21 and βKlotho are coexpressed in TECs and maintain T-cell diversity in models of aging and hematopoietic stem cell transplantation (HSCT) by enhancing thymic function.     

Spleen supports a pool of innate-like B cells in white adipose tissue that protects against obesity-associated insulin resistance

Lipid accumulation in obesity triggers a low-grade inflammation that results from an imbalance between pro- and anti-inflammatory components of the immune system and acts as the major underlying mechanism for the development of obesity-associated diseases, notably insulin resistance and type 2 diabetes. Innate-like B cells are a subgroup of B cells that respond to innate signals and modulate inflammatory responses through production of immunomodulatory mediators such as the anti-inflammatory cytokine IL-10. In this study, we examined innate-like B cells in visceral white adipose tissue (VAT) and the relationship of these cells with their counterparts in the peritoneal cavity and spleen during diet-induced obesity (DIO) in mice. We show that a considerable number of innate-like B cells bearing a surface phenotype distinct from the recently identified “adipose natural regulatory B cells” populate VAT of lean animals, and that spleen represents a source for the recruitment of these cells in VAT during DIO. However, demand for these cells in the expanding VAT outpaces their recruitment during DIO, and the obese environment in VAT further impairs their function. We further show that removal of splenic precursors of innate-like B cells through splenectomy exacerbates, whereas supplementation of these cells via adoptive transfer ameliorates, DIO-associated insulin resistance. Additional adoptive transfer experiments pointed toward a dominant role of IL-10 in mediating the protective effects of innate-like B cells against DIO-induced insulin resistance. These findings identify spleen-supplied innate-like B cells in VAT as previously unrecognized players and therapeutic targets for obesity-associated diseases.The current obesity epidemic has led to an increase in the incidence of a variety of disorders that are collectively referred to as obesity-associated diseases. Changes in diet and lifestyle, especially the abundance of energy-dense high-fat foods, have played a central role in the emergence of this epidemic. Lipid accumulation in obesity triggers a low-grade inflammation that results from an imbalance between pro- and anti-inflammatory components of the immune system. This chronic inflammation acts as the major underlying mechanism for the development of obesity-associated diseases, notably insulin resistance and type 2 diabetes (T2D) (1–5). Both the innate and adaptive branches of the immune system are activated during obesity and participate in the induction and maintenance of obesity-triggered inflammation (1–7). In metabolic organs, most notably visceral white adipose tissue (VAT), increases in proinflammatory cells and decreases in anti-inflammatory cells create an insulin-antagonizing environment that interferes with normal metabolic pathways (1–5). Whereas it is now clear that multiple immune cells play a role in this process, the full spectrum of cellular and molecular signals that initiate and sustain the chronic inflammation in obesity remains to be delineated.Lymphocytes of the T- and B-cell lineages are traditionally categorized as components of the adaptive immune system, as they recognize specific pathogen-derived antigens and are capable of developing long-lasting immune memory. Among these conventional adaptive lymphocytes, CD8⁺ T cells and follicular (B-2) B cells have been shown to exacerbate, whereas CD4⁺Foxp3⁺ regulatory T (T_reg) cells have been shown to protect against, obesity-triggered inflammation and insulin resistance (8–11). Over the past few decades, a growing family of lymphocyte subsets with innate-like properties and functions has been identified (12–17). These cells, through recognition of nonspecific innate immune signals and production of immunomodulatory cytokines, interact with and influence the function of multiple cell types of the innate and adaptive branches of the immune system and thus shape subsequent immune and inflammatory responses and impact disease outcomes. In the T-cell lineage, several research groups have investigated the role of natural killer T (NKT) cells in obesity and insulin resistance (18). A recent report identified a subset of B cells in white adipose tissue that expressed a unique surface phenotype and protected mice against obesity-induced inflammation (19). However, whether other subsets of innate-like B cells capable of influencing insulin sensitivity also populate VAT is currently unknown. Additionally, the source(s) for the recruitment of these cells in VAT during obesity remains unclear.Several subsets of innate-like B cells, including B-1a and B-1b B cells, marginal zone (MZ) B cells, regulatory B cells, and innate response activator (IRA) B cells have been identified in lymphoid organs and in peritoneal cavity (PerC) of mice (12, 14–16, 19–21). These innate-like B-cell subsets share several phenotypic and functional characteristics but also display important differences. Compared with conventional adaptive B-2 B cells, innate-like B cells exhibit increased responsiveness to innate signals through a variety of innate receptors such as toll-like receptors (TLRs) (12, 14–16, 20, 21). Although these cells only constitute a small subpopulation of B cells in spleen and mainly reside in the PerC under steady-state conditions (12), adult mouse spleen houses progenitors and precursors of these cells and this organ therefore plays a critical role in maintaining a functional pool of innate-like B cells (22–24). Among innate-like B cells, IL-10–producing B cells, often referred to as regulatory B cells or B10 cells, modulate inflammatory responses primarily through production of the anti-inflammatory cytokine IL-10 (14–16). Furthermore, B-1a B cells are an important source of natural IgM antibodies that protect against atherosclerosis, a chronic inflammatory disease that shares several mechanistic features with obesity-associated diseases (25, 26).Several recent studies are consistent with a protective role of spleen-derived IL-10–producing B cells in obesity-induced inflammation and insulin resistance. Removal of spleen in diet-induced obesity (DIO) mice exacerbated VAT inflammation, which could be ameliorated by supplementation of IL-10 (27). Unfractionated B cells of DIO mice and of patients with T2D produced reduced levels of IL-10 in response to in vitro stimulation (10, 28). Whereas these findings suggest a critical role of spleen for provision of IL-10, possibly through IL-10–producing B cells, in protecting against obesity-associated insulin resistance, the cellular mechanisms of action remain unclear.We show here that a substantial number of B cells with a surface phenotype resembling innate-like B-1a and regulatory B10 B cells (12, 16, 29) but distinct from recently identified “adipose natural regulatory B cells” (19) populate VAT under steady-state conditions. Similar to their counterparts in spleen and PerC, these cells in VAT spontaneously produce IgM antibodies and constitute the majority of IL-10–competent B cells at this anatomic location. Whereas the spleen supports a pool of innate-like B cells in VAT, the demand for these cells in the expanding VAT during DIO outpaces their recruitment and the obese environment in VAT further impairs their IL-10 competence. Consequently, splenectomy exacerbates, whereas supplementation with these innate-like B cells via adoptive transfer ameliorates, DIO-induced systemic insulin resistance. Overall, our findings have identified a previously unrecognized subset of IL-10–competent innate-like B cells in VAT and provided evidence for a critical role of spleen in supplying these cells to VAT for protection against obesity-associated insulin resistance.     

Numb-deficient satellite cells have regeneration and proliferation defects

The adaptor protein Numb has been implicated in the switch between cell proliferation and differentiation made by satellite cells during muscle repair. Using two genetic approaches to ablate Numb, we determined that, in its absence, muscle regeneration in response to injury was impaired. Single myofiber cultures demonstrated a lack of satellite cell proliferation in the absence of Numb, and the proliferation defect was confirmed in satellite cell cultures. Quantitative RT-PCR from Numb-deficient satellite cells demonstrated highly up-regulated expression of p21 and Myostatin, both inhibitors of myoblast proliferation. Transfection with Myostatin-specific siRNA rescued the proliferation defect of Numb-deficient satellite cells. Furthermore, overexpression of Numb in satellite cells inhibited Myostatin expression. These data indicate a unique function for Numb during the initial activation and proliferation of satellite cells in response to muscle injury.Satellite cells represent a muscle-specific stem cell population that allows for muscle growth postnatally and is necessary for muscle repair (1). In response to muscle-fiber damage, quiescent satellite cells that lie along the myofibers under the plasmalemma are activated and proliferate. Proliferating satellite cells have a binary fate decision to make—they can differentiate into myoblasts and intercalate into myofibers by fusion to repair the damaged muscle or they can renew the satellite cell population and return to a quiescent state (2–4). Quiescent satellite cells express paired box 7 (Pax7), but low or undetectable levels of the myogenic regulatory factors Myf5 and MyoD (5, 6). Activated satellite cells robustly express Pax7 and MyoD/Myf5, but a subset will subsequently down-regulate the myogenic regulatory factors in the process of satellite cell self-renewal (7). Recent studies have demonstrated that, in vivo, Pax7-positive cells are necessary for muscle repair (8, 9).Notch signaling is an important regulator of satellite cell function; it is implicated in satellite cell activation, proliferation (2, 10, 11), and maintenance of quiescence (12, 13). Expression of constitutively active Notch1 results in maintenance of Pax7 expression and down-regulation of Myod/Myf5 whereas inhibition of Notch signaling leads to myogenic differentiation (10, 14). In fact, conditional ablation of Rbpj embryonically results in hypotrophic muscle (15), and, if ablated in the adult, satellite cells undergo spontaneous activation and precocious differentiation with a failure of self-renewal (12, 13). In adult muscle, the Notch ligand, Delta-like1 (Dll1), is expressed on satellite cells, myofibers, and newly differentiating myoblasts and is necessary for repair (10, 11, 16). In aged muscle, impairment of regeneration is due, in part, to a failure of Dll1 expression (17).Numb en`s four proteins with molecular masses of 65, 66, 71, and 72 kDa by alternative splicing of two exons (18, 19). The Numb proteins are cytoplasmic adaptors that direct ubiquitination and degradation of Notch1 by recruiting the E3 ubiquitin ligase Itch to the receptor (18–22). Numb is a cell-fate determinant that mediates asymmetric cell division, leading to selective Notch inhibition in one daughter cell and its subsequent differentiation whereas the other daughter has active Notch signaling and remains proliferative (10). Embryonically, Numb is expressed in the myotome whereas Notch1 is limited to the dermomyotome (23, 24). This pattern suggests that the expression of Numb in one daughter cell allows entry into the myogenic lineage. Indeed, overexpression of Numb embryonically increases the number of myogenic progenitors in the somite (25, 26).Numb expression increases during the activation and proliferative expansion of satellite cells, becoming asymmetrically segregated in transit-amplifying cells and leading to asymmetric cell divisions (10, 27). These observations led to a model in which Numb inhibits Notch signaling in one daughter satellite cell, allowing it to undergo myogenic differentiation. The molecular switch that controls the decision of satellite cell progeny to continue proliferating or to differentiate is not well understood. This process seems to be controlled by a decrease of Notch signaling due to increased expression of Numb and an increase in Wnt signaling (10–14, 17, 28). In these studies, we examined the role of Numb in satellite cell function by genetic deletion of Numb from myogenic progenitors and satellite cells. Our observations reveal that Numb is necessary for satellite cell-mediated repair. Furthermore, Numb-deficient satellite cells have an unexpected proliferation defect due to an up-regulation of Myostatin. These data indicate a unique role for Numb in regulating the activation and proliferation of satellite cells.     

Efficient germ-line transmission obtained with transgene-free induced pluripotent stem cells

Induced pluripotent stem (iPS) cells hold great promise for regenerative medicine. To overcome potential problems associated with transgene insertions, efforts have been directed over the past several years to generate transgene-free iPS cells by using non-viral-vector approaches. To date, however, cells generated through such procedures have had problems producing reproductively competent animals, suggesting that their quality needed further improvement. Here we report the use of optimized assemblies of reprogramming factors and selection markers incorporated into single plasmids as nonintegrating episomes to generate germ-line–competent iPS cells. In particular, the pMaster12 episome can produce transgene-free iPS cells that, when grown in 2i medium, recapitulate good mouse ES cells, in terms of their competency for generating germ-line chimeras.Although induced pluripotent stem (iPS) cells hold enormous promise for cell-based therapies (1, 2), their use in humans is still problematic because of their potential to also do harm to the patient. High among these risk factors is their potential to induce cancer. For example, use of viral vectors to randomly integrate genes into somatic cells used for human gene therapy trials has resulted in the induction of cancer in the recipient patients (3). Also, random integration of the exogenous reprograming genes in iPS cells increases tumor formation, morbidity, and mortality in mice generated from these cells (4, 5). Therefore, the safety and quality of iPS cells are of critical importance to their anticipated use for human cell-based therapies. To circumvent some of these safety issues, safer methods, especially nonviral methods, for the introduction of the reprogramming factors into somatic cells are being developed. These methods have included the use of plasmids (6, 7), the piggyBac (PB) transposon (8, 9), nonintegrating episomes (10–13), protein transduction (14, 15), transfection of mRNA and microRNAs (16–18), and small molecule inhibitors (19). Although these approaches have yielded transgene-free iPS cells, the competency of the resulting iPS cells to contribute to a functional germ line has not been adequately demonstrated.     

CD14-expressing cancer cells establish the inflammatory and proliferative tumor microenvironment in bladder cancer

Nonresolving chronic inflammation at the neoplastic site is consistently associated with promoting tumor progression and poor patient outcomes. However, many aspects behind the mechanisms that establish this tumor-promoting inflammatory microenvironment remain undefined. Using bladder cancer (BC) as a model, we found that CD14-high cancer cells express higher levels of numerous inflammation mediators and form larger tumors compared with CD14-low cells. CD14 antigen is a glycosyl-phosphatidylinositol (GPI)-linked glycoprotein and has been shown to be critically important in the signaling pathways of Toll-like receptor (TLR). CD14 expression in this BC subpopulation of cancer cells is required for increased cytokine production and increased tumor growth. Furthermore, tumors formed by CD14-high cells are more highly vascularized with higher myeloid cell infiltration. Inflammatory factors produced by CD14-high BC cells recruit and polarize monocytes and macrophages to acquire immune-suppressive characteristics. In contrast, CD14-low BC cells have a higher baseline cell division rate than CD14-high cells. Importantly, CD14-high cells produce factors that further increase the proliferation of CD14-low cells. Collectively, we demonstrate that CD14-high BC cells may orchestrate tumor-promoting inflammation and drive tumor cell proliferation to promote tumor growth.Solid tumors represent a complex mass of tissue composed of multiple distinct cell types (1, 2). Cells within the tumor produce a range of soluble factors to create a complex of signaling networks within the tumor microenvironment (3–7). One of the outcomes of this crosstalk is tumor-promoting inflammation (TPI) (8, 9). TPI can modulate the functions of tumor-infiltrating myeloid lineage cells including macrophages (10–12). Tumor-associated macrophages (TAMs) consistently display an alternatively activated phenotype (M2) commonly found in sites of wound healing (13–18). These macrophages promote tumor growth while suppressing the host immune response locally (19–22). Polarization and subversion of tumor-infiltrating macrophages is accomplished via immune mediators in the tumor microenvironment (23, 24). Adding to the complexity of solid tumors is the heterogeneity of the cancer cells (2). Tumor cells of varying differentiation states and different characteristics coexist within a tumor (25–29). However, the different roles of each tumor cell subset during cancer progression remain undefined.Bladder cancer (BC) represents a growing number of solid tumors characterized by the infiltration of a significant number of myeloid cells in the neoplastic lesion (30, 31). We have previously determined that keratin 14 (KRT14) expression marks the most primitive differentiation state in BC cells (32). KRT14 expression is significantly associated with poor overall patient survival. However, the mechanisms used by KRT14-expressing cells to promote tumor growth remain unclear. In the current study, we found that KRT14+ basal BC cells also express higher levels of CD14. Here, we investigate the strategies used by KRT14+ CD14-high BC cells to promote tumor growth.     

TRAF3 enforces the requirement for T cell cross-talk in thymic medullary epithelial development

