Therapeutic Targeting of TIM-4-L with Engineered T Cells for Acute Myeloid Leukemia.

PURPOSE: Disruption of lipid bilayer asymmetry is a common feature observed in cancer cells and offers novel routes for therapeutic targeting. We used the natural immune receptor TIM-4 to interrogate for loss of plasma membrane phospholipid polarity in primary acute myelogenous leukemia (AML) samples and evaluated the anti-leukemic activity of TIM-4-L-directed T-cell therapy in preclinical AML models. EXPERIMENTAL DESIGN: We performed FACS analysis on 33 primary AML bone marrow specimens and correlated TIM-4-L expression frequency and intensity with molecular disease characteristics. Using Kasumi-1 and MV-4-11 AML cell lines, we further tested the anti-leukemic effects of TIM-4-L-directed engineered T cells in vitro and in vivo. RESULTS: We found that 86% of untreated AML blasts displayed upregulation of cell surface TIM-4-L. These observations were agnostic to AML genetic classification, as samples with mutations in TP53, ASXL1, and RUNX1 displayed TIM-4-L upregulation similar to that seen in favorable and intermediate subtypes. TIM-4-L dysregulation was also stably present in AML cell lines. To evaluate the potential of targeting upregulated TIM-4-L with adoptive T-cell therapy, we constructed TIM-4-L-directed engineered T cells, which demonstrated potent anti-leukemic effects, effectively eliminating AML cell lines with a range of endogenous TIM-4-L expression levels both in vitro and in vivo. CONCLUSIONS: These results highlight TIM-4-L as a highly prevalent target on AML across a range of genetic classifications and novel target for T-cell-based therapy in AML. Further investigations into the role of TIM-4-L in AML pathogenesis and its potential as an anti-leukemic target for clinical development are warranted.

Chimeric TIM-4 receptor-modified T cells targeting phosphatidylserine mediates both cytotoxic anti-tumor responses and phagocytic uptake of tumor-associated antigen for T cell cross-presentation.

To leverage complementary mechanisms for cancer cell removal, we developed a novel cell engineering and therapeutic strategy co-opting phagocytic clearance and antigen presentation activity into T cells. We engineered a chimeric engulfment receptor (CER)-1236, which combines the extracellular domain of TIM-4, a phagocytic receptor recognizing the "eat me" signal phosphatidylserine, with intracellular signaling domains (TLR2/TIR, CD28, and CD3ζ) to enhance both TIM-4-mediated phagocytosis and T cell cytotoxic function. CER-1236 T cells demonstrate target-dependent phagocytic function and induce transcriptional signatures of key regulators responsible for phagocytic recognition and uptake, along with cytotoxic mediators. Pre-clinical models of mantle cell lymphoma (MCL) and EGFR mutation-positive non-small cell lung cancer (NSCLC) demonstrate collaborative innate-adaptive anti-tumor immune responses both in vitro and in vivo. Treatment with BTK (MCL) and EGFR (NSCLC) inhibitors increased target ligand, conditionally driving CER-1236 function to augment anti-tumor responses. We also show that activated CER-1236 T cells exhibit superior cross-presentation ability compared with conventional T cells, triggering E7-specific TCR T responses in an HLA class I- and TLR-2-dependent manner, thereby overcoming the limited antigen presentation capacity of conventional T cells. Therefore, CER-1236 T cells have the potential to achieve tumor control by eliciting both direct cytotoxic effects and indirect-mediated cross-priming.

Preclinical Development and Evaluation of Allogeneic CAR T Cells Targeting CD70 for the Treatment of Renal Cell Carcinoma.

CD70 is highly expressed in renal cell carcinoma (RCC), with limited expression in normal tissue, making it an attractive CAR T target for an immunogenic solid tumor indication. Here we generated and characterized a panel of anti-CD70 single-chain fragment variable (scFv)-based CAR T cells. Despite the expression of CD70 on T cells, production of CAR T cells from a subset of scFvs with potent in vitro activity was achieved. Expression of CD70 CARs masked CD70 detection in cis and provided protection from CD70 CAR T cell-mediated fratricide. Two distinct classes of CAR T cells were identified with differing memory phenotype, activation status, and cytotoxic activity. Epitope mapping revealed that the two classes of CARs bind unique regions of CD70. CD70 CAR T cells displayed robust antitumor activity against RCC cell lines and patient-derived xenograft mouse models. Tissue cross-reactivity studies identified membrane staining in lymphocytes, thus matching the known expression pattern of CD70. In a cynomolgus monkey CD3-CD70 bispecific toxicity study, expected findings related to T-cell activation and elimination of CD70-expressing cells were observed, including cytokine release and loss of cellularity in lymphoid tissues. Finally, highly functional CD70 allogeneic CAR T cells were produced at large scale through elimination of the T-cell receptor by TALEN-based gene editing. Taken together, these efficacy and safety data support the evaluation of CD70 CAR T cells for the treatment of RCC and has led to the advancement of an allogeneic CD70 CAR T-cell candidate into phase I clinical trials. SIGNIFICANCE: These findings demonstrate the efficacy and safety of fratricide-resistant, allogeneic anti-CD70 CAR T cells targeting renal cell carcinoma and the impact of CAR epitope on functional activity. See related commentary by Adotévi and Galaine, p. 2517.

Treatment of stress urinary incontinence with low-intensity extracorporeal shock wave therapy in a vaginal balloon dilation induced rat model.

BACKGROUND: To investigate the outcomes and mechanisms of low-intensity extracorporeal shock wave therapy (Li-ESWT) on stress urinary incontinence (SUI) in a vaginal balloon dilation (VBD) rat model. METHODS: Thirty Sprague-Dawley rats were randomly grouped into normal controls, VBD only, and VBD with Li-ESWT. Li-ESWT was administered twice per week for 3 weeks. Afterward, all 30 rats were assessed with functional and histological studies. To explore the acute effect of Li-ESWT, another 25 rats, given intraperitoneal 5-ethynyl-2-deoxyuridine (EdU) at birth, were treated with Li-ESWT followed by assessment of vascular endothelial growth factor (VEGF) expression and endogenous progenitor cells distribution at 24 hours or 1 week after the last Li-ESWT therapy. Additionally, rat myoblast L6 cells were used for myotube formation assay in vitro. RESULTS: Functional analysis with leak-point pressure (LPP) testing showed that rats treated with Li-ESWT following VBD had significantly higher LPP relative to those receiving VBD only (44.8±3.2 versus 27.0±2.9 cmH2O, P<0.01). Histological examinations showed increased urethral sphincter regeneration in Li-ESWT group. The rats treated with Li-ESWT also had increased vascularity, which was confirmed by immunohistochemistry of rat endothelial cell antigen, while reverse-transcriptase polymerase chain reaction (RT-PCR) showed VEGF expression was significantly enhanced. Additionally, there were significantly increased EdU+ cells in Li-ESWT treated rats at 24 hours. In vitro, Li-ESWT promoted myotube formation from L6 cells. CONCLUSIONS: Li-ESWT ameliorated SUI by promoting angiogenesis, progenitor cell recruitment, and urethral sphincter regeneration in a rat model induced by VBD. Li-ESWT represents a potential novel non-invasive therapy for SUI.

Low-intensity extracorporeal shock wave therapy promotes myogenesis through PERK/ATF4 pathway.

