S-nitrosoglutathione reductase–dependent PPARγ denitrosylation participates in MSC-derived adipogenesis and osteogenesis

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Abstract

Bone marrow–derived mesenchymal stem cells (MSCs) are a common precursor of both adipocytes and osteoblasts. While it is appreciated that PPARγ regulates the balance between adipogenesis and osteogenesis, the roles of additional regulators of this process remain controversial. Here, we show that MSCs isolated from mice lacking S-nitrosoglutathione reductase, a denitrosylase that regulates protein S-nitrosylation, exhibited decreased adipogenesis and increased osteoblastogenesis compared with WT MSCs. Consistent with this cellular phenotype, S-nitrosoglutathione reductase–deficient mice were smaller, with reduced fat mass and increased bone formation that was accompanied by elevated bone resorption. WT and S-nitrosoglutathione reductase–deficient MSCs exhibited equivalent PPARγ expression; however, S-nitrosylation of PPARγ was elevated in S-nitrosoglutathione reductase–deficient MSCs, diminishing binding to its downstream target fatty acid–binding protein 4 (FABP4). We further identified Cys 139 of PPARγ as an S-nitrosylation site and demonstrated that S-nitrosylation of PPARγ inhibits its transcriptional activity, suggesting a feedback regulation of PPARγ transcriptional activity by NO-mediated S-nitrosylation. Together, these results reveal that S-nitrosoglutathione reductase–dependent modification of PPARγ alters the balance between adipocyte and osteoblast differentiation and provides checkpoint regulation of the lineage bifurcation of these 2 lineages. Moreover, these findings provide pathophysiological and therapeutic insights regarding MSC participation in adipogenesis and osteogenesis.

Authors

Yenong Cao, Samirah A. Gomes, Erika B. Rangel, Ellena C. Paulino, Tatiana L. Fonseca, Jinliang Li, Marilia B. Teixeira, Cecilia H. Gouveia, Antonio C. Bianco, Michael S. Kapiloff, Wayne Balkan, Joshua M. Hare

Vascular niche promotes hematopoietic multipotent progenitor formation from pluripotent stem cells

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Abstract

Pluripotent stem cells (PSCs) represent an alternative hematopoietic stem cell (HSC) source for treating hematopoietic disease. The limited engraftment of human PSC–derived (hPSC-derived) multipotent progenitor cells (MPP) has hampered the clinical application of these cells and suggests that MPP require additional cues for definitive hematopoiesis. We hypothesized that the presence of a vascular niche that produces Notch ligands jagged-1 (JAG1) and delta-like ligand-4 (DLL4) drives definitive hematopoiesis. We differentiated hes2 human embryonic stem cells (hESC) and Macaca nemestrina–induced PSC (iPSC) line-7 with cytokines in the presence or absence of endothelial cells (ECs) that express JAG1 and DLL4. Cells cocultured with ECs generated substantially more CD34⁺CD45⁺ hematopoietic progenitors compared with cells cocultured without ECs or with ECs lacking JAG1 or DLL4. EC-induced cells exhibited Notch activation and expressed HSC-specific Notch targets RUNX1 and GATA2. EC-induced PSC-MPP engrafted at a markedly higher level in NOD/SCID/IL-2 receptor γ chain–null (NSG) mice compared with cytokine-induced cells, and low-dose chemotherapy-based selection further increased engraftment. Long-term engraftment and the myeloid-to-lymphoid ratio achieved with vascular niche induction were similar to levels achieved for cord blood–derived MPP and up to 20-fold higher than those achieved with hPSC-derived MPP engraftment. Our findings indicate that endothelial Notch ligands promote PSC-definitive hematopoiesis and production of long-term engrafting CD34⁺ cells, suggesting these ligands are critical for HSC emergence.

Authors

Jennifer L. Gori, Jason M. Butler, Yan-Yi Chan, Devikha Chandrasekaran, Michael G. Poulos, Michael Ginsberg, Daniel J. Nolan, Olivier Elemento, Brent L. Wood, Jennifer E. Adair, Shahin Rafii, Hans-Peter Kiem

Pluripotent stem cells reveal erythroid-specific activities of the GATA1 N-terminus

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Abstract

Germline GATA1 mutations that result in the production of an amino-truncated protein termed GATA1s (where s indicates short) cause congenital hypoplastic anemia. In patients with trisomy 21, similar somatic GATA1s-producing mutations promote transient myeloproliferative disease and acute megakaryoblastic leukemia. Here, we demonstrate that induced pluripotent stem cells (iPSCs) from patients with GATA1-truncating mutations exhibit impaired erythroid potential, but enhanced megakaryopoiesis and myelopoiesis, recapitulating the major phenotypes of the associated diseases. Similarly, in developmentally arrested GATA1-deficient murine megakaryocyte-erythroid progenitors derived from murine embryonic stem cells (ESCs), expression of GATA1s promoted megakaryopoiesis, but not erythropoiesis. Transcriptome analysis revealed a selective deficiency in the ability of GATA1s to activate erythroid-specific genes within populations of hematopoietic progenitors. Although its DNA-binding domain was intact, chromatin immunoprecipitation studies showed that GATA1s binding at specific erythroid regulatory regions was impaired, while binding at many nonerythroid sites, including megakaryocytic and myeloid target genes, was normal. Together, these observations indicate that lineage-specific GATA1 cofactor associations are essential for normal chromatin occupancy and provide mechanistic insights into how GATA1s mutations cause human disease. More broadly, our studies underscore the value of ESCs and iPSCs to recapitulate and study disease phenotypes.

Authors

Marta Byrska-Bishop, Daniel VanDorn, Amy E. Campbell, Marisol Betensky, Philip R. Arca, Yu Yao, Paul Gadue, Fernando F. Costa, Richard L. Nemiroff, Gerd A. Blobel, Deborah L. French, Ross C. Hardison, Mitchell J. Weiss, Stella T. Chou

Differentiation of hypothalamic-like neurons from human pluripotent stem cells

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Abstract

The hypothalamus is the central regulator of systemic energy homeostasis, and its dysfunction can result in extreme body weight alterations. Insights into the complex cellular physiology of this region are critical to the understanding of obesity pathogenesis; however, human hypothalamic cells are largely inaccessible for direct study. Here, we developed a protocol for efficient generation of hypothalamic neurons from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) obtained from patients with monogenetic forms of obesity. Combined early activation of sonic hedgehog signaling followed by timed NOTCH inhibition in human ESCs/iPSCs resulted in efficient conversion into hypothalamic NKX2.1⁺ precursors. Application of a NOTCH inhibitor and brain-derived neurotrophic factor (BDNF) further directed the cells into arcuate nucleus hypothalamic-like neurons that express hypothalamic neuron markers proopiomelanocortin (POMC), neuropeptide Y (NPY), agouti-related peptide (AGRP), somatostatin, and dopamine. These hypothalamic-like neurons accounted for over 90% of differentiated cells and exhibited transcriptional profiles defined by a hypothalamic-specific gene expression signature that lacked pituitary markers. Importantly, these cells displayed hypothalamic neuron characteristics, including production and secretion of neuropeptides and increased p-AKT and p-STAT3 in response to insulin and leptin. Our results suggest that these hypothalamic-like neurons have potential for further investigation of the neurophysiology of body weight regulation and evaluation of therapeutic targets for obesity.

Authors

Liheng Wang, Kana Meece, Damian J. Williams, Kinyui Alice Lo, Matthew Zimmer, Garrett Heinrich, Jayne Martin Carli, Charles A. Leduc, Lei Sun, Lori M. Zeltser, Matthew Freeby, Robin Goland, Stephen H. Tsang, Sharon L. Wardlaw, Dieter Egli, Rudolph L. Leibel

Transient vascularization of transplanted human adult–derived progenitors promotes self-organizing cartilage

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Abstract

Millions of patients worldwide are affected by craniofacial deformations caused by congenital defects or trauma. Current surgical interventions have limited therapeutic outcomes; therefore, methods that would allow cartilage restoration are of great interest. A number of studies on embryonic limb development have shown that chondrogenesis is initiated by cellular condensation, during which mesenchymal progenitors aggregate and form 3D structures. Here, we demonstrated efficient regeneration of avascular elastic cartilage from in vitro–grown mesenchymal condensation, which recapitulated the early stages of chondrogenesis, including transient vascularization. After transplantation of vascularized condensed progenitors into immunodeficient mice, we used an intravital imaging approach to follow cartilage maturation. We determined that endothelial cells are present inside rudimentary cartilage (mesenchymal condensation) prior to cartilage maturation. Recreation of endothelial interactions in culture enabled a recently identified population of adult elastic cartilage progenitors to generate mesenchymal condensation in a self-driven manner, without requiring the support of exogenous inductive factors or scaffold materials. Moreover, the culture-grown 3D condensed adult–derived progenitors were amenable to storage via simple freezing methods and efficiently reconstructed 3D elastic cartilage upon transplantation. Together, our results indicate that transplantation of endothelialized and condensed progenitors represents a promising approach to realizing a regenerative medicine treatment for craniofacial deformations.