Induction of self-tolerance in developing T cells depends on medullary thymic epithelial cells (mTECs), whose development, in turn, requires signals from single-positive (SP) thymocytes. Thus, the absence of SP thymocytes in Tcra^−/− mice results in a profound deficiency in mTECs. Here, we have probed the mechanism that underlies this requirement for cross-talk with thymocytes in medullary development. Previous studies have implicated nonclassical NF-κB as a pathway important in the development of mTECs, because mice lacking RelB, NIK, or IKKα, critical components of this pathway, have an almost complete absence of mTECs, with resulting autoimmune pathology. We therefore assessed the effect of selective deletion in TEC of TNF receptor-associated factor 3 (TRAF3), an inhibitor of nonclassical NF-κB signaling. Deletion of TRAF3 in thymic epithelial cells allowed RelB-dependent development of normal numbers of AIRE-expressing mTECs in the complete absence of SP thymocytes. Thus, mTEC development can occur in the absence of cross-talk with SP thymocytes, and signals provided by SP T cells are needed to overcome TRAF3-imposed arrest in mTEC development mediated by inhibition of nonclassical NF-κB. We further observed that TRAF3 deletion is also capable of overcoming all requirements for LTβR and CD40, which are otherwise necessary for mTEC development, but is not sufficient to overcome the requirement for RANKL, indicating a role for RANKL that is distinct from the signals provided by SP thymocytes. We conclude that TRAF3 plays a central role in regulation of mTEC development by imposing requirements for SP T cells and costimulation-mediated cross-talk in generation of the medullary compartment.A major role of the thymus is the generation of a functional T-cell repertoire that is broadly responsive to foreign antigens but is self-tolerant. Through their role in exposing developing thymocytes to a spectrum of self-antigens, the stromal cells of the thymus are integral to this tolerization. Of particular importance in this process are the epithelial cells comprising the thymic medulla, the region of the thymus where thymocytes selected into the CD4 and CD8 single-positive (SP) lineages reside before emigrating to the periphery (reviewed in refs. 1–3). The importance of thymic medullary epithelial cells (mTECs) in the maintenance of self-tolerance is illustrated by the destructive autoreactivity that results from disruption of mTEC development (reviewed in ref. 4).Just as mTECs have a central role in shaping the developing T-cell repertoire, thymocytes, in turn, are vital to the development and maintenance of the mTEC compartment, a bidirectional interaction that has been termed cross-talk (5–7). A number of recent reports have characterized the CD4 SP thymocyte–stromal cell interactions that are critical for mTEC development (5–11). However, the mechanism that enforces the requirement for SP thymocytes in mTEC development has not been fully identified.We therefore addressed the signaling requirements that mediate the cross-talk required for mTEC development. Previous studies have implicated nonclassical NF-κB as a pathway important in the development of mTECs. It has been shown that mice lacking RelB, NIK, or IKKα, components of the nonclassical NF-κB pathway, have an almost complete absence of mTECs and exhibit resulting autoimmune pathology (12–16). Engagement of TNF receptor (TNFR) family members including CD40, LTβR, and RANK has been shown to activate nonclassical NF-κB signaling via a pathway regulated by several members of the TRAF family of adaptor/ubiquitin ligase proteins. TRAF3 has a unique role in inhibiting nonclassical NF-κB signaling in resting cells (17), and it has been demonstrated that deletion of TRAF3 in B cells results in constitutive activation of the alternative NF-κB pathway (18, 19). We therefore tested the possibility that TRAF3-mediated inhibition of alternative NF-κB is responsible for the failure of mTEC development in the absence of signals from SP thymocytes. We made the striking observation that deletion of TRAF3 in thymic epithelium is sufficient to allow RelB-dependent mTEC development in the complete absence of TCRαβ SP thymocytes. TRAF3 deletion is also capable of overcoming all requirements for LTβR and CD40 during mTEC development, but is not sufficient to overcome the requirement for RANKL, indicating an essential role for RANKL that is distinct from the signals provided by SP thymocytes. Together, these results demonstrate that mTECs can develop in the complete absence of SP thymocytes and that TRAF3 plays a critical role in imposing the requirement for cross-talk with SP thymocytes, thus linking the appearance of mature thymocytes to the development of the thymic medullary environment necessary for imposing self-tolerance.     

Dopamine release from transplanted neural stem cells in Parkinsonian rat striatum in vivo

Embryonic stem cell-based therapies exhibit great potential for the treatment of Parkinson’s disease (PD) because they can significantly rescue PD-like behaviors. However, whether the transplanted cells themselves release dopamine in vivo remains elusive. We and others have recently induced human embryonic stem cells into primitive neural stem cells (pNSCs) that are self-renewable for massive/transplantable production and can efficiently differentiate into dopamine-like neurons (pNSC–DAn) in culture. Here, we showed that after the striatal transplantation of pNSC–DAn, (i) pNSC–DAn retained tyrosine hydroxylase expression and reduced PD-like asymmetric rotation; (ii) depolarization-evoked dopamine release and reuptake were significantly rescued in the striatum both in vitro (brain slices) and in vivo, as determined jointly by microdialysis-based HPLC and electrochemical carbon fiber electrodes; and (iii) the rescued dopamine was released directly from the grafted pNSC–DAn (and not from injured original cells). Thus, pNSC–DAn grafts release and reuptake dopamine in the striatum in vivo and alleviate PD symptoms in rats, providing proof-of-concept for human clinical translation.Parkinson’s disease (PD) is a chronic progressive neurodegenerative disorder characterized by the specific loss of dopaminergic neurons in the substantia nigra pars compacta and their projecting axons, resulting in loss of dopamine (DA) release in the striatum (1). During the last two decades, cell-replacement therapy has proven, at least experimentally, to be a potential treatment for PD patients (2–7) and in animal models (8–15). The basic principle of cell therapy is to restore the DA release by transplanting new DA-like cells. Until recently, obtaining enough transplantable cells was a major bottleneck in the practicability of cell therapy for PD. One possible source is embryonic stem cells (ESCs), which can develop infinitely into self-renewable pluripotent cells with the potential to generate any type of cell, including DA neurons (DAns) (16, 17).Recently, several groups including us have introduced rapid and efficient ways to generate primitive neural stem cells (pNSCs) from human ESCs using small-molecule inhibitors under chemically defined conditions (12, 18, 19). These cells are nonpolarized neuroepithelia and retain plasticity upon treatment with neuronal developmental morphogens. Importantly, pNSCs differentiate into DAns (pNSC–DAn) with high efficiency (∼65%) after patterning by sonic hedgehog (SHH) and fibroblast growth factor 8 (FGF8) in vitro, providing an immediate and renewable source of DAns for PD treatment. Importantly, the striatal transplantation of human ESC-derived DA-like neurons, including pNSC–DAn, are able to relieve the motor defects in a PD rat model (11–13, 15, 19–23). Before attempting clinical translation of pNSC–DAn, however, there are two fundamental open questions. (i) Can pNSC–DAn functionally restore the striatal DA levels in vivo? (ii) What cells release the restored DA, pNSC–DAn themselves or resident neurons/cells repaired by the transplants?Regarding question 1, a recent study using nafion-coated carbon fiber electrodes (CFEs) reported that the amperometric current is rescued in vivo by ESC (pNSC–DAn-like) therapy (19). Both norepinephrine (NE) and serotonin are present in the striatum (24, 25). However, CFE amperometry/chronoamperometry alone cannot distinguish DA from other monoamines in vivo, such as NE and serotonin (Fig. S1) (see also refs. 26–28). Considering that the compounds released from grafted ESC-derived cells are unknown, the work of Kirkeby et al. was unable to determine whether DA or other monoamines are responsible for the restored amperometric signal. Thus, the key question of whether pNSC–DAn can rescue DA release needs to be reexamined for the identity of the restored amperometric signal in vivo.Regarding question 2, many studies have proposed that DA is probably released from the grafted cells (8, 12, 13, 20), whereas others have proposed that the grafted stem cells might restore striatal DA levels by rescuing injured original cells (29, 30). Thus, whether the grafted cells are actually capable of synthesizing and releasing DA in vivo must be investigated to determine the future cellular targets (residual cells versus pNSC–DAn) of treatment.To address these two mechanistic questions, advanced in vivo methods of DA identification and DA recording at high spatiotemporal resolution are required. Currently, microdialysis-based HPLC (HPLC) (31–33) and CFE amperometric recordings (34, 35) have been used independently by different laboratories to assess evoked DA release from the striatum in vivo. The major advantage of microdialysis-based HPLC is to identify the substances secreted in the cell-grafted striatum (33), but its spatiotemporal resolution is too low to distinguish the DA release site (residual cells or pNSC–DAn). In contrast, the major advantage of CFE-based amperometry is its very high temporal (ms) and spatial (μm) resolution, making it possible to distinguish the DA release site (residual cells or pNSC–DAn) in cultured cells, brain slices, and in vivo (34–39), but it is unable to distinguish between low-level endogenous oxidizable substances (DA versus serotonin and NE) in vivo.In the present study, we developed a challenging experimental paradigm of combining the two in vivo methods, microdialysis-based HPLC and CFE amperometry, to identify the evoked substance as DA and its release site as pNSC–DAn in the striatum of PD rats.     

Bimodal rheotactic behavior reflects flagellar beat asymmetry in human sperm cells

Rheotaxis, the directed response to fluid velocity gradients, has been shown to facilitate stable upstream swimming of mammalian sperm cells along solid surfaces, suggesting a robust physical mechanism for long-distance navigation during fertilization. However, the dynamics by which a human sperm orients itself relative to an ambient flow is poorly understood. Here, we combine microfluidic experiments with mathematical modeling and 3D flagellar beat reconstruction to quantify the response of individual sperm cells in time-varying flow fields. Single-cell tracking reveals two kinematically distinct swimming states that entail opposite turning behaviors under flow reversal. We constrain an effective 2D model for the turning dynamics through systematic large-scale parameter scans, and find good quantitative agreement with experiments at different shear rates and viscosities. Using a 3D reconstruction algorithm to identify the flagellar beat patterns causing left or right turning, we present comprehensive 3D data demonstrating the rolling dynamics of freely swimming sperm cells around their longitudinal axis. Contrary to current beliefs, this 3D analysis uncovers ambidextrous flagellar waveforms and shows that the cell’s turning direction is not defined by the rolling direction. Instead, the different rheotactic turning behaviors are linked to a broken mirror symmetry in the midpiece section, likely arising from a buckling instability. These results challenge current theoretical models of sperm locomotion.Taxis, the directed kinematic response to external signals, is a defining feature of living things that affects their reproduction, foraging, migration, and survival strategies (1–4). Higher organisms rely on sophisticated networks of finely tuned sensory mechanisms to move efficiently in the presence of chemical or physical stimuli. However, various fundamental forms of taxis are already manifest at the unicellular level, ranging from chemotaxis in bacteria (5) and phototaxis in unicellular green algae (2) to the mechanical response (durotaxis) of fibroblasts (6) and rheotaxis (7, 8) in spermatozoa (3, 9–12). Over the last few decades, much progress has been made in deciphering chemotactic, phototactic, and durotactic pathways in prokaryotic and eukaryotic model systems. In contrast, comparatively little is known about the physical mechanisms that enable flow gradient sensing in sperm cells (3, 9–13). Recent studies (3, 12) suggest that mammalian sperm use rheotaxis for long-distance navigation, but it remains unclear how shear flows alter flagellar beat patterns in the vicinity of surfaces and, in particular, how such changes in the beat dynamics affect the steering process. Answering these questions will be essential for evaluating the importance of chemical (14) and physical (4) signals during mammalian fertilization (15–17).A necessary requirement for any form of directed kinematic response is the ability to change the direction of locomotion. Multiflagellate bacteria achieve this feat by varying their motor activity, resulting in alternating phases of entangled and disentangled flagellar dynamics that give rise to run-and-tumble motion (5). A similar mechanism was recently discovered in the biflagellate eukaryote Chlamydomonas reinhardtii (18). This unicellular green alga actively redirects its swimming motion through occasional desynchronization of its two cilia (19), although it is still debated whether this effect is of mechanical (20) or hydrodynamic (21, 22) origin. Experiments (23) show that the alga’s reorientation dynamics can lead to localization in shear flow (24, 25), with potentially profound implications in marine ecology. In contrast to taxis in multiflagellate organisms (2, 5, 18, 26, 27), the navigation strategies of uniflagellate cells are less well understood. For instance, it was discovered only recently that uniflagellate marine bacteria, such as Vibrio alginolyticus and Pseudoalteromonas haloplanktis, use a buckling instability in their lone flagellum to change their swimming direction (28). However, as passive prokaryotic flagella differ fundamentally from their active eukaryotic counterparts, it is unclear to what extent such insights translate to spermatozoa.Earlier studies of human sperm locomotion have identified several potential steering and transport mechanisms, including thermotaxis (4), uterine peristalsis (29, 30), and chemotaxis (14, 16, 31), but their relative importance has yet to be quantified. Recent experiments (3, 32, 33) demonstrate that rheotaxis, combined with steric surface alignment (12, 34), enables robust long-distance navigation by turning sperm cells preferentially against an externally imposed flow direction (9, 10), but how exactly this realignment process happens is unknown. It has been suggested (32, 35, 36) that the intrinsic curvature or chiral beat dynamics (37, 38) of the flagellum could play an essential role in rheotactic steering, but this remains to be confirmed in experiments. Similarly, an increasing number of theoretical models (36, 39–47) still await empirical validation, because 3D data for the beat pattern of sperm swimming close to surfaces has been lacking.To examine the dynamics of human sperm rheotaxis quantitatively, we here combine microfluidic experiments with mathematical modeling and 3D flagellar beat reconstruction. Single-cell tracking reveals the existence of two kinematically distinct swimming states that result in opposite turning behaviors under flow reversal. We quantify this effect for a range of viscosities and shear rates, and use these comprehensive data to constrain an effective 2D model through a systematic large-scale scan ( > 6,000 parameter combinations). To identify the details of the flagellar beat dynamics during rheotaxis, we developed an algorithm that translates 2D intensity profiles into 3D positional data. Our 3D analysis confirms that human sperm perform a rolling motion (48), characterized by weakly nonplanar beat patterns and a rotating beat plane. However, contrary to current beliefs, we find that neither the rolling direction nor beat helicity determine the turning direction after flow reversal. Instead, the rheotactic turning behavior correlates with a previously unrecognized asymmetry in the midpiece, likely caused by a buckling instability. These findings call for a revision and extension of current models (36, 39–44, 46).