A Novel Human Myoblast Chimeric Cells Therapy for Restoration of Muscle Function

Siemionow, Maria; Cwykiel, Joanna; Marchese, Enza; Rafidi, George; Heydemann, Ahlke; Garcı́a, Jesús; Futoma, Katarzyna; Szilàgyi, Erzsèbet

doi:10.1097/01.tp.0000543097.21030.13

Cited by 5 publications

(2 citation statements)

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“…Next, the PKH26- and PKH67-stained UCB cells were washed and suspended in serum-free RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA), mixed in a ratio of 1:1, and fused using 500 µL of PEG 4000 solution (EMD, Burlington, MA, USA). The cell fusion procedure was performed sixteen times as previously reported ( 37 , 38 ). Fused HUDC cells were centrifuged, and suspended in DPBS-based fluorescence-activated cell sorting (FACS) buffer containing 25 mM HEPES, 2 mM ethylenediaminetetraacetic acid (EDTA) and 1% FBS in preparation for sorting (BD FACSAria TM II cell sorter, Becton Dickinson, Franklin Lakes, NJ, USA) based on PKH26-red and PKH67-green fluorescent cell labeling.…”

Section: Methodsmentioning

confidence: 99%

Novel Human Umbilical Di-Chimeric (HUDC) cell therapy for transplantation without life-long immunosuppression

Siemionow,

Cwykiel,

Chambily

et al. 2023

Stem Cell Investig

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Background Cell-based therapies are promising for tolerance induction in bone marrow (BM), solid organs, and vascularized composite allotransplantation (VCA). The toxicity of bone marrow transplantation (BMT) protocols precludes this approach from routine clinical applications. To address this problem, we developed a new therapy of Human Umbilical Di-Chimeric (HUDC) cells for tolerance induction in transplantation. This study established in vitro characterization of the created HUDC cells. Methods We performed sixteen ex vivo polyethylene glycol (PEG)-mediated fusions of human umbilical cord blood (UCB) cells from two unrelated donors. Fusion feasibility was confirmed in vitro by flow cytometry (FC) and confocal microscopy (CM). The HUDC cells’ genotype was assessed by lymphocytotoxicity test and short tandem repeat-polymerase chain reaction (STR-PCR) analysis, phenotype by FC, viability by LIVE/DEAD ® assay, and apoptosis level by Annexin V staining. We used COMET assay to assess HUDC cells’ genotoxicity after the fusion procedure. Clonogenic properties of HUDC cells were evaluated by colony forming unit (CFU) assay. Mixed lymphocyte reaction (MLR) assay assessed immunogenic and tolerogenic properties of HUDC cells. Results We confirmed the creation of HUDC cells from two unrelated human donors of UCB cells by FC and CM. Human leukocyte antigen (HLA) class I and II typing, and STR-PCR analysis of HUDC cells confirmed the presence of alleles and loci from both unrelated UCB donors (donor chimerism: 49%±8.3%, n=4). FC confirmed the hematopoietic phenotype of HUDC cells. We confirmed high HUDC cells’ viability (0.47% of dead cells) and a low apoptosis level of fused HUDC cells (15.9%) compared to positive control of PKH-stained UCB cells (20.4%) before fusion. COMET assay of HUDC cells revealed a lack of DNA damage. CFU assay confirmed clonogenic properties of HUDC cells, and MLR assay revealed a low immunogenicity of HUDC cells. Conclusions This study confirmed creation of a novel HUDC cell line by ex vivo PEG-mediated fusion of UCB cells from two unrelated donors. The unique concept of creating a HUDC cell line, representing the genotype and phenotype of both, transplant donor and the recipient, introduces a promising approach for tolerance induction in BM, solid organs, and VCA transplantation.

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Section: Methodsmentioning

confidence: 99%

Novel Human Umbilical Di-Chimeric (HUDC) cell therapy for transplantation without life-long immunosuppression

Siemionow,

Cwykiel,

Chambily

et al. 2023

Stem Cell Investig

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“…Recently, in an attempt to increase engraftment, limit rejection and restore dystrophin expression, fused myoblasts from healthy donors and DMDaffected donors were i.m. injected into mdx/scid mice [34]. SCs are able to maintain their stemness due to their selfrenewal capacity, implying that SCs are preferred over myoblasts for a successful therapy.…”

Section: Myoblasts and Satellite Cells: How The Mighty Have Fallen?mentioning

confidence: 99%

Systemic cell therapy for muscular dystrophies

Ausems

Engelen

Bokhoven

et al. 2020

Stem Cell Rev and Rep

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The intrinsic regenerative capacity of skeletal muscle makes it an excellent target for cell therapy. However, the potential of muscle tissue to renew is typically exhausted and insufficient in muscular dystrophies (MDs), a large group of heterogeneous genetic disorders showing progressive loss of skeletal muscle fibers. Cell therapy for MDs has to rely on suppletion with donor cells with high myogenic regenerative capacity. Here, we provide an overview on stem cell lineages employed for strategies in MDs, with a focus on adult stem cells and progenitor cells resident in skeletal muscle. In the early days, the potential of myoblasts and satellite cells was explored, but after disappointing clinical results the field moved to other muscle progenitor cells, each with its own advantages and disadvantages. Most recently, mesoangioblasts and pericytes have been pursued for muscle cell therapy, leading to a handful of preclinical studies and a clinical trial. The current status of (pre)clinical work for the most common forms of MD illustrates the existing challenges and bottlenecks. Besides the intrinsic properties of transplantable cells, we discuss issues relating to cell expansion and cell viability after transplantation, optimal dosage, and route and timing of administration. Since MDs are genetic conditions, autologous cell therapy and gene therapy will need to go hand-in-hand, bringing in additional complications. Finally, we discuss determinants for optimization of future clinical trials for muscle cell therapