|
1
|
Chen Y, Banie L, Breyer BN, Tan Y, Wang Z, Zhou F, Wang G, Lin G, Liu J, Qi LS, Lue TF. Enhanced Myogenesis by Silencing Myostatin with Nonviral Delivery of dCas9 Ribonucleoprotein Complex. CRISPR J 2022; 5:598-608. [PMID: 35758824 DOI: 10.1089/crispr.2022.0009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Stress urinary incontinence (SUI) and pelvic floor disorder (PFD) are common conditions with limited treatment options in women worldwide. Regenerative therapy to restore urethral striated and pelvic floor muscles represents a valuable therapeutic approach. We aim to determine the CRISPR interference-mediated gene silencing effect of the nonviral delivery of nuclease-deactivated dCas9 ribonucleoprotein (RNP) complex on muscle regeneration at the cellular and molecular level. We designed four myostatin (MSTN)-targeting sgRNAs and transfected them into rat myoblast L6 cells together with the dCas9 protein. Myogenesis assay and immunofluorescence staining were performed to evaluate muscle differentiation, while CCK8 assay, cell cycle assay, and 5-ethynyl-2'-deoxyuridine staining were used to measure muscle proliferation. Reverse transcription-polymerase chain reaction and Western blotting were also performed to examine cellular signaling. Myogenic factors (including myosin heavy chain, MSTN, myocardin, and serum response factor) increased significantly after day 5 during myogenesis. MSTN was efficiently silenced after transfecting the dCas9 RNP complex, which significantly promoted more myotube formation and a higher fusion index for L6 cells. In cellular signaling, MSTN repression enhanced the expression of MyoG and MyoD, phosphorylation of Smad2, and the activity of Wnt1/GSK-3β/β-catenin pathway. Moreover, MSTN repression accelerated L6 cell growth with a higher cell proliferation index as well as a higher expression of cyclin D1 and cyclin E. Nonviral delivery of the dCas9 RNP complex significantly promoted myoblast differentiation and proliferation, providing a promising approach to improve muscle regeneration for SUI and PFD. Further characterization and validation of this approach in vivo are needed.
Collapse
Affiliation(s)
- Yinwei Chen
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, San Francisco, California, USA.,Reproductive Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Lia Banie
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, San Francisco, California, USA
| | - Benjamin N Breyer
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, San Francisco, California, USA
| | - Yan Tan
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, San Francisco, California, USA
| | - Zhao Wang
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, San Francisco, California, USA
| | - Feng Zhou
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, San Francisco, California, USA
| | - Guifang Wang
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, San Francisco, California, USA
| | - Guiting Lin
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, San Francisco, California, USA
| | - Jihong Liu
- Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Lei S Qi
- Department of Bioengineering, Stanford University, Stanford, California, USA.,ChEM-H, Stanford University, Stanford, California, USA
| | - Tom F Lue
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, San Francisco, California, USA
| |
Collapse
|
|
2
|
Yuan H, Ruan Y, Tan Y, Reed-Maldonado AB, Chen Y, Zhao D, Wang Z, Zhou F, Peng D, Banie L, Wang G, Liu J, Lin G, Qi LS, Lue TF. Regenerating Urethral Striated Muscle by CRISPRi/dCas9-KRAB-Mediated Myostatin Silencing for Obesity-Associated Stress Urinary Incontinence. CRISPR J 2020; 3:562-572. [PMID: 33346712 PMCID: PMC7757699 DOI: 10.1089/crispr.2020.0077] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Overweight females are prone to obesity-associated stress urinary incontinence (OA-SUI), and there are no definitive medical therapies for this common urologic condition. This study was designed to test the hypothesis that regenerative therapy to restore urethral striated muscle (stM) and pelvic floor muscles might represent a valuable therapeutic approach. For the in vitro experiment, single-guide RNAs targeting myostatin (MSTN) were used for CRISPRi/dCas9-Kruppel associated box (KRAB)-mediated gene silencing. For the in vivo experiment, a total of 14 female lean ZUC-Leprfa 186 and 14 fatty ZUC-Leprfa 185 rats were used as control and CRISPRi-MSTN treated groups, respectively. The results indicated that lentivirus-mediated expression of MSTN CRISPRi/dCas9-KRAB caused sustained downregulation of MSTN in rat L6 myoblast cells and significantly enhanced myogenesis in vitro. In vivo, the urethral sphincter injection of lentiviral-MSTN sgRNA and lentiviral-dCas9-KRAB significantly increased the leak point pressure, the thickness of the stM layer, the ratio of stM to smooth muscle, and the number of neuromuscular junctions. Downregulation of MSTN with CRISPRi/dCas9-KRAB-mediated gene silencing significantly enhanced myogenesis in vitro and in vivo. It also improved urethral continence in the OA-SUI rat model.
Collapse
Affiliation(s)
- Huixing Yuan
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, California, USA; Department of Chemical and Systems Biology, ChEM-H, Stanford University, Stanford, California, USA
- Department of Urology, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, PR China; Department of Chemical and Systems Biology, ChEM-H, Stanford University, Stanford, California, USA
| | - Yajun Ruan
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, California, USA; Department of Chemical and Systems Biology, ChEM-H, Stanford University, Stanford, California, USA
- Department of Urology, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, PR China; Department of Chemical and Systems Biology, ChEM-H, Stanford University, Stanford, California, USA
| | - Yan Tan
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, California, USA; Department of Chemical and Systems Biology, ChEM-H, Stanford University, Stanford, California, USA
| | - Amanda B. Reed-Maldonado
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, California, USA; Department of Chemical and Systems Biology, ChEM-H, Stanford University, Stanford, California, USA
- Department of Urology, Tripler Army Medical Center, 1 Jarrett White Road, Honolulu, Hawaii, USA; and Department of Chemical and Systems Biology, ChEM-H, Stanford University, Stanford, California, USA
| | - Yinwei Chen
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, California, USA; Department of Chemical and Systems Biology, ChEM-H, Stanford University, Stanford, California, USA
| | - Dehua Zhao
- Department of Bioengineering, Department of Chemical and Systems Biology, ChEM-H, Stanford University, Stanford, California, USA
| | - Zhao Wang
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, California, USA; Department of Chemical and Systems Biology, ChEM-H, Stanford University, Stanford, California, USA
| | - Feng Zhou
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, California, USA; Department of Chemical and Systems Biology, ChEM-H, Stanford University, Stanford, California, USA
| | - Dongyi Peng
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, California, USA; Department of Chemical and Systems Biology, ChEM-H, Stanford University, Stanford, California, USA
| | - Lia Banie
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, California, USA; Department of Chemical and Systems Biology, ChEM-H, Stanford University, Stanford, California, USA
| | - Guifang Wang
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, California, USA; Department of Chemical and Systems Biology, ChEM-H, Stanford University, Stanford, California, USA
| | - Jihong Liu
- Department of Urology, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, PR China; Department of Chemical and Systems Biology, ChEM-H, Stanford University, Stanford, California, USA
| | - Guiting Lin
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, California, USA; Department of Chemical and Systems Biology, ChEM-H, Stanford University, Stanford, California, USA
| | - Lei S. Qi
- Department of Bioengineering, Department of Chemical and Systems Biology, ChEM-H, Stanford University, Stanford, California, USA
| | - Tom F. Lue
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, California, USA; Department of Chemical and Systems Biology, ChEM-H, Stanford University, Stanford, California, USA
| |
Collapse
|
|
3
|
Peng D, Reed-Maldonado AB, Zhou F, Tan Y, Yuan H, Banie L, Wang G, Tang Y, He L, Lin G, Lue TF. Exosome Released From Schwann Cells May Be Involved in Microenergy Acoustic Pulse-Associated Cavernous Nerve Regeneration. J Sex Med 2020; 17:1618-1628. [PMID: 32669249 DOI: 10.1016/j.jsxm.2020.05.018] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2019] [Revised: 05/07/2020] [Accepted: 05/13/2020] [Indexed: 12/13/2022]
Abstract
BACKGROUND Neurogenic erectile dysfunction (ED) is often refractory to treatment because of insufficient functional nerve recovery after injury or insult. Noninvasive mechano-biological intervention, such as microenergy acoustic pulse (MAP), low-intensity pulsed ultrasound, and low-intensity extracorporeal shockwave treatment, is an optimal approach to stimulate nerve regeneration. AIM To establish a new model in vitro to simulate nerve injury in neurogenic ED and to explore the mechanisms of MAP in vitro. METHODS Sprague-Dawley rats were used to isolate Schwann cells (SCs), major pelvic ganglion (MPG), and cavernous nerve with MPG (CN/MPG). SCs were then treated with MAP (0.033 mJ/mm2, 1 Hz, 100 pulses), and SC exosomes were isolated. The MPG and CN/MPG were treated with MAP (0.033 mJ/mm2, 1 Hz) at different dosages (25, 50, 100, 200, or 300 pulses) or exosomes derived from MAP-treated SCs in vitro. OUTCOMES Neurite growth from the MPG fragments and CN was photographed and measured. Expression of neurotropic factors (brain-derived neurotrophic factor, nerve growth factor, and neurotrophin-3) was checked. RESULTS Neurite outgrowth from MPG and CN/MPG was enhanced by MAP in a dosage response manner, peaking at 100 pulses. MAP promoted SC proliferation, neurotropic factor (brain-derived neurotrophic factor, nerve growth factor, and neurotrophin-3) expression, and exosome secretion. SC-derived exosomes significantly enhanced neurite outgrowth from MPG in vitro. CLINICAL IMPLICATIONS MAP may have utility in the treatment of neurogenic ED by SC-derived exosomes. STRENGTH & LIMITATIONS We confirmed that MAP enhances penile nerve regeneration through exsomes. Limitations of this study include that our study did not explore the exact mechanisms of how MAP increases SC exosome secretion nor whether MAP modulates the content of exosomes. CONCLUSION This study revealed that neurite outgrowth from MPG was enhanced by MAP and by SC-derived exosomes which were isolated after MAP treatment. Our findings indicate that one mechanism by which MAP induces nerve regeneration is by stimulation of SCs to secrete exosomes. Peng D, Reed-Maldonado AB, Zhou F, et al. Exosome Released From Schwann Cells May Be Involved in Microenergy Acoustic Pulse-Associated Cavernous Nerve Regeneration. J Sex Med 2020;17:1618-1628.