AIM: Stress urinary incontinence (SUI) is a significant health problem for women. Treatments employing muscle derived stem cells (MDSCs) may be a promising approach to this prevalent, bothersome condition, but these treatments are invasive and require collection of cells from one site for injection into another. It is also unknown whether or not these cells establish themselves and function as muscle cells in the target tissues. Alternatively, low-intensity extracorporeal shock wave therapy (Li-ESWT) is non-invasive and has shown positive outcomes in the treatment of multiple musculoskeletal disorders, but the biological effects responsible for clinical success are not yet well understood. The aim of this study is to explore the possibility of employing Li-ESWT for activation of MDSCs in situ and to further elucidate the underlying biological effects and mechanisms of action in urethral muscle. METHODS: Urethral muscle derived stem cells (uMDSCs) were harvest from Zucker Lean (ZUC-LEAN) (ZUC-Leprfa 186) rats and characterized with flow cytometry. Li-ESWT (0.02 mJ/mm2 , 3 Hz, 200 pulses) and GSK2656157, an inhibitor of PERK pathway, were applied to L6 rat myoblast cells. To assess for myotube formation, we used immunofluorescence staining and western blot analysis in uMDSCs and L6 cells. RESULTS: The results indicate that uMDSCs could form myotubes. Myotube formation was significantly increased by the Li-ESWT as was the expression of muscle heavy chain (MHC) and myogenic factor 5 (Myf5) in L6 cells in vitro. Li-ESWT activated protein kinase RNA-like ER kinase (PERK) pathway by increasing the phosphorylation levels of PERK and eukaryotic initiation factor 2a (eIF2α) and by increasing activating transcription factor 4 (ATF4). In addition, GSK2656157, an inhibitor of PERK, effectively inhibited the myotube formation in L6 rat myoblast cells. Furthermore, GSK2656157 also attenuated myotube formation induced by Li-ESWT. CONCLUSION: In conclusion, this experiment reveals that rat uMDSCs can be isolated successfully and can form myotubes in vitro. PERK/ATF4 pathway was involved in myotube formation, and L6 rat myoblast cells were activated by Li-ESWT to form myotubes. These findings suggest that PERK/ATF4 pathway is activated by Li-ESWT. This study elucidates one of the biochemical pathways responsible for the clinical improvements seen after Li-ESWT. It is possible that this information will help to establish Li-ESWT as an acceptable treatment modality and may help to further refine the use of Li-ESWT in the clinical practice of medicine.

Low-Intensity Extracorporeal Shock Wave Therapy Enhances Brain-Derived Neurotrophic Factor Expression through PERK/ATF4 Signaling Pathway.

Low-intensity extracorporeal shock wave therapy (Li-ESWT) is used in the treatment of erectile dysfunction, but its mechanisms are not well understood. Previously, we found that Li-ESWT increased the expression of brain-derived neurotrophic factor (BDNF). Here we assessed the underlying signaling pathways in Schwann cells in vitro and in penis tissue in vivo after nerve injury. The result indicated that BDNF were significantly increased by the Li-ESWT after nerve injury, as well as the expression of BDNF in Schwann cells (SCs, RT4-D6P2T) in vitro. Li-ESWT activated the protein kinase RNA-like endoplasmic reticulum (ER) kinase (PERK) pathway by increasing the phosphorylation levels of PERK and eukaryotic initiation factor 2a (eIF2α), and enhanced activating transcription factor 4 (ATF4) in an energy-dependent manner. In addition, GSK2656157-an inhibitor of PERK-effectively inhibited the effect of Li-ESWT on the phosphorylation of PERK, eIF2α, and the expression of ATF4. Furthermore, silencing ATF4 dramatically attenuated the effect of Li-ESWT on the expression of BDNF, but had no effect on hypoxia-inducible factor (HIF)1α or glial cell-derived neurotrophic factor (GDNF) in Schwann cells. In conclusion, our findings shed new light on the underlying mechanisms by which Li-ESWT may stimulate the expression of BDNF through activation of PERK/ATF4 signaling pathway. This information may help to refine the use of Li-ESWT to further improve its clinical efficacy.

Low-energy Shock Wave Therapy Ameliorates Erectile Dysfunction in a Pelvic Neurovascular Injuries Rat Model.

INTRODUCTION: Erectile dysfunction (ED) caused by pelvic injuries is a common complication of civil and battlefield trauma with multiple neurovascular factors involved, and no effective therapeutic approach is available. AIMS: To test the effect and mechanisms of low-energy shock wave (LESW) therapy in a rat ED model induced by pelvic neurovascular injuries. METHODS: Thirty-two male Sprague-Dawley rats injected with 5-ethynyl-2'-deoxyuridine (EdU) at newborn were divided into 4 groups: sham surgery (Sham), pelvic neurovascular injury by bilateral cavernous nerve injury and internal pudendal bundle injury (PVNI), PVNI treated with LESW at low energy (Low), and PVNI treated with LESW at high energy (High). After LESW treatment, rats underwent erectile function measurement and the tissues were harvested for histologic and molecular study. To examine the effect of LESW on Schwann cells, in vitro studies were conducted. MAIN OUTCOME MEASUREMENTS: The intracavernous pressure (ICP) measurement, histological examination, and Western blot (WB) were conducted. Cell cycle, Schwann cell activation-related markers were examined in in vitro experiments. RESULTS: LESW treatment improves erectile function in a rat model of pelvic neurovascular injury by leading to angiogenesis, tissue restoration, and nerve generation with more endogenous EdU(+) progenitor cells recruited to the damaged area and activation of Schwann cells. LESW facilitates more complete re-innervation of penile tissue with regeneration of neuronal nitric oxide synthase (nNOS)-positive nerves from the MPG to the penis. In vitro experiments demonstrated that LESW has a direct effect on Schwann cell proliferation. Schwann cell activation-related markers including p-Erk1/2 and p75 were upregulated after LESW treatment. CONCLUSION: LESW-induced endogenous progenitor cell recruitment and Schwann cell activation coincides with angiogenesis, tissue, and nerve generation in a rat model of pelvic neurovascular injuries.

Urethral musculature and innervation in the female rat.

AIMS: The urethral sphincter and urethral muscle innervation are critically involved in maintaining continence, especially in the female. However, the urethral muscle type and distribution, as well as the urethral nerves are far from being well documented. Our aim was to clearly identify the distribution of urethral striated muscle, smooth muscle, and urethral nerves. METHODS: In a cohort analysis of 3-month-old female Sprague-Dawley rats, cross and longitudinal sections of female rat urethra were extensively investigated using morphological techniques. Urethras were harvested to the sections, in order to provide both global and detailed visions of the urethra. H&E, Masson's Trichrome, phalloidin and immunoflourence stains were used. The cytoarchitecture, nitrergic, and cholinergic innervations were mainly investigated. Different layers of the segments of urethra were traced to draw curve graphs that represent the thickness of each muscle layer of urethral wall. RESULTS: The results showed that the primary peak of striated muscle is in the middle urethra. The inner layer close to mucosa was found to contain longitudinal smooth muscle. Near the bladder orifice, the circular smooth muscle dominates, which becomes thinner distally throughout the rest of urethra. In the middle urethra the vast majority of the urethral muscle are circularly oriented striated muscle cells. Typical nerve endings were present in high power images to show the different characteristic features of nerve innervation. CONCLUSIONS: This study has illustrated the detailed morphological structure and innervations of the normal female rat urethra and can serve as a basis for further study of stress urinary incontinence (SUI).

Novel therapeutic approach for neurogenic erectile dysfunction: effect of neurotrophic tyrosine kinase receptor type 1 monoclonal antibody.

BACKGROUND: Erectile dysfunction (ED) is a major health issue in aged populations, and neurogenic ED is particularly difficult to treat. Novel therapeutic approaches are needed for treatment of neurogenic ED of peripheral origin. OBJECTIVE: To investigate the therapeutic effects of a neurotrophic tyrosine kinase receptor type 1 monoclonal antibody (TrkA-mAb) on erectile function and sexual behavior in a rat model of cavernous nerve injury (CNI). DESIGN, SETTING, AND PARTICIPANTS: In one experiment, 84 male rats were randomly assigned to seven groups. The groups underwent either CNI or sham surgery, subsequent injection into the major pelvic ganglion (IMPG) of phosphate-buffered saline (PBS), an immunoglobulin G (IgG) control, or TrkA-mAb, and then intracavernosal (IC) injection of either PBS or varying TrkA-mAb concentrations immediately after surgery and then 1 wk later. Erectile function was assessed and histologic/molecular analyses were performed at 6 wk after surgery. In a second experiment, 36 male rats were randomly divided into three groups. The groups underwent CNI or sham surgery and then IC injection of PBS, IgG, or TrkA-mAb immediately after surgery and for 5 wk thereafter. At 6 wk after surgery, the performance of the rats in sexual behavior tests was videotaped. INTERVENTION: CNI or sham surgery; IMPG of PBS, IgG, or TrkA-mAb; IC injection of PBS or TrkA-mAb. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The intracavernous pressure response to cavernous nerve electrostimulation was measured and midpenile cross-sections were histologically examined. Western blotting (WB) of cavernous tissue protein was performed. Rats were assessed for chasing, mounting, intromission, and ejaculation behaviors during sexual behavior tests. The data were analyzed using one-way analysis of variance followed by the Tukey-Kramer t test. RESULTS AND LIMITATIONS: Recovery of erectile function of varying degrees was observed in the TrkA-mAb groups. TrkA-mAb treatment significantly suppressed tyrosine hydroxylase-positive nerve fibers in the corpus cavernosum and enhanced neuronal nitric oxide synthase-positive fibers in the dorsal nerve. The ratio of smooth muscle to collagen in the corpus cavernosum was significantly improved in TrkA-mAb treatment groups compared to PBS vehicle and IgG control groups. WB confirmed these biological changes. There was a nonsignificant increase in the average number of intromissions and ejaculations in the TrkA-mAb group. The study limitations include small sample size, variability in sexual behavior, lack of data on the neuromuscular mechanism involved, and lack of information of the role of neurotrophins or cytokines in regeneration. CONCLUSIONS: TrkA-mAb successfully inhibits sympathetic nerve regeneration, leads to parasympathetic nerve regeneration, and has therapeutic effects on ED and sexual behavior disorder in a rat model of CNI. PATIENT SUMMARY: This report provides strong evidence that a neurotrophic tyrosine kinase receptor type 1 monoclonal antibody (TrkA-mAb) inhibits sympathetic nerve regeneration, leads to parasympathetic nerve regeneration, and has therapeutic effects on erectile dysfunction and sexual behavior disorder in a rat model of cavernous nerve injury. The results raise the possibility that human patients with neurogenic erectile dysfunction may respond to TrkA-mAb in a manner that parallels the response seen in our rodent study.

Presence of stem/progenitor cells in the rat penis.

Tissue resident stem cells are believed to exist in every organ, and their identification is commonly done using a combination of immunostaining for putative stem cell markers and label-retaining cell (LRC) strategy. In this study, we employed these approaches to identify potential stem cells in the penis. Newborn rats were intraperitoneally injected with thymidine analog, 5-ethynyl-2-deoxyuridine (EdU), and their penis was harvested at 7 h, 3 days, 1 week, and 4 weeks. It was processed for EdU stains and immunofluorescence staining for stem cell markers A2B5, PCNA, and c-kit. EdU-positive cells were counted for each time point and co-localized with each stem cell marker, then isolated and cultured in vitro followed by their characterization using flowcytometry and immunofluorescence. At 7 h post-EdU injection, 410 ± 105.3 penile corporal cells were labeled in each cross-section (∼28%). The number of EdU-positive cells at 3 days increased to 536 ± 115.6, while their percentage dropped to 25%. Progressively fewer EdU-positive cells were present in the sacrificed rat penis at longer time points (1 and 4 weeks). They were mainly distributed in the subtunic and perisinusoidal spaces, and defined as subtunic penile progenitor cells (STPCs) and perisinusoidal penile progenitor cells (PPCs). These cells expressed c-kit, A2B5, and PCNA. After culturing in vitro, only ∼0.324% corporal cells were EdU-labeled LRCs and expressed A2B5/PCNA. Therefore, labeling of penis cells by EdU occurred randomly, and label retaining was not associated with expression of c-kit, A2B5, or PCNA. The penile LRCs are mainly distributed within the subtunic and perisinusoidal space.

Estrogen attenuates TGF-ß1 induced elastogenesis in rat urethral smooth muscle cells by inhibiting Smad response elements.

PURPOSE: We investigated the effect and mechanism of estrogen on elastogenesis in urethral smooth muscle cells in vitro. MATERIALS AND METHODS: Urethral smooth muscle cells were isolated from normal adult female rats. For elastogenesis assay cells were treated with TGF-ß1, the potent TGF-ß1 receptor inhibitor SB431542 and estrogen for 2 weeks. Real-time polymerase chain reaction was performed to assay gene expression during this process. Activity of the TGF-ß1 responsive elements CAGA(12)-Luc and GCCG(12)-Luc were also assayed. Estrogen receptor and Smad2/3 interaction was evaluated by immunoprecipitation and Western blot. RESULTS: TGF-ß1 induced elastogenesis in rat urethral smooth muscle cells. This effect was partially blocked by estrogen and completely abrogated by SB431542. SB431542 completely inhibited activation of the Smad2/3 response element CAGA(12)-Luc and estrogen significantly inhibited activation. The Smad1/4 response element GCCG(12)-Luc was not affected by SB431542 treatment but estrogen partially inhibited the activation of GCCG(12)-Luc induced by TGF-ß1. Estrogen receptor bound to Smad 2 and 3 in vitro. CONCLUSIONS: Estrogen attenuated TGF-ß1 induced elastogenesis via binding of its activated receptor to Smad2/3 to inhibit the TGF-ß1 response element in rat urethral smooth muscle cells.

Conversion of adipose-derived stem cells into natural killer-like cells with anti-tumor activities in nude mice.

Efforts to develop peripheral blood-derived nature killer (NK) cells into therapeutic products have been hampered by these cells' low abundance and histoincompatibility. On the other hand, derivation of NK-like cells from more abundant cell sources such as embryonic stem cells (ESCs) and umbilical cord blood (UCB) requires the selection of rare CD34+ cells. Thus, we sought to convert adipose-derived stem cells (ADSCs), which are abundant and natively CD34+, into NK-like cells. When grown in hematopoietic induction medium, ADSCs formed sphere clusters and expressed hematopoietic markers CD34, CD45, and KDR. Further induction in NK cell-specific medium resulted in a population of cells that expressed NK cell marker CD56, and thus termed ADSC-NK. Alternatively, the hematopoietically induced ADSCs were transduced with NK cell-specific transcription factor E4BP4 prior to induction in NK cell-specific medium. This latter population of cells, termed ADSC-NKE, expressed CD56 and additional NK cell markers such as CD16, CD94, CD158, CD314, FasL, and NKp46. ADSC-NKE was as potent as NK leukemia cell NKL in killing breast cancer cell MCF7 and prostate cancer cells DU145, PC3, LnCap, DuPro, C4-2 and CWR22, but exhibited no killing activity toward normal endothelial and smooth muscle cells. In nude mice test ADSC-NKE was able to significantly delay the progression of tumors formed by MCF7 and PC3. When injected into immunocompetent rats, ADSC-NKE was detectable in bone marrow and spleen for at least 5 weeks. Together, these results suggest that ADSCs can be converted into NK-like cells with anti-tumor activities.

GdX/UBL4A specifically stabilizes the TC45/STAT3 association and promotes dephosphorylation of STAT3 to repress tumorigenesis.

Impaired phosphatase activity contributes to the persistent activation of STAT3 in tumors. Given that STAT family members with various or even opposite functions are often phosphorylated or dephosphorylated by the same enzymes, the mechanism for STAT3-specific dephosphorylation in cells remains largely unknown. Here, we report that GdX (UBL4A) promotes STAT3 dephosphorylation via mediating the interaction between TC45 (the nuclear isoform of TC-PTP) and STAT3 specifically. GdX stabilizes the TC45-STAT3 complex to bestow upon STAT3 an efficient dephosphorylation by TC45. Inasmuch, GdX suppresses tumorigenesis and tumor development by reducing the level of phospho-STAT3 (p-STAT3), whereas deletion of GdX results in a high level of p-STAT3 and accelerated colorectal tumorigenesis induced by AOM/DSS. Thus, GdX converts TC45, a nonspecific phosphatase, into a STAT3-specific phosphatase by bridging an association between TC45 and STAT3.

SIPAR negatively regulates STAT3 signaling and inhibits progression of melanoma.

Persistently activated STAT3 is important for tumorigenesis in a variety of cancers, including melanoma. Although many co-factors in the regulation of STAT3 activity have been identified, it remains unclear how STAT3 phosphorylation is negatively regulated. Here, we report that SIPAR (STAT3-Interacting Protein As a Repressor) inhibits STAT3 activity by accelerating its dephosphorylation. We observed that SIPAR directly interacted with STAT3 upon IL-6 stimulation. Moreover, over-expression of SIPAR reduced, whereas depletion enhanced, the level of phosphorylated STAT3. We further demonstrated that SIPAR inhibited the growth of melanoma cells by decreasing the level of phosphorylated STAT3 and the expression of its target genes. These results suggest that SIPAR, functioning as a new negative regulator, inhibits STAT3 activity by enhancing its dephosphorylation and represses melanoma progression.

Effects of EdU labeling on mesenchymal stem cells.

BACKGROUND: Thymidine analog 5-ethynyl-2-deoxyuridine (EdU) has recently been used for tracking mesenchymal stem cells (MSCs). In the present study, we tested whether EdU was cytotoxic and whether it interfered with differentiation, cytokine secretion and migration of MSCs. METHODS: EdU labeling was performed by incubating adipose-derived stem cells (ADSCs) with 10(-8) mol/L of EdU for 48 h. Incorporation of EdU was detected by reaction with azide-conjugated Alexa594. The labeled and unlabeled ADSCs were compared for proliferation and apoptosis as determined by CellTiter and comet assays, respectively. They were also compared for neuron-like and endothelial differentiation as determined by morphology, marker expression and function. Comparison of their secreted cytokine profile was performed by cytokine antibody array. Comparison of their response to homing factor SDF-1 was performed by migration assay. RESULTS: EdU was incorporated into the nucleus in approximately 70% of ADSCs. No significant differences in proliferation and apoptosis rates were observed between EdU-labeled and unlabeled ADSCs. Isobutylmethylxanthine induced both EdU-labeled and unlabeled ADSCs to assume a neuron-like morphology and to express ß-III tubulin. Endothelial growth medium-2 (EGM2) induced endothelial differentiation in both EdU-labeled and unlabeled ADSCs, including the ability to uptake low-density lipoprotein and to form capillary-like structures as well as the expression of vWF, eNOS and CD31. EdU-labeled and unlabeled ADSCs exhibited identical secreted cytokine profile and identical migratory response to SDF-1. DISCUSSION: At the recommended dosage of 10(-8) mol/L, EdU is non-toxic to ADSCs. EdU label did not interfere with differentiation, cytokine secretion or migratory response to SDF-1 by ADSCs.

Is CD34 truly a negative marker for mesenchymal stromal cells?

The prevailing school of thought is that mesenchymal stromal cells (MSC) do not express CD34, and this sets MSC apart from hematopoietic stem cells (HSC), which do express CD34. However, the evidence for MSC being CD34(-) is largely based on cultured MSC, not tissue-resident MSC, and the existence of CD34(-) HSC is in fact well documented. Furthermore, the Stro-1 antibody, which has been used extensively for the identification/isolation of MSC, was generated by using CD34(+) bone marrow cells as immunogen. Thus, neither MSC being CD34(-) nor HSC being CD34(+) is entirely correct. In particular, two studies that analyzed CD34 expression in uncultured human bone marrow nucleated cells found that MSC (BMSC) existed in the CD34(+) fraction. Several studies have also found that freshly isolated adipose-derived MSC (ADSC) express CD34. In addition, all of these ADSC studies and several other MSC studies have observed a disappearance of CD34 expression when the cells are propagated in culture. Thus the available evidence points to CD34 being expressed in tissue-resident MSC, and its negative finding being a consequence of cell culturing.

Effects of high glucose on human cavernous endothelial cells.

OBJECTIVE: To obtain experimental evidence for a causal effect of high glucose (HG) on cavernous endothelial dysfunction. METHODS: Cavernous tissues were obtained from patients undergoing surgery for penile prosthesis implantation. Endothelial cells were isolated by binding to anti-CD31 antibody, followed by magnetic capture. Their endothelial identity was verified by flow cytometry and immunofluorescence staining for endothelial markers CD31, von Willebrand factor, and endothelial nitric oxide synthase, and by their ability to form tube-like structures in matrigel (tube formation) and to endocytose acetylated low-density lipoprotein (low-density lipoprotein uptake). The cells were then cultured under normal glucose (NG) (5 mM) or HG (25 mM) conditions, followed by analysis for endothelial gene expression, function, proliferation, apoptosis, and mitochondrial fragmentation. RESULTS: Human cavernous endothelial cell (HCEC) strains were established and determined to be nearly 100% pure endothelial cells. In the HG culture condition, HCECs expressed approximately 50% less CD31, von Willebrand factor, and endothelial nitric oxide synthase, but nearly twice as much collagen IV compared with HCECs grown in NG medium. HG also suppressed low-density lipoprotein uptake and tube formation by approximately 50%. HCECs grew significantly slower in the high-glucose medium than in the NG medium. Approximately 3 times as many cells exhibited apoptosis in the HG medium as in the NG medium. Approximately 4 times as many cells contained fragmented mitochondria in the HG medium as in the NG medium. CONCLUSION: HG caused a decrease in endothelial proliferation, function, and marker expression. It also caused an increase in endothelial collagen IV expression, apoptosis, and mitochondrial fragmentation. Together, these HG-induced changes in cavernous endothelial cells provide an explanation for hyperglycemia's detrimental effects on erectile function.

Bone marrow cells stained by azide-conjugated Alexa fluors in the absence of an alkyne label.

Thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) has recently been introduced as an alternative to 5-bromo-2-deoxyuridine (BrdU) for cell labeling and tracking. Incorporation of EdU into replicating DNA can be detected by azide-conjugated fluors (eg, Alexa-azide) through a Cu(i)-catalyzed click reaction between EdU's alkyne moiety and azide. While this cell labeling method has proven to be valuable for tracking transplanted stem cells in various tissues, we have found that some bone marrow cells could be stained by Alexa-azide in the absence of EdU label. In intact rat femoral bone marrow, ~3% of nucleated cells were false-positively stained, and in isolated bone marrow cells, ~13%. In contrast to true-positive stains, which localize in the nucleus, the false-positive stains were cytoplasmic. Furthermore, while true-positive staining requires Cu(i), false-positive staining does not. Reducing the click reaction time or reducing the Alexa-azide concentration failed to improve the distinction between true- and false-positive staining. Hematopoietic and mesenchymal stem cell markers CD34 and Stro-1 did not co-localize with the false-positively stained cells, and these cells' identity remains unknown.

miR-764-5p promotes osteoblast differentiation through inhibition of CHIP/STUB1 expression.

Differentiation of committed precursor cells into the osteoblast lineage is tightly regulated by several factors, including Runx2 and BMP2. We previously reported that C terminus of Hsc70-interacting protein/STIP1 homology and U-Box containing protein 1 (CHIP/STUB1) negatively regulated osteoblast differentiation through promoting Runx2 protein degradation. However, how CHIP is regulated during osteoblast differentiation remains unknown. In this study, we found that miR-764-5p is up-expressed during the osteoblast differentiation in calvarial and osteoblast progenitor cells, coupled with down-expression of CHIP protein. We observed that forced expression or inhibition of miR-764-5p decreased or increased the CHIP protein level through affecting its translation by targeting the 3'-UTR region. Perturbation of miR-764-5p resulted in altered differentiation fate of osteoblast progenitor cells and the role of miR-764-5p was reversed by overexpression of CHIP, whereas depletion of CHIP impaired the effect of miR-764-5p. Our data showed that miR-764-5p positively regulates osteoblast differentiation from osteoblast progenitor cells by repressing the translation of CHIP protein.

Adipose tissue-derived stem cells ameliorate diabetic bladder dysfunction in a type II diabetic rat model.

Diabetes mellitus is associated with a broad constellation of voiding complaints that are often multifactorial and resistant to currently available therapies. The leading causes of diabetic bladder dysfunction (DBD) include alterations in the bladder smooth muscle, neuronal degeneration, and urothelial dysfunction. Adipose tissue-derived stem cells (ADSCs), a type of mesenchymal stromal cells, have shown promise as a novel tissue regenerative technique that may have utility in DBD. The aim of this study is to determine the efficacy and mechanism by which ADSCs may ameliorate DBD in rats fed a high-fat diet and treated with low-dose streptozotocin to induce type II diabetes. Improved voiding function was noted in ADSCs-treated rats as compared with phosphate-buffered saline-treated rats. Though some ADSCs differentiated into smooth muscle cells, paracrine pathway seems to play a main role in this process, thus resulting in reduction of apoptosis and preservation of "suburothelial capillaries network."