Increasing injection frequency enhances the survival of injected bone marrow derived mesenchymal stem cells in a critical limb ischemia animal model

Abstract Introduction Critical limb ischemia (CLI) is the most advanced form of peripheral arterial disease (PAD) characterized by ischemic rest pain and non-healing ulcers. Currently, the standard therapy for CLI is the surgical reconstruction and endovascular therapy or limb amputation for patients with no treatment options. Neovasculogenesis induced by mesenchymal stem cells (MSCs) therapy is a promising approach to improve CLI. Owing to their angiogenic and immunomodulatory potential, MSCs are perfect candidates for the treatment of CLI. The purpose of this study was to determine and compare the in vitro and in vivo effects of allogeneic bone marrow mesenchymal stem cells (BM-MSCs) and adipose tissue mesenchymal stem cells (AT-MSCs) on CLI treatment. Methods For the first step, BM-MSCs and AT-MSCs were isolated and characterized for the characteristic MSC phenotypes. Then, femoral artery ligation and total excision of the femoral artery were performed on C57BL/6 mice to create a CLI model. The cells were evaluated for their in vitro and in vivo biological characteristics for CLI cell therapy. In order to determine these characteristics, the following tests were performed: morphology, flow cytometry, differentiation to osteocyte and adipocyte, wound healing assay, and behavioral tests including Tarlov, Ischemia, Modified ischemia, Function and the grade of limb necrosis scores, donor cell survival assay, and histological analysis. Results Our cellular and functional tests indicated that during 28 days after cell transplantation, BM-MSCs had a great effect on endothelial cell migration, muscle restructure, functional improvements, and neovascularization in ischemic tissues compared with AT-MSCs and control groups. Conclusions Allogeneic BM-MSC transplantation resulted in a more effective recovery from critical limb ischemia compared to AT-MSCs transplantation. In fact, BM-MSC transplantation could be considered as a promising therapy for diseases with insufficient angiogenesis including hindlimb ischemia.

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Characterization of Mesenchymal Stem Cells of “No-Options” Patients with Critical Limb Ischemia Treated by Autologous Bone Marrow Mononuclear Cells

PLoS ONE ◽

10.1371/journal.pone.0073722 ◽

2013 ◽

Vol 8 (9) ◽

pp. e73722 ◽

Cited By ~ 27

Author(s):

Cestmir Altaner ◽

Veronika Altanerova ◽

Marina Cihova ◽

Lubica Hunakova ◽

Katarina Kaiserova ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Critical Limb Ischemia ◽

Mononuclear Cells ◽

Autologous Bone Marrow ◽

Limb Ischemia ◽

Bone Marrow Mononuclear Cells ◽

Autologous Bone

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Abstract 231: Bone Marrow-Derived Mesenchymal Stem Cells from Amputated Limbs of Patients with Critical Limb Ischemia Provide Stromal Support for Endothelial Cells

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.33.suppl_1.a231 ◽

2013 ◽

Vol 33 (suppl_1) ◽

Author(s):

Ruoya Wang ◽

Ian Copland ◽

Haiyan Li ◽

Alexandra Peister ◽

Todd McDevitt ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Critical Limb Ischemia ◽

Healthy Donor ◽

Cellular Therapy ◽

Limb Ischemia ◽

Growth Media ◽

Stromal Support ◽

The Difference

Critical limb ischemia (CLI) often results in amputation, but autologous cellular therapy using bone marrow-derived mesenchymal stem cells (MSCs) may be able to prevent this outcome. However, the quality of MSCs in the CLI population is not clear. Amputated CLI limbs contain reservoirs of MSCs that can be used to characterize their stromal capacity. We hypothesize that MSC stromal capacity is relatively preserved in ischemic limbs compared to ones from healthy donors. Methods Healthy donor MSCs (hdMSCs, n=2) and ischemic MSCs (iMSCs, n=3) from amputated limbs of non-diabetic CLI patients were cultured. Human microdermal ECs were commercially obtained. Co-culture pellets (1:1 ratio) of hdMSC:EC, iMSC:EC, and EC alone were formed and cultured in 3-D fibrin hydrogels in EC growth media. Images of the pellets and invasion areas were captured up to 5 days. The invasion area was quantified as the difference between the total and pellet area normalized to initial pellet area. Experiments were performed in quadruplicates and co-culture experiments were duplicated. Results iMSC:EC co-culture provided similar stromal support as the healthy donor cohort. Both MSC groups were significantly greater than the ECs alone following day 2 (see figure, *P<0.0005, ANOVA). Both MSC groups also exhibited significant increase in invasion area with time (P<0.01) while the ECs did not (P>0.5). Conclusion This is the first study to demonstrate that iMSCs provide similar stromal support to ECs as healthy hdMSCs, thus iMSCs may be of sufficient quality for use in autologous therapy. We are currently quantifying secretome expressions of the iMSCs to provide insights into mechanisms of the stromal support.

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Deciphering the in vivo Dynamic Proteomics of Mesenchymal Stem Cells in Critical Limb Ischemia

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.682476 ◽

2021 ◽

Vol 9 ◽

Author(s):

Yipeng Du ◽

Xiaoting Li ◽

Wenying Yan ◽

Zhaohua Zeng ◽

Dunzheng Han ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Protein Synthesis ◽

Critical Limb Ischemia ◽

Click Reaction ◽

Limb Ischemia ◽

Trna Synthetase ◽

Doppler Imaging

ObjectiveRegenerative therapy using mesenchymal stem cells (MSC) is a promising therapeutic method for critical limb ischemia (CLI). To understand how the cells are involved in the regenerative process of limb ischemia locally, we proposed a metabolic protein labeling method to label cell proteomes in situ and then decipher the proteome dynamics of MSCs in ischemic hind limb.Methods and ResultsIn this study, we overexpressed mutant methionyl-tRNA synthetase (MetRS), which could utilize azidonorleucine (ANL) instead of methionine (Met) during protein synthesis in MSCs. Fluorescent non-canonical amino-acid tagging (FUNCAT) was performed to detect the utilization of ANL in mutant MSCs. Mice with hindlimb ischemia (HLI) or Sham surgery were treated with MetRSmut MSCs or PBS, followed by i.p. administration of ANL at days 0, 2 6, and 13 after surgery. FUNCAT was also performed in hindlimb tissue sections to demonstrate the incorporation of ANL in transplanted cells in situ. At days 1, 3, 7, and 14 after the surgery, laser doppler imaging were performed to detect the blood reperfusion of ischemic limbs. Ischemic tissues were also collected at these four time points for histological analysis including HE staining and vessel staining, and processed for click reaction based protein enrichment followed by mass spectrometry and bioinformatics analysis. The MetRSmut MSCs showed strong green signal in cell culture and in HLI muscles as well, indicating efficient incorporation of ANL in nascent protein synthesis. By 14 days post-treatment, MSCs significantly increased blood reperfusion and vessel density, while reducing inflammation in HLI model compared to PBS. Proteins enriched by click reaction were distinctive in the HLI group vs. the Sham group. 34, 31, 49, and 26 proteins were significantly up-regulated whereas 28, 32, 62, and 27 proteins were significantly down-regulated in HLI vs. Sham at days 1, 3, 7, and 14, respectively. The differentially expressed proteins were more pronounced in the pathways of apoptosis and energy metabolism.ConclusionIn conclusion, mutant MetRS allows efficient and specific identification of dynamic cell proteomics in situ, which reflect the functions and adaptive changes of MSCs that may be leveraged to understand and improve stem cell therapy in critical limb ischemia.

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Abstract No. 175: Endovascular Treatment of Critical Limb Ischemia by Autologous Bone-Marrow Derived Stem Cells

Journal of Vascular and Interventional Radiology ◽

10.1016/j.jvir.2007.12.194 ◽

2008 ◽

Vol 19 (2) ◽

pp. S67

Author(s):

S. Sharma ◽

G.S. Gulati ◽

P. Jagia ◽

V.K. Sheorain ◽

S. Mohanty

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Endovascular Treatment ◽

Critical Limb Ischemia ◽

Autologous Bone Marrow ◽

Limb Ischemia ◽

Autologous Bone

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Brazilian minipig as a large-animal model for basic research and stem cell-based tissue engineering. Characterization and in vitro differentiation of bone marrow-derived mesenchymal stem cells

Journal of Applied Oral Science ◽

10.1590/1678-775720130526 ◽

2014 ◽

Vol 22 (3) ◽

pp. 218-227 ◽

Cited By ~ 8

Author(s):

Roberta Targa STRAMANDINOLI-ZANICOTTI ◽

André Lopes CARVALHO ◽

Carmen Lúcia Kuniyoshi REBELATTO ◽

Laurindo Moacir SASSI ◽

Maria Fernanda TORRES ◽

...

Keyword(s):

Tissue Engineering ◽

Stem Cells ◽

Bone Marrow ◽

Animal Model ◽

Mesenchymal Stem Cells ◽

Basic Research ◽

Large Animal Model ◽

Large Animal ◽

In Vitro Differentiation

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Feasibility of autologous mesenchymal stem cells therapy for patients with no-option critical limb ischemia

Cytotherapy ◽

10.1016/j.jcyt.2017.02.263 ◽

2017 ◽

Vol 19 (5) ◽

pp. S186-S187

Author(s):

S. Mohamed ◽

L. Howard ◽

A. Duffy ◽

A. Finnerty ◽

M. Holohan ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Critical Limb Ischemia ◽

Limb Ischemia ◽

Stem Cells Therapy ◽

Autologous Mesenchymal Stem Cells

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In vivo tracking of intravenously injected mesenchymal stem cells in an Alzheimer’s animal model

Cell Transplantation ◽

10.1177/0963689718788067 ◽

2018 ◽

Vol 27 (8) ◽

pp. 1203-1209

Author(s):

Bok-Nam Park ◽

Tae Sung Lim ◽

Joon-Kee Yoon ◽

Young-Sil An

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Animal Model ◽

Mesenchymal Stem Cells ◽

Gamma Camera ◽

The Body ◽

Control Group ◽

Indium 111 ◽

The Brain

Purpose: The purpose of this study was to investigate how intravenously injected bone marrow-derived mesenchymal stem cells (BMSCs) are distributed in the body of an Alzheimer’s disease (AD) animal model. Methods: Stem cells were collected from bone marrow of mice and labeled with Indium-111 (111In). The 111In-labeled BMSCs were infused intravenously into 3×Tg-AD mice in the AD group and non-transgenic mice (B6129SF2/J) as controls. Biodistribution was evaluated with a gamma counter and gamma camera 24 and 48 h after injecting the stem cells. Results: A gamma count of the brain showed a higher distribution of labeled cells in the AD model than in the control group at 24 (p = .0004) and 48 h (p = .0016) after injection of the BMSCs. Similar results were observed by gamma camera imaging (i.e., brain uptake in the AD model was significantly higher than that in the control group). Among the other organs, uptake by the spleen was the highest in both groups. More BMSCs were found in the lungs of the control group than in those of the AD group. Conclusions: These results suggest that more intravenously infused BMSCs reached the brain in the AD model than in the control group, but the numbers of stem cells reaching the brain was very small.

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Bone marrow stem cells for the critical limb ischemia treatment: biological aspects and clinical application

Genes and Cells ◽

10.23868/201805002 ◽

2018 ◽

Vol XIII (1) ◽

Author(s):

P. Orekhov ◽

M. Konoplyannikov ◽

V. Baklaushev ◽

V. Kalsin ◽

A. Averyanov ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Clinical Application ◽

Critical Limb Ischemia ◽

Limb Ischemia ◽

Bone Marrow Stem Cells ◽

Biological Aspects

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Abstract 182: Increased Adipose-Derived Mesenchymal Stem Cells Counts and Pro-B-Type Natriuretic Peptide in Patients With Critical Limb Ischemia

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.34.suppl_1.182 ◽

2014 ◽

Vol 34 (suppl_1) ◽

Author(s):

Houssam Farres ◽

Caroline Sutton ◽

Abba Zubair ◽

John D Dortch ◽

Albert Hakaim

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Natriuretic Peptide ◽

Critical Limb Ischemia ◽

Limb Ischemia ◽

Human Model ◽

Differentiation Potential ◽

Paracrine Effects ◽

Ischemic Group ◽

Vascular Regeneration

Mesenchymal stem cells (MSCs) have been shown to improve regeneration of injured tissues in vivo. Several in vitro studies and animal models have demonstrated improvement in MSCs paracrine effects under hypoxic conditions. Moreover, several studies suggested that the pro B-type natriuretic peptide (pro-BNP) could be involved in the stimulation of postischemic vascular regeneration. The purpose of this study was to investigate the effect of critical limb ischemia, in a human model, on in-situ adipose derived mesenchymal stem cells (ADMSCs) and to determine whether serum levels of N-terminal pro-BNP correlate with ADMSCs counts and associated paracrine effects. Lipoaspirate samples of ≥ 10mL were collected from ischemic limbs (ischemic group) and compared to control (without ischemia). MSCs were characterized by frequency, viability, differentiation potential, cytokines expression, and cell surface markers. Serum NT pro-BNP was measured as well. MSCs counts were 9-to-10-fold higher in patients with ischemic limbs (mean 7952 MSC/mL ± 542) than controls (mean 790 MSC/mL ± 65). Pro-BNP levels (1878-4757 pg/mL) were approximately 8-to-26-fold higher than in age- and sex-matched controls. Furthermore, there were positive correlations between pro-BNP levels and MSCs counts in the ischemic group. In conclusion, patients with critical limb ischemia (CLI) have higher levels of pro-BNP and MSCs counts than controls. Increased levels of pro-BNP and MSCs counts can be considered humoral and cellular surrogates of ischemia and hypoxia in patients with CLI. This supports recent studies that suggest that the increase production of peripheral BNP may be a stem cells-mediated response to stimulate angiogenesis in the ischemic skeletal muscles.

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