scholarly journals Co-Transplantation of Bone Marrow-MSCs and Myogenic Stem/Progenitor Cells from Adult Donors Improves Muscle Function of Patients with Duchenne Muscular Dystrophy

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1119
Author(s):  
Aleksandra Klimczak ◽  
Agnieszka Zimna ◽  
Agnieszka Malcher ◽  
Urszula Kozlowska ◽  
Katarzyna Futoma ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Bone Marrow ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Progenitor Cells ◽  
Muscle Function ◽  
Progenitor Cell ◽  
Genetic Disorder ◽  
Cell Populations ◽  
Muscle Fibres

Duchenne muscular dystrophy (DMD) is a genetic disorder associated with a progressive deficiency of dystrophin that leads to skeletal muscle degeneration. In this study, we tested the hypothesis that a co-transplantation of two stem/progenitor cell populations, namely bone marrow-derived mesenchymal stem cells (BM-MSCs) and skeletal muscle-derived stem/progenitor cells (SM-SPCs), directly into the dystrophic muscle can improve the skeletal muscle function of DMD patients. Three patients diagnosed with DMD, confirmed by the dystrophin gene mutation, were enrolled into a study approved by the local Bioethics Committee (no. 79/2015). Stem/progenitor cells collected from bone marrow and skeletal muscles of related healthy donors, based on HLA matched antigens, were expanded in a closed MC3 cell culture system. A simultaneous co-transplantation of BM-MSCs and SM-SPCs was performed directly into the biceps brachii (two patients) and gastrocnemius (one patient). During a six-month follow-up, the patients were examined with electromyography (EMG) and monitored for blood kinase creatine level. Muscle biopsies were examined with histology and assessed for dystrophin at the mRNA and protein level. A panel of 27 cytokines was analysed with multiplex ELISA. We did not observe any adverse effects after the intramuscular administration of cells. The efficacy of BM-MSC and SM-SPC application was confirmed through an EMG assessment by an increase in motor unit parameters, especially in terms of duration, amplitude range, area, and size index. The beneficial effect of cellular therapy was confirmed by a decrease in creatine kinase levels and a normalised profile of pro-inflammatory cytokines. BM-MSCs may support the pro-regenerative potential of SM-SPCs thanks to their trophic, paracrine, and immunomodulatory activity. Both applied cell populations may fuse with degenerating skeletal muscle fibres in situ, facilitating skeletal muscle recovery. However, further studies are required to optimise the dose and timing of stem/progenitor cell delivery.

Bone Marrow Niches for Skeletal Progenitor Cells and their Inhabitants in Health and Disease

2019 ◽  
Vol 14 (4) ◽  
pp. 305-319 ◽  
Author(s):  
Marietta Herrmann ◽  
Franz Jakob
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Adult Stem Cells ◽  
Current Knowledge ◽  
Cell Populations ◽  
Surface Markers ◽  
Bone Marrow Niches

The bone marrow hosts skeletal progenitor cells which have most widely been referred to as Mesenchymal Stem or Stromal Cells (MSCs), a heterogeneous population of adult stem cells possessing the potential for self-renewal and multilineage differentiation. A consensus agreement on minimal criteria has been suggested to define MSCs in vitro, including adhesion to plastic, expression of typical surface markers and the ability to differentiate towards the adipogenic, osteogenic and chondrogenic lineages but they are critically discussed since the differentiation capability of cells could not always be confirmed by stringent assays in vivo. However, these in vitro characteristics have led to the notion that progenitor cell populations, similar to MSCs in bone marrow, reside in various tissues. MSCs are in the focus of numerous (pre)clinical studies on tissue regeneration and repair.Recent advances in terms of genetic animal models enabled a couple of studies targeting skeletal progenitor cells in vivo. Accordingly, different skeletal progenitor cell populations could be identified by the expression of surface markers including nestin and leptin receptor. While there are still issues with the identity of, and the overlap between different cell populations, these studies suggested that specific microenvironments, referred to as niches, host and maintain skeletal progenitor cells in the bone marrow. Dynamic mutual interactions through biological and physical cues between niche constituting cells and niche inhabitants control dormancy, symmetric and asymmetric cell division and lineage commitment. Niche constituting cells, inhabitant cells and their extracellular matrix are subject to influences of aging and disease e.g. via cellular modulators. Protective niches can be hijacked and abused by metastasizing tumor cells, and may even be adapted via mutual education. Here, we summarize the current knowledge on bone marrow skeletal progenitor cell niches in physiology and pathophysiology. We discuss the plasticity and dynamics of bone marrow niches as well as future perspectives of targeting niches for therapeutic strategies.

Mass Cytometric Analysis of AML Stem and Early Progenitor Cells Reveals Karyotype and Genotype-Specific Cell Cycle Properties That Correlate with Known Responses to Chemotherapy

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2359-2359
Author(s):  
Gregory K. Behbehani ◽  
Wendy J. Fantl ◽  
Bruno C Medeiros ◽  
Garry P. Nolan
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
S Phase ◽  
The Other ◽  
Cytotoxic Agents ◽  
Phase Fraction ◽  
Cell Populations

Abstract Introduction: Leukemic stem cells (LSCs) are recognized as important mediators of chemotherapy resistance and leukemia relapse. The postulated mechanism for this is the relative quiescence of these cell populations that renders them resistant to cytotoxic agents. This simple hypothesis, however, is supported almost entirely by indirect evidence, and fails to explain the large differences in relapse rates across different AML subtypes. To address this question, we have developed a mass cytometry (MCM) approach to assess the cell cycle of immunophenotypically complex primary samples from patients with AML. By processing samples immediately upon bone marrow harvest, we could determine if AML stem cells were quiescent in vivo and if the cell cycle properties of these cells varied between chemotherapy-responsive versus resistant AML subtypes. Methods: Bone marrow aspirates from 33 AML patients, 3 with APL, 2 with high-risk MDS, 5 with AML who achieved a CR with chemotherapy treatment, and 5 healthy donors (48 total samples) were incubated at 37°C for 15 minutes with 20uM Iodo-deoxyuridine (IdU) immediately after aspiration (<1 min), followed by fixation and storage. Samples were then analyzed with two overlapping 39-antibody MCM panels (50 markers total). Cellular barcoding was utilized to stain and analyze cells in tubes of 20 samples each, enabling direct comparison of samples to each other and to the healthy controls. Results: The high dimensionality of MCM enabled the simultaneous measurement of 25 surface markers and the identification of almost all immunophenotypic populations in human bone marrow. The use of barcoding, and the resultant ability to directly compare samples, enabled the detection of aberrant marker expression at very high resolution (2-3 fold changes). At least one surface marker aberrancy was detected in each AML sample. Unexpectedly, cell cycle analysis revealed that, compared to immunophenotypically similar normal cells, the average fraction of S-phase cells in AML samples was significantly lower. In both AML and healthy samples, the lowest S-phase fraction was found in fully differentiated populations and in hematopoietic stem cells (HSCs) while committed progenitor populations (myelo-monoblasts, promyelocytes, erythroblasts) exhibited the highest S-phase fraction. The HSC and early progenitor cell populations from patients with CBF AML (t(8;21) and inv(16)) demonstrated a significantly higher S-phase fraction than the same cell populations from the other AML samples (7.76% vs. 2.66%; p=0.0014). Furthermore, samples with FLT3-ITD mutations exhibited the lowest S-phase fraction in the HSC and early progenitor cell populations (0.63%), which was significantly lower than the S-phase fraction of the other AML samples (4.37%; p=9.3x10-4). Finally, a subset of patients (n=10) was being treated with hydroxyurea (HU) at the time of their bone marrow aspiration. The effect of HU treatment was manifest as a reduction in the IdU incorporation rate (with no change in S-phase fraction) in the cells of the treated patients. However, neither cell cycle arrest nor apoptosis were observed in these samples. This is in contrast with the commonly observed occurrence of both in leukemic cell lines treated in vitro with HU. Conclusions: By combining fresh sample processing with high-dimensional MCM analysis, we developed an innovative approach for the analysis of hematologic malignancies. Our results suggest that the relative sensitivity of CBF AML to cytotoxic chemotherapy may be the result of the increased fraction of S-phase cells within the HSC and early progenitor cell populations. Conversely, HSC and early progenitor cell populations from patients with FLT3-ITD mutations would be expected to be particularly resistant to cytarabine-based consolidation therapy due to the very low frequency of S-phase cells within these populations. This finding, combined with our observation that the stem and early progenitor cells from the FLT3-ITD samples have high expression of CD33, may provide a mechanistic explanation for the improved disease-free survival recently reported for FLT3-ITD AML patients treated with fractioned gemtuzumab ozogamicin in combination with standard therapy. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures Behbehani: Fluidigm: Consultancy. Medeiros:Agios: Consulting - Ad board Other. Nolan:Fluidigm, Inc: Consultancy, Equity Ownership.

scholarly journals DOCK3 is a dosage-sensitive regulator of skeletal muscle and Duchenne muscular dystrophy-associated pathologies

2020 ◽  
Vol 29 (17) ◽  
pp. 2855-2871
Author(s):  
Andrea L Reid ◽  
Yimin Wang ◽  
Adrienne Samani ◽  
Rylie M Hightower ◽  
Michael A Lopez ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Muscle Function ◽  
Myogenic Differentiation ◽  
Nucleotide Exchange ◽  
Dystrophic Muscle ◽  
Guanine Nucleotide Exchange ◽  
Myogenic Factors

Abstract DOCK3 is a member of the DOCK family of guanine nucleotide exchange factors that regulate cell migration, fusion and viability. Previously, we identified a dysregulated miR-486/DOCK3 signaling cascade in dystrophin-deficient muscle, which resulted in the overexpression of DOCK3; however, little is known about the role of DOCK3 in muscle. Here, we characterize the functional role of DOCK3 in normal and dystrophic skeletal muscle. Utilizing Dock3 global knockout (Dock3 KO) mice, we found that the haploinsufficiency of Dock3 in Duchenne muscular dystrophy mice improved dystrophic muscle pathologies; however, complete loss of Dock3 worsened muscle function. Adult Dock3 KO mice have impaired muscle function and Dock3 KO myoblasts are defective for myogenic differentiation. Transcriptomic analyses of Dock3 KO muscles reveal a decrease in myogenic factors and pathways involved in muscle differentiation. These studies identify DOCK3 as a novel modulator of muscle health and may yield therapeutic targets for treating dystrophic muscle symptoms.

Hematopoietic stem cell transplantation does not restore dystrophin expression in Duchenne muscular dystrophy dogs

Blood ◽  
2004 ◽  
Vol 104 (13) ◽  
pp. 4311-4318 ◽  
Author(s):  
Chiara Dell'Agnola ◽  
Zejing Wang ◽  
Rainer Storb ◽  
Stephen J. Tapscott ◽  
Christian S. Kuhr ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Bone Marrow ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Cell Transplantation ◽  
Hematopoietic Cell ◽  
Mdx Mouse ◽  
Hematopoietic Stem ◽  
Bone Marrow Derived Cells ◽  
Donor Muscle

Abstract Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene on the X-chromosome that result in skeletal and cardiac muscle damage and premature death. Studies in mice, including the mdx mouse model of DMD, have demonstrated that circulating bone marrow–derived cells can participate in skeletal muscle regeneration, but the potential clinical utility of treating human DMD by allogeneic marrow transplantation from a healthy donor remains unknown. To assess whether allogeneic hematopoietic cell transplantation (HCT) provides clinically relevant levels of donor muscle cell contribution in dogs with canine X-linked muscular dystrophy (c-xmd), 7 xmd dogs were given hematopoietic cell (HC) transplants from nonaffected littermates. Compared with the pretransplantation baseline, the number of dystrophin-positive fibers and the amount of wild-type dystrophin RNA did not increase after HCT, with observation periods ranging from 28 to 417 days. Similar results were obtained when the recipient dogs were given granulocyte colony-stimulating factor (G-CSF) after their initial transplantation to mobilize the cells. Despite successful allogeneic HCT and a permissive environment for donor muscle engraftment, there was no detectable contribution of bone marrow–derived cells to either skeletal muscle or muscle precursor cells assayed by clonal analyses at a level of sensitivity that should detect as little as 0.1% donor contribution.

Caveolin-3 in skeletal muscle fibres of Duchenne muscular dystrophy and mdx mouse

1997 ◽  
Vol 7 (6-7) ◽  
pp. 436
Author(s):  
Y. Hagiwara ◽  
Y. Nishina ◽  
M. Imamura ◽  
M. Yoshida ◽  
T. Kikuchi ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Mdx Mouse ◽  
Muscle Fibres ◽  
Skeletal Muscle Fibres

scholarly journals Assessing the Use of the sGC Stimulator BAY-747, as a Potential Treatment for Duchenne Muscular Dystrophy

2021 ◽  
Vol 22 (15) ◽  
pp. 8016
Author(s):  
Shalini Murali Krishnan ◽  
Johannes Nordlohne ◽  
Lisa Dietz ◽  
Alexandros Vakalopoulos ◽  
Petra Haning ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Grip Strength ◽  
Muscle Function ◽  
Phase Iii ◽  
Potential Treatment ◽  
Skeletal Muscle Function ◽  
Therapeutic Benefits ◽  
Sgc Stimulators

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder, affecting one in 3500 to 5000 boys worldwide. The NO-sGC-cGMP pathway plays an important role in skeletal muscle function, primarily by improving blood flow and oxygen supply to the muscles during exercise. In fact, PDE5 inhibitors have previously been investigated as a potential therapy for DMD, however, a large-scale Phase III clinical trial did not meet its primary endpoint. Since the efficacy of PDE5i is dependent on sufficient endogenous NO production, which might be impaired in DMD, we investigated if NO-independent sGC stimulators, could have therapeutic benefits in a mouse model of DMD. Male mdx/mTRG2 mice aged six weeks were given food supplemented with the sGC stimulator, BAY-747 (150 mg/kg of food) or food alone (untreated) ad libitum for 16 weeks. Untreated C57BL6/J mice were used as wild type (WT) controls. Assessments of the four-limb hang, grip strength, running wheel and serum creatine kinase (CK) levels showed that mdx/mTRG2 mice had significantly reduced skeletal muscle function and severe muscle damage compared to WT mice. Treatment with BAY-747 improved grip strength and running speed, and these mice also had reduced CK levels compared to untreated mdx/mTRG2 mice. We also observed increased inflammation and fibrosis in the skeletal muscle of mdx/mTRG2 mice compared to WT. While gene expression of pro-inflammatory cytokines and some pro-fibrotic markers in the skeletal muscle was reduced following BAY-747 treatment, there was no reduction in infiltration of myeloid immune cells nor collagen deposition. In conclusion, treatment with BAY-747 significantly improves several functional and pathological parameters of the skeletal muscle in mdx/mTRG2 mice. However, the effect size was moderate and therefore, more studies are needed to fully understand the potential treatment benefit of sGC stimulators in DMD.

scholarly journals Changes in Myonuclear Number During Postnatal Growth –Implications for AAV Gene Therapy for Muscular Dystrophy

2021 ◽  
pp. 1-8
Author(s):  
Jennifer Morgan ◽  
Francesco Muntoni
Keyword(s):  
Skeletal Muscle ◽  
Gene Therapy ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Muscle Growth ◽  
Postnatal Growth ◽  
Muscle Fibres ◽  
Postnatal Life ◽  
Adult Skeletal Muscle ◽  
Dividing Cells

Adult skeletal muscle is a relatively stable tissue, as the multinucleated muscle fibres contain post-mitotic myonuclei. During early postnatal life, muscle growth occurs by the addition of skeletal muscle stem cells (satellite cells) or their progeny to growing muscle fibres. In Duchenne muscular dystrophy, which we shall use as an example of muscular dystrophies, the muscle fibres lack dystrophin and undergo necrosis. Satellite-cell mediated regeneration occurs, to repair and replace the necrotic muscle fibres, but as the regenerated muscle fibres still lack dystrophin, they undergo further cycles of degeneration and regeneration. AAV gene therapy is a promising approach for treating Duchenne muscular dystrophy. But for a single dose of, for example, AAV coding for dystrophin, to be effective, the treated myonuclei must persist, produce sufficient dystrophin and a sufficient number of nuclei must be targeted. This latter point is crucial as AAV vector remains episomal and does not replicate in dividing cells. Here, we describe and compare the growth of skeletal muscle in rodents and in humans and discuss the evidence that myofibre necrosis and regeneration leads to the loss of viral genomes within skeletal muscle. In addition, muscle growth is expected to lead to the dilution of the transduced nuclei especially in case of very early intervention, but it is not clear if growth could result in insufficient dystrophin to prevent muscle fibre breakdown. This should be the focus of future studies.

Abstract 15440: Dynamics of Adipocyte and Endothelial Progenitor Cell Pools in Expanding Adipose Tissue

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Candice R Holden ◽  
Marcin Wysoczynski ◽  
Brian Sansbury ◽  
Jason Hellmann ◽  
Nagma Zafar ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Adipose Tissue ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Stromal Vascular Fraction ◽  
Cell Populations ◽  
Adipose Organ ◽  
The Impact

Objective: Obesity is a major risk factor for the development of several chronic diseases including type 2 diabetes and cardiovascular disease. Proper fat storage in white adipose tissue (WAT) is required to maintain insulin sensitivity and to preserve (cardio)vascular health. We hypothesize that endothelial and adipocyte progenitor cell populations (EPCs and APCs, respectively) must be appropriately balanced for physiological, as opposed to pathological, remodeling of WAT. Methods and Results: To determine the impact of nutrient excess on stem/progenitor cells in epididymal WAT, male C57BL/6J mice were placed on a high fat diet (HFD; 60% fat) for 12 weeks and changes in WAT stem cell populations were measured in the stromal vascular fraction by flow cytometry. Although the APC (CD24+/CD29+/Sca+/CD14-/CD45-) population, which has the capacity to differentiate into adipocytes both in vitro and in vivo , was not significantly changed with diet, Flk+/Sca+ EPCs were diminished, promoting a 4-fold decrease in the EPC/APC ratio (p <0.05, n = 6/group). To determine whether this deficit may be due to poor stem cell recruitment, mice were irradiated, and the bone marrow was repopulated with GFP+ donor marrow. The transplanted mice were then placed on a low fat diet (LFD; 10% fat) or HFD for 12 weeks, and WAT progenitor cells were again measured. Greater than 95% of the putative APCs in the WAT of HF-fed mice were GFP+ (p<0.0001, n=7-8/group), indicating a bone marrow-derived origin. Unexpectedly, less than 1% of the EPCs were GFP+ (p<0.001, n=7-8/group), which suggests that EPCs present in WAT are not derived from bone marrow in adult mice. Confocal analysis of WAT from HF-fed, bone marrow-transplanted mice showed little evidence of significant APC differentiation into triglyceride-laden adipocytes, suggesting that conditions associated with nutrient excess may impair the ability of the adipose organ to store fat properly. Conclusions: These results demonstrate that putative APCs, and not EPCs, in epididymal WAT are derived from bone marrow. Furthermore, our data suggest that conditions of nutrient excess promote an imbalance in EPCs and APCs, the stoichiometry of which may be critical for the development of new adipocytes and for proper storage of fat.

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