Authors

Takanori Takebe, Shinji Kobayashi, Hiromu Suzuki, Mitsuru Mizuno, Yu-Min Chang, Emi Yoshizawa, Masaki Kimura, Ayaka Hori, Jun Asano, Jiro Maegawa, Hideki Taniguchi

Human muscle–derived stem/progenitor cells promote functional murine peripheral nerve regeneration

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Abstract

Peripheral nerve injuries and neuropathies lead to profound functional deficits. Here, we have demonstrated that muscle-derived stem/progenitor cells (MDSPCs) isolated from adult human skeletal muscle (hMDSPCs) can adopt neuronal and glial phenotypes in vitro and ameliorate a critical-sized sciatic nerve injury and its associated defects in a murine model. Transplanted hMDSPCs surrounded the axonal growth cone, while hMDSPCs infiltrating the regenerating nerve differentiated into myelinating Schwann cells. Engraftment of hMDSPCs into the area of the damaged nerve promoted axonal regeneration, which led to functional recovery as measured by sustained gait improvement. Furthermore, no adverse effects were observed in these animals up to 18 months after transplantation. Following hMDSPC therapy, gastrocnemius muscles from mice exhibited substantially less muscle atrophy, an increase in muscle mass after denervation, and reorganization of motor endplates at the postsynaptic sites compared with those from PBS-treated mice. Evaluation of nerve defects in animals transplanted with vehicle-only or myoblast-like cells did not reveal histological or functional recovery. These data demonstrate the efficacy of hMDSPC-based therapy for peripheral nerve injury and suggest that hMDSPC transplantation has potential to be translated for use in human neuropathies.

Authors

Mitra Lavasani, Seth D. Thompson, Jonathan B. Pollett, Arvydas Usas, Aiping Lu, Donna B. Stolz, Katherine A. Clark, Bin Sun, Bruno Péault, Johnny Huard

LRIG1 inhibits STAT3-dependent inflammation to maintain corneal homeostasis

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Abstract

Corneal integrity and transparency are indispensable for good vision. Cornea homeostasis is entirely dependent upon corneal stem cells, which are required for complex wound-healing processes that restore corneal integrity following epithelial damage. Here, we found that leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is highly expressed in the human holoclone-type corneal epithelial stem cell population and sporadically expressed in the basal cells of ocular-surface epithelium. In murine models, LRIG1 regulated corneal epithelial cell fate during wound repair. Deletion of Lrig1 resulted in impaired stem cell recruitment following injury and promoted a cell-fate switch from transparent epithelium to keratinized skin-like epidermis, which led to corneal blindness. In addition, we determined that LRIG1 is a negative regulator of the STAT3-dependent inflammatory pathway. Inhibition of STAT3 in corneas of Lrig1^–/– mice rescued pathological phenotypes and prevented corneal opacity. Additionally, transgenic mice that expressed a constitutively active form of STAT3 in the corneal epithelium had abnormal features, including corneal plaques and neovascularization similar to that found in Lrig1^–/– mice. Bone marrow chimera experiments indicated that LRIG1 also coordinates the function of bone marrow–derived inflammatory cells. Together, our data indicate that LRIG1 orchestrates corneal-tissue transparency and cell fate during repair, and identify LRIG1 as a key regulator of tissue homeostasis.

Authors

Takahiro Nakamura, Junji Hamuro, Mikiro Takaishi, Szandor Simmons, Kazuichi Maruyama, Andrea Zaffalon, Adam J. Bentley, Satoshi Kawasaki, Maho Nagata-Takaoka, Nigel J. Fullwood, Satoshi Itami, Shigetoshi Sano, Masaru Ishii, Yann Barrandon, Shigeru Kinoshita

Eliminating malignant contamination from therapeutic human spermatogonial stem cells

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Abstract

Spermatogonial stem cell (SSC) transplantation has been shown to restore fertility in several species and may have application for treating some cases of male infertility (e.g., secondary to gonadotoxic therapy for cancer). To ensure safety of this fertility preservation strategy, methods are needed to isolate and enrich SSCs from human testis cell suspensions and also remove malignant contamination. We used flow cytometry to characterize cell surface antigen expression on human testicular cells and leukemic cells (MOLT-4 and TF-1a). We demonstrated via FACS that EpCAM is expressed by human spermatogonia but not MOLT-4 cells. In contrast, HLA-ABC and CD49e marked >95% of MOLT-4 cells but were not expressed on human spermatogonia. A multiparameter sort of MOLT-4–contaminated human testicular cell suspensions was performed to isolate EpCAM⁺/HLA-ABC^–/CD49e^– (putative spermatogonia) and EpCAM^–/HLA-ABC⁺/CD49e⁺ (putative MOLT-4) cell fractions. The EpCAM⁺/HLA-ABC^–/CD49e^– fraction was enriched for spermatogonial colonizing activity and did not form tumors following human-to–nude mouse xenotransplantation. The EpCAM^–/HLA-ABC⁺/CD49e⁺ fraction produced tumors following xenotransplantation. This approach could be generalized with slight modification to also remove contaminating TF-1a leukemia cells. Thus, FACS provides a method to isolate and enrich human spermatogonia and remove malignant contamination by exploiting differences in cell surface antigen expression.

Authors

Serena L. Dovey, Hanna Valli, Brian P. Hermann, Meena Sukhwani, Julia Donohue, Carlos A. Castro, Tianjiao Chu, Joseph S. Sanfilippo, Kyle E. Orwig

Identification of embryonic stem cell–derived midbrain dopaminergic neurons for engraftment

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Abstract

Embryonic stem cells (ESCs) represent a promising source of midbrain dopaminergic (DA) neurons for applications in Parkinson disease. However, ESC-based transplantation paradigms carry a risk of introducing inappropriate or tumorigenic cells. Cell purification before transplantation may alleviate these concerns and enable identification of the specific DA neuron stage most suitable for cell therapy. Here, we used 3 transgenic mouse ESC reporter lines to mark DA neurons at 3 stages of differentiation (early, middle, and late) following induction of differentiation using Hes5::GFP, Nurr1::GFP, and Pitx3::YFP transgenes, respectively. Transplantation of FACS-purified cells from each line resulted in DA neuron engraftment, with the mid-stage and late-stage neuron grafts being composed almost exclusively of midbrain DA neurons. Mid-stage neuron cell grafts had the greatest amount of DA neuron survival and robustly induced recovery of motor deficits in hemiparkinsonian mice. Our data suggest that the Nurr1+ stage (middle stage) of neuronal differentiation is particularly suitable for grafting ESC-derived DA neurons. Moreover, global transcriptome analysis of progeny from each of the ESC reporter lines revealed expression of known midbrain DA neuron genes and also uncovered previously uncharacterized midbrain genes. These data demonstrate remarkable fate specificity of ESC-derived DA neurons and outline a sequential stage-specific ESC reporter line paradigm for in vivo gene discovery.

Authors

Yosif M. Ganat, Elizabeth L. Calder, Sonja Kriks, Jenny Nelander, Edmund Y. Tu, Fan Jia, Daniela Battista, Neil Harrison, Malin Parmar, Mark J. Tomishima, Urs Rutishauser, Lorenz Studer

Human dental pulp-derived stem cells promote locomotor recovery after complete transection of the rat spinal cord by multiple neuro-regenerative mechanisms

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Abstract

Spinal cord injury (SCI) often leads to persistent functional deficits due to loss of neurons and glia and to limited axonal regeneration after injury. Here we report that transplantation of human dental pulp stem cells into the completely transected adult rat spinal cord resulted in marked recovery of hind limb locomotor functions. Transplantation of human bone marrow stromal cells or skin-derived fibroblasts led to substantially less recovery of locomotor function. The human dental pulp stem cells exhibited three major neuroregenerative activities. First, they inhibited the SCI-induced apoptosis of neurons, astrocytes, and oligodendrocytes, which improved the preservation of neuronal filaments and myelin sheaths. Second, they promoted the regeneration of transected axons by directly inhibiting multiple axon growth inhibitors, including chondroitin sulfate proteoglycan and myelin-associated glycoprotein, via paracrine mechanisms. Last, they replaced lost cells by differentiating into mature oligodendrocytes under the extreme conditions of SCI. Our data demonstrate that tooth-derived stem cells may provide therapeutic benefits for treating SCI through both cell-autonomous and paracrine neuroregenerative activities.

Authors

Kiyoshi Sakai, Akihito Yamamoto, Kohki Matsubara, Shoko Nakamura, Mami Naruse, Mari Yamagata, Kazuma Sakamoto, Ryoji Tauchi, Norimitsu Wakao, Shiro Imagama, Hideharu Hibi, Kenji Kadomatsu, Naoki Ishiguro, Minoru Ueda