Collapse
Affiliation(s)
- Dongyi Peng
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, CA, USA; Department of Urology, Third Xiangya Hospital of Central South University, Changsha, China
| | - Amanda B Reed-Maldonado
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, CA, USA
| | - Feng Zhou
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, CA, USA
| | - Yan Tan
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, CA, USA
| | - Huixing Yuan
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, CA, USA
| | - Lia Banie
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, CA, USA
| | - Guifang Wang
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, CA, USA
| | - Yuxin Tang
- Department of Urology, Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, China
| | - Leye He
- Department of Urology, Third Xiangya Hospital of Central South University, Changsha, China
| | - Guiting Lin
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, CA, USA
| | - Tom F Lue
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, CA, USA.
| |
Collapse
|
|
4
|
Peng D, Yuan H, Liu T, Wang T, Reed-Maldonado AB, Kang N, Banie L, Wang G, Tang Y, He L, Lin G, Lue TF. Smooth Muscle Differentiation of Penile Stem/Progenitor Cells Induced by Microenergy Acoustic Pulses In Vitro. J Sex Med 2019; 16:1874-1884. [PMID: 31585805 DOI: 10.1016/j.jsxm.2019.08.020] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2019] [Revised: 08/08/2019] [Accepted: 08/19/2019] [Indexed: 12/28/2022]
Abstract
INTRODUCTION Modulating tissue-resident stem and progenitor cells with a non-invasive, mechanobiological intervention is an optimal approach for tissue regeneration. Stem cell antigen-1 (Sca-1) has been identified as a stem cell marker within many organs but never within the penis. AIM To localize and isolate penile stem/progenitor cells (PSPCs) and to evaluate cellular differentiation after exposure to induction medium and microenergy acoustic pulse (MAP) therapy. METHODS Six male Sprague-Dawley rats were used to isolate PSPCs. Isolation was followed by stem cell characterization and differentiation assays. The PSPCs were then treated with MAP (0.033 mJ/mm2, 1 Hz) at various dosages (25, 50, 100, and 200 pulses) and for different durations (1, 2, 4, 6, or 8 hours) in vitro. MAIN OUTCOME MEASURE The PSPCs (Sca-1-positive cells) were isolated using the magnetic-activated cell sorting system. PSPC cellular differentiation was assessed after induction with induction medium and with MAP in vitro. Wnt/β-catenin signaling was also assayed. RESULTS The PSPCs were successfully localized within the penile subtunic and perisinusoidal spaces, and they were successfully isolated using magnetic-activated cell sorting. The stemness of the cells was confirmed by stem cell marker characterization and by multiple differentiation into smooth muscle cells, endothelial cells, adipocytes, and neurons. MAP-induced PSPCs differentiated into smooth muscle cells by activating the Wnt/β-catenin signaling pathway in a time- and dosage-dependent manner. CLINICAL IMPLICATIONS By modulating resident PSPCs, MAP may have utility in the treatment of erectile dysfunction (ED). STRENGTHS & LIMITATIONS This study provides solid evidence in support of microenergy therapies, including both MAP and low-intensity extracorporeal shock wave therapy, for the treatment of ED. Additional studies are needed and should include additional stem cells markers. Furthermore, studies exploring the underling mechanisms for PSPC activation and differentiation are required. CONCLUSION PSPCs were successfully identified, localized, and isolated. Additionally, MAP provoked PSPCs to differentiate into smooth muscle cells via the Wnt/β-catenin signaling pathway. As such, MAP provides a novel method for activating endogenous tissue-resident stem/progenitor cells and might facilitate stem cell regenerative therapy targeting ED. Peng D, Yuan H, Liu T, et al. Smooth Muscle Differentiation of Penile Stem/Progenitor Cells Induced by Microenergy Acoustic Pulses In Vitro. J Sex Med 2019; 16:1874-1884.
Collapse
Affiliation(s)
- Dongyi Peng
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, CA, USA; Department of Urology, Third Xiangya Hospital of Central South University, Changsha, China
| | - Huixing Yuan
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, CA, USA
| | - Tianshu Liu
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, CA, USA
| | - Tianyu Wang
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, CA, USA
| | - Amanda B Reed-Maldonado
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, CA, USA
| | - Ning Kang
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, CA, USA
| | - Lia Banie
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, CA, USA
| | - Guifang Wang
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, CA, USA
| | - Yuxin Tang
- Department of Urology, Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, China
| | - Leye He
- Department of Urology, Third Xiangya Hospital of Central South University, Changsha, China
| | - Guiting Lin
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, CA, USA
| | - Tom F Lue
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, CA, USA.
| |
Collapse
|
|
5
|
Zhang H, Zhao Y, Wang M, Song W, Sun P, Jin X. A promising therapeutic option for diabetic bladder dysfunction: Adipose tissue-derived stem cells pretreated by defocused low-energy shock wave. J Tissue Eng Regen Med 2019; 13:986-996. [PMID: 30811857 DOI: 10.1002/term.2844] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2018] [Revised: 01/18/2019] [Accepted: 02/21/2019] [Indexed: 12/19/2022]
Abstract
Adipose tissue-derived stem cells (ADSCs) have shown effectiveness in treating diabetic bladder dysfunction (DBD). In the present study, ADSCs pretreated by defocused low-energy shock wave (DLSW) were first used to achieve better therapeutic effect. ADSCs were treated by DLSW prior to each passage. Secretions of vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) were tested. Proliferation ability was examined by staining 5-ethynyl-2-deoxyuridine (EdU) and assessing expressions of proliferating cell nuclear antigen (PCNA) and Ki67. DBD rat model was created and subgrouped via therapeutic options of phosphate-buffered saline, ADSCs, pretreated ADSCs, and ADSCs lysate. Afterward, voiding functions were evaluated, and tissues were examined by histology. Neonatal rats received intraperitoneal injection of EdU. All rats were subgrouped and treated as narrated above. Bladder tissues were stained with EdU, Stro-1, and CD34. Results showed that shocked ADSCs were activated by secreting more VEGF and NGF, by higher EdU-retaining cells ratios, and by higher expressions of PCNA and Ki67 compared with unshocked ADSCs. Shocked ADSCs had the most effective efficacy in treating DBD by secreting the most VEGF and NGF to accelerate regenerations of revascularization and innervation. Migrations of EdU+ Stro-1+ CD34- endogenous stem cells to bladders were enhanced by injecting ADSCs. In conclusion, ADSCs pretreated by DLSW had potent therapeutic effect in treating DBD by secreting VEGF and NGF. Recruitment of endogenous stem cells was considered as an important mechanism in this regenerative process.
Collapse
Affiliation(s)
- Haiyang Zhang
- School of Basic Medical Sciences, Shandong University, Jinan, China.,Department of Urology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, China.,Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, California, USA
| | - Yong Zhao
- Department of Urology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, China
| | - Muwen Wang
- Department of Urology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, China
| | - Wei Song
- Department of Urology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, China
| | - Peng Sun
- Department of Urology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, China
| | - Xunbo Jin
- Department of Urology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, China
| |
Collapse
|
|
6
|
Hodges WM, O'Brien F, Fulzele S, Hamrick MW. Function of microRNAs in the Osteogenic Differentiation and Therapeutic Application of Adipose-Derived Stem Cells (ASCs). Int J Mol Sci 2017; 18:ijms18122597. [PMID: 29207475 PMCID: PMC5751200 DOI: 10.3390/ijms18122597] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2017] [Revised: 11/27/2017] [Accepted: 11/28/2017] [Indexed: 02/08/2023] Open
Abstract
Traumatic wounds with segmental bone defects represent substantial reconstructive challenges. Autologous bone grafting is considered the gold standard for surgical treatment in many cases, but donor site morbidity and associated post-operative complications remain a concern. Advances in regenerative techniques utilizing mesenchymal stem cell populations from bone and adipose tissue have opened the door to improving bone repair in the limbs, spine, and craniofacial skeleton. The widespread availability, ease of extraction, and lack of immunogenicity have made adipose-derived stem cells (ASCs) particularly attractive as a stem cell source for regenerative strategies. Recently it has been shown that small, non-coding miRNAs are involved in the osteogenic differentiation of ASCs. Specifically, microRNAs such as miR-17, miR-23a, and miR-31 are expressed during the osteogenic differentiation of ASCs, and appear to play a role in inhibiting various steps in bone morphogenetic protein-2 (BMP2) mediated osteogenesis. Importantly, a number of microRNAs including miR-17 and miR-31 that act to attenuate the osteogenic differentiation of ASCs are themselves stimulated by transforming growth factor β-1 (TGFβ-1). In addition, transforming growth factor β-1 is also known to suppress the expression of microRNAs involved in myogenic differentiation. These data suggest that preconditioning strategies to reduce TGFβ-1 activity in ASCs may improve the therapeutic potential of ASCs for musculoskeletal application. Moreover, these findings support the isolation of ASCs from subcutaneous fat depots that tend to have low endogenous levels of TGFβ-1 expression.
Collapse
Affiliation(s)
- Walter M Hodges
- Department of Cellular Biology & Anatomy, Medical College of Georgia, Augusta University, Augusta, GA 30912, USA.
| | - Frederick O'Brien
- Dwight D. Eisenhower Army Medical Center, Fort Gordon, Augusta, GA 30912, USA.
| | - Sadanand Fulzele
- Department of Cellular Biology & Anatomy, Medical College of Georgia, Augusta University, Augusta, GA 30912, USA.
| | - Mark W Hamrick
- Department of Cellular Biology & Anatomy, Medical College of Georgia, Augusta University, Augusta, GA 30912, USA.
| |
Collapse
|
|
7
|
Responses of adventitial CD34 + vascular wall-resident stem/progenitor cells and medial smooth muscle cells to carotid injury in rats. Exp Mol Pathol 2016; 101:332-340. [PMID: 27856167 DOI: 10.1016/j.yexmp.2016.11.004] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2016] [Revised: 09/19/2016] [Accepted: 11/11/2016] [Indexed: 12/21/2022]
Abstract
Cell culture and carotid injury studies with SD rats were performed to investigate the roles of CD34+ vascular wall-resident stem/progenitor cells (VRS/Pcs) and vascular smooth muscle cells (SMCs) in neointimal formation. In vitro, the media-isolated SM MHC+ SMCs occupied 93.92±8.62% of total BrdU+ cells, whereas the CD34+ cells, only 2.61±0.82%, indicating that the cell expansion in SMC culture was attributed to SM MHC+ SMCs. The adventitia-isolated CD34+ VRS/Pcs responded to PDGF-BB by differentiating into SMC-like cells which expressed SM22α (an early stage SMC marker), but seldom SM MHC (a late stage SMC marker). In carotid injury model, the CD34+ VRS/Pcs differentiated SMC-like cells migrated in very few numbers into only the outer layer of the media, and this was further confirmed by a cell tracking analysis. While the neointimal cells were consistently SM MHC+ and CD34- SMCs during whole course of the post-injury remodeling. Thus it is speculated that the adventitial CD34+ VRS/Pcs, at least in rat model, do not directly participate in neointimal formation, but function to maintain homeostasis of the media during injury-induced vascular wall remodeling.
Collapse
|
|
8
|
Wang J, Lin G, Alwaal A, Zhang X, Wang G, Jia X, Banie L, Villalta J, Lin CS, Lue TF. Kinetics of Label Retaining Cells in the Developing Rat Kidneys. PLoS One 2015; 10:e0144734. [PMID: 26650841 PMCID: PMC4674088 DOI: 10.1371/journal.pone.0144734] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2015] [Accepted: 11/23/2015] [Indexed: 12/23/2022] Open
Abstract
Background The kidney is a specialized low-regenerative organ with several different types of cellular lineages. The BrdU label-retaining cell (LRCs) approach has been used as part of a strategy to identify tissue-specific stem cells in the kidney; however, because the complementary base pairing in double-stranded DNA blocks the access of the anti-BrdU antibody to BrdU subunits, the stem cell marker expression in BrdU-labeled cells are often difficult to detect. In this study, we introduced a new cell labeling and detection method in which BrdU was replaced with 5-ethynyl-2-deoxyuridine (EdU) and examined the time-dependent dynamic changes of EdU-labeled cells and potential stem/progenitor markers in the development of kidney. Methods Newborn rats were intraperitoneally injected with EdU, and their kidneys were harvested respectively at different time points at 1 day, 3 days, 1 week, 2 weeks, and 6 weeks post-injection. The kidney tissues were processed for EdU and cellular markers by immunofluorescence staining. Results At the early stage, LRCs labeled by EdU were 2176.0 ± 355.6 cells at day one in each renal tissue section, but dropped to 168 ± 48.4 cells by week 6. As time increased, the numbers of LRCs were differentially expressed in the renal cortex and papilla. At the postnatal day one, nearly twice as many cells in the cortex were EdU-labeled as compared to the papilla (28.6 ± 3.6% vs. 15.6 ± 3.4%, P<0.05), while there were more LRCs within the renal papilla since the postnatal week one, and at the postnatal week 6, one third as many cells in the cortex were EdU-labeled as compared to the papilla (2.5 ± 0.1% vs. 7.7 ± 2.7%, P<0.05). The long-term LRCs at 6-week time point were associated exclusively with the glomeruli in the cortex and the renal tubules in the papilla. At 6 weeks, the EdU-labeled LRCs combined with expression of CD34, RECA-1, Nestin, and Synaptopodin were discretely but widely distributed within the glomeruli; Stro-1 around the glomeruli; and α-smooth muscle actin (SMA) in arteries. Conversely, co-expression of CD34, RECA-1, and Nestin with the long term EdU-labeled LRCs was significantly lower in renal tubules (P<0.01), while Stro-1 and Synaptopodin were not detected. Conclusion Our data found that at 6-week time point, EdU-labeled LRCs existing in the glomeruli expressed undifferentiated podocyte and endothelial markers at high rates, while those in the renal tubules expressed Nestin and vascular markers at low rates. To understand the characterization and localization of these EdU-LRCs, further studies will be needed to test cell lineage tracing, clonogenicity and differentiation potency, and the contributions to the regeneration of the kidney in response to renal injury/repair.
Collapse
Affiliation(s)
- Jianwen Wang
- Department of Urology, Beijing ChaoYang Hospital, Capital Medical University, 8 Gongtinanlu, Beijing, 100020, China
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California San Francisco, San Francisco, CA, 94143-0738, United States of America
- * E-mail:
| | - Guiting Lin
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California San Francisco, San Francisco, CA, 94143-0738, United States of America
| | - Amjad Alwaal
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California San Francisco, San Francisco, CA, 94143-0738, United States of America
| | - Xiaoyu Zhang
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California San Francisco, San Francisco, CA, 94143-0738, United States of America
| | - Guifang Wang
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California San Francisco, San Francisco, CA, 94143-0738, United States of America
| | - Xingyuan Jia
- Department of Urology, Beijing ChaoYang Hospital, Capital Medical University, 8 Gongtinanlu, Beijing, 100020, China
| | - Lia Banie
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California San Francisco, San Francisco, CA, 94143-0738, United States of America
| | - Jacqueline Villalta
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California San Francisco, San Francisco, CA, 94143-0738, United States of America
| | - Ching-Shwun Lin
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California San Francisco, San Francisco, CA, 94143-0738, United States of America
| | - Tom F. Lue
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California San Francisco, San Francisco, CA, 94143-0738, United States of America
| |
Collapse
|
|
9
|
Tsai SY, Huang YC, Chueh LL, Yeh LS, Lin CS. Intra-articular transplantation of porcine adipose-derived stem cells for the treatment of canine osteoarthritis: A pilot study. World J Transplant 2014; 4:196-205. [PMID: 25346893 PMCID: PMC4208083 DOI: 10.5500/wjt.v4.i3.196] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/21/2013] [Revised: 03/16/2014] [Accepted: 06/27/2014] [Indexed: 02/05/2023] Open
Abstract
AIM: To test whether intra-articular injection of porcine adipose-derived stem cells (ADSCs) can treat canine osteoarthritis (OA).
METHODS: To enroll in this study dogs must have stifle joint OA that had lasted ≥ 3 mo and been treated with OA medication without significant improvement. Three dogs fulfilled these criteria and were thus subjects for ADSCs treatment. ADSCs were isolated from abdominal adipose tissue of a 2-mo-old female Yorkshire pig. Their stem cell marker expression was examined by immunofluorescence staining. For treatment, 5 million ADSCs were injected into the diseased joint of each dog. In the next 48 h, the patient was observed for signs of inflammatory and allergic reactions. The patient was then discharged to the owner and, at 2, 6, and 12 wk, followed up with orthopedic assessment, owner questionnaire, X-ray imaging, and force-plate gait analysis.
RESULTS: Porcine ADSCs expressed mesenchymal stem cell markers CD90 and CD105. Injection of porcine ADSCs into canine stifle joints did not cause any inflammatory or allergic reactions. Orthopedic evaluation found improvements in two dogs, particularly at the longest time point. Owners’ evaluation found increased capacity and decreased pain in all three dogs’ activities such as walking and running. Radiographic evaluation did not find statistically significant differences before and after treatment. Force-plate analysis found significant improvements in all three dogs after treatment.
CONCLUSION: Xenotransplantation of ADSCs for the treatment of OA is feasible. Further studies are needed to validate this novel treatment modality, which can then be implemented for the routine treatment of OA in veterinary medicine.
Collapse
|
|
10
|
Conversion of adipose-derived stem cells into natural killer-like cells with anti-tumor activities in nude mice. PLoS One 2014; 9:e106246. [PMID: 25162225 PMCID: PMC4146612 DOI: 10.1371/journal.pone.0106246] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2014] [Accepted: 07/30/2014] [Indexed: 01/29/2023] Open
Abstract
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.
Collapse
|
|
11
|
Baer PC. Adipose-derived mesenchymal stromal/stem cells: An update on their phenotype in vivo and in vitro. World J Stem Cells 2014; 6:256-265. [PMID: 25126376 PMCID: PMC4131268 DOI: 10.4252/wjsc.v6.i3.256] [Citation(s) in RCA: 131] [Impact Index Per Article: 11.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/26/2013] [Revised: 03/10/2014] [Accepted: 06/16/2014] [Indexed: 02/06/2023] Open
Abstract
Adipose tissue is a rich, ubiquitous and easily accessible source for multipotent stromal/stem cells and has, therefore, several advantages compared to other sources of mesenchymal stromal/stem cells. Several studies have tried to identify the origin of the stromal/stem cell population within adipose tissue in situ. This is a complicated attempt because no marker has currently been described which unambiguously identifies native adipose-derived stromal/stem cells (ASCs). Isolated and cultured ASCs are a non-uniform preparation consisting of several subsets of stem and precursor cells. Cultured ASCs are characterized by their expression of a panel of markers (and the absence of others), whereas their in vitro phenotype is dynamic. Some markers were expressed de novo during culture, the expression of some markers is lost. For a long time, CD34 expression was solely used to characterize haematopoietic stem and progenitor cells, but now it has become evident that it is also a potential marker to identify an ASC subpopulation in situ and after a short culture time. Nevertheless, long-term cultured ASCs do not express CD34, perhaps due to the artificial environment. This review gives an update of the recently published data on the origin and phenotype of ASCs both in vivo and in vitro. In addition, the composition of ASCs (or their subpopulations) seems to vary between different laboratories and preparations. This heterogeneity of ASC preparations may result from different reasons. One of the main problems in comparing results from different laboratories is the lack of a standardized isolation and culture protocol for ASCs. Since many aspects of ASCs, such as the differential potential or the current use in clinical trials, are fully described in other recent reviews, this review further updates the more basic research issues concerning ASCs’ subpopulations, heterogeneity and culture standardization.
Collapse
|
|
12
|
Abstract
Stem cell (SC) therapy for erectile dysfunction (ED) has been investigated in 35 published studies, with one being a small-scale clinical trial. Out of these 35 studies, 19 are concerned with cavernous nerve (CN) injury-associated ED while 10 with diabetes mellitus- (DM-) associated ED. Adipose-derived SCs (ADSCs) were employed in 18 studies while bone marrow SCs (BMSCs) in 9. Transplantation of SCs was done mostly by intracavernous (IC) injection, as seen in 25 studies. Allogeneic and xenogeneic transplantations have increasingly been performed but their immune-incompatibility issues were rarely discussed. More recent studies also tend to use combinatory therapies by modifying or supplementing SCs with angiogenic or neurotrophic genes or proteins. All studies reported better erectile function with SC transplantation, and the majority also reported improved muscle, endothelium, and/or nerve in the erectile tissue. However, differentiation or engraftment of transplanted SCs has rarely been observed; thus, paracrine action is generally believed to be responsible for SC’s therapeutic effects. But still, few studies actually investigated and none proved paracrine action as a therapeutic mechanism. Thus, based exclusively on functional outcome data shown in preclinical studies, two clinical trials are currently recruiting patients for treatment with IC injection of ADSC and BMSC, respectively.
Collapse
|
|
13
|
Lin CS, Ning H, Lin G, Lue TF. Is CD34 truly a negative marker for mesenchymal stromal cells? Cytotherapy 2013; 14:1159-63. [PMID: 23066784 DOI: 10.3109/14653249.2012.729817] [Citation(s) in RCA: 165] [Impact Index Per Article: 13.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
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.
Collapse
Affiliation(s)
- Ching-Shwun Lin
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California , San Francisco, California 94143 – 0738, USA.
| | | | | | | |
Collapse
|
|
14
|
Pericytes derived from adipose-derived stem cells protect against retinal vasculopathy. PLoS One 2013; 8:e65691. [PMID: 23741506 PMCID: PMC3669216 DOI: 10.1371/journal.pone.0065691] [Citation(s) in RCA: 110] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2012] [Accepted: 04/26/2013] [Indexed: 12/21/2022] Open
Abstract
Background Retinal vasculopathies, including diabetic retinopathy (DR), threaten the vision of over 100 million people. Retinal pericytes are critical for microvascular control, supporting retinal endothelial cells via direct contact and paracrine mechanisms. With pericyte death or loss, endothelial dysfunction ensues, resulting in hypoxic insult, pathologic angiogenesis, and ultimately blindness. Adipose-derived stem cells (ASCs) differentiate into pericytes, suggesting they may be useful as a protective and regenerative cellular therapy for retinal vascular disease. In this study, we examine the ability of ASCs to differentiate into pericytes that can stabilize retinal vessels in multiple pre-clinical models of retinal vasculopathy. Methodology/Principal Findings We found that ASCs express pericyte-specific markers in vitro. When injected intravitreally into the murine eye subjected to oxygen-induced retinopathy (OIR), ASCs were capable of migrating to and integrating with the retinal vasculature. Integrated ASCs maintained marker expression and pericyte-like morphology in vivo for at least 2 months. ASCs injected after OIR vessel destabilization and ablation enhanced vessel regrowth (16% reduction in avascular area). ASCs injected intravitreally before OIR vessel destabilization prevented retinal capillary dropout (53% reduction). Treatment of ASCs with transforming growth factor beta (TGF-β1) enhanced hASC pericyte function, in a manner similar to native retinal pericytes, with increased marker expression of smooth muscle actin, cellular contractility, endothelial stabilization, and microvascular protection in OIR. Finally, injected ASCs prevented capillary loss in the diabetic retinopathic Akimba mouse (79% reduction 2 months after injection). Conclusions/Significance ASC-derived pericytes can integrate with retinal vasculature, adopting both pericyte morphology and marker expression, and provide functional vascular protection in multiple murine models of retinal vasculopathy. The pericyte phenotype demonstrated by ASCs is enhanced with TGF-β1 treatment, as seen with native retinal pericytes. ASCs may represent an innovative cellular therapy for protection against and repair of DR and other retinal vascular diseases.
Collapse
|
|
15
|
Lin CS, Xin ZC, Dai J, Lue TF. Commonly used mesenchymal stem cell markers and tracking labels: Limitations and challenges. Histol Histopathol 2013; 28:1109-16. [PMID: 23588700 DOI: 10.14670/hh-28.1109] [Citation(s) in RCA: 91] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Early observations that cultured mesenchymal stem cells (MSCs) could be induced to exhibit certain characteristics of osteocytes and chondrocytes led to the proposal that they could be transplanted for tissue repair through cellular differentiation. Therefore, many subsequent preclinical studies with transplanted MSCs have strived to demonstrate that cellular differentiation was the underlying mechanism for the therapeutic effect. These studies generally followed the minimal criteria set by The International Society for Cellular Therapy in assuring MSC identity by using CD70, CD90, and CD105 as positive markers and CD34 as a negative marker. However, the three positive markers are co-expressed in a wide variety of cells, and therefore, even when used in combination, they are certainly incapable of identifying MSCs in vivo. Another frequently used MSC marker, Stro-1, has been shown to be an endothelial antigen and whether it can identify MSCs in vivo remains unknown. On the other hand, the proposed negative marker CD34 has increasingly been shown to be expressed in native MSCs, such as in the adipose tissue. It has also helped establish that MSCs are likely vascular stem cells (VSCs) that reside in the capillaries and in the adventitia of larger blood vessels. These cells do not express CD31, CD104b, or α-SMA, and therefore are designated as CD34+CD31-CD140b-SMA-. Many preclinical MSC transplantation studies have also attempted to demonstrate cellular differentiation by using labeled MSCs. However, all commonly used labels have shortcomings that often complicate data interpretation. The β-gal (LacZ) gene as a label is problematic because many mammalian tissues have endogenous β-gal activities. The GFP gene is similarly problematic because many mammalian tissues are endogenously fluorescent. The cell membrane label DiI can be adsorbed by host cells, and nuclear stains Hoechst dyes and DAPI can be transferred to host cells. Thymidine analog BrdU is associated with loss of cellular protein antigenicity due to harsh histological conditions. Newer thymidine analog EdU is easier to detect by chemical reaction to azide-conjugated Alexa fluors, but certain bone marrow cells are reactive to these fluors in the absence of EdU. These caveats need to be taken into consideration when designing or interpreting MSC transplantation experiments.
Collapse
Affiliation(s)
- Ching-Shwun Lin
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, California 94143-0738, USA.
| | | | | | | |
Collapse
|
|
16
|
Abstract
Mesenchymal stem cells (MSCs) exist in most adult tissues and have been located near or within blood vessels. Although "perivascular" has been commonly used to describe such locations, increasing evidence points at the vessel wall as the exact location. Thus, "vascular stem cells (VSCs)" is recommended as a more accurate term for MSCs. Furthermore, 2 cell populations, namely pericytes and adventitial progenitor cells (APCs), are the likely VSCs. The pericyte evidence relies on the so-called pericyte-specific markers, but none of these markers is pericyte specific. In addition, pericytes appear to be too functionally diverse and sophisticated to have a large differentiation capacity. On the other hand, APCs are more naïve functionally and, therefore, more akin to being VSCs. In vitro, these cells spontaneously differentiate into pericytes, and can be induced to differentiate into vascular cells (endothelial and smooth muscle cells) and mesenchymal cells (e.g., bone, cartilage, and fat). In vivo, indirect evidence also points to their ability to differentiate into mesenchymal cells of their native tissue (e.g., fat). Moreover, they possess a large paracrine capacity and, therefore, can help maintain tissue homeostasis by encouraging the replication and differentiation of mesenchymal cells locally. These proposed in vivo functions are areas of interest for future research on VSCs.
Collapse
Affiliation(s)
- Ching-Shwun Lin
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, California 94143-0738, USA.
| | | |
Collapse
|
|
17
|
Ning H, Albersen M, Lin G, Lue TF, Lin CS. Effects of EdU labeling on mesenchymal stem cells. Cytotherapy 2013; 15:57-63. [PMID: 23260086 PMCID: PMC3535288 DOI: 10.1016/j.jcyt.2012.10.010] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2012] [Accepted: 07/27/2012] [Indexed: 12/11/2022]
Abstract
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 β-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.
Collapse
Affiliation(s)
- Hongxiu Ning
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, CA 94143-0738, USA
| | - Maarten Albersen
- Department of Urology, University Hospitals Leuven, Leuven, Belgium
| | - Guiting Lin
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, CA 94143-0738, USA
| | - Tom F. Lue
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, CA 94143-0738, USA
| | - Ching-Shwun Lin
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, CA 94143-0738, USA
| |
Collapse
|
|
18
|
Qiu X, Lin G, Xin Z, Ferretti L, Zhang H, Lue TF, Lin CS. Effects of low-energy shockwave therapy on the erectile function and tissue of a diabetic rat model. J Sex Med 2012; 10:738-46. [PMID: 23253086 DOI: 10.1111/jsm.12024] [Citation(s) in RCA: 127] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
Introduction. Low-energy shockwave therapy (LESWT) has been shown to improve erectile function in patients suffering from diabetes mellitus (DM)-associated erectile dysfunction (ED). However, the underlying mechanism remains unknown. Aim. The aim of this study is to investigate whether LESWT can ameliorate DM-associated ED in a rat model and examine the associated changes in the erectile tissues. Methods. Newborn male rats were intraperitoneally injected with 5-ethynyl-2-deoxyuridine (EdU; 50 mg/kg) for the purpose of tracking endogenous mesenchymal stem cells (MSCs). Eight weeks later, eight of these rats were randomly chosen to serve as normal control (N group). The remaining rats were injected intraperitoneally with 60 mg/kg of streptozotocin (STZ) to induce DM. Eight of these rats were randomly chosen to serve as DM control (DM group), whereas another eight rats were subject to shockwave (SW) treatment (DM+SW group). Each rat in the DM+SW group received 300 shocks at energy level of 0.1 mJ/mm(2) and frequency of 120/minute. This procedure was repeated three times a week for 2 weeks. Another 2 weeks later, all 24 rats were evaluated for erectile function by intracavernous pressure (ICP) measurement. Afterward, their penile tissues were examined by histology. Main Outcome Measures. Erectile function was measured by ICP. Neuronal nitric oxide synthase (nNOS)-positive nerves and the endothelium were examined by immunofluorescence staining. Smooth muscle and MSCs were examined by phalloidin and EdU staining, respectively. Results. STZ treatment caused a significant decrease in erectile function and in the number of nNOS-positive nerves and in endothelial and smooth muscle contents. These DM-associated deficits were all partially but significantly reversed by LESWT. MSCs (EdU-positive cells) were significantly more numerous in DM+SW than in DM rats. Conclusion. LESWT can partially ameliorate DM-associated ED by promoting regeneration of nNOS-positive nerves, endothelium, and smooth muscle in the penis. These beneficial effects appear to be mediated by recruitment of endogenous MSCs. Qiu X, Lin G, Xin Z, Ferretti L, Zhang H, Lue TF, and Lin C-S. Effects of low-energy shockwave therapy on the erectile function and tissue of a diabetic rat model. J Sex Med 2013;10:738-746.
Collapse
Affiliation(s)
- Xuefeng Qiu
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, CA 94143-0738, USA
| | | | | | | | | | | | | |
Collapse
|
|
19
|
Lin G, Ning H, Banie L, Qiu X, Zhang H, Lue TF, Lin CS. Bone marrow cells stained by azide-conjugated Alexa fluors in the absence of an alkyne label. Stem Cells Dev 2012; 21:2552-9. [PMID: 22380729 DOI: 10.1089/scd.2012.0092] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
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.
Collapse
Affiliation(s)
- Guiting Lin
- Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, California 94143-0738, USA
| | | | | | | | | | | | | |
Collapse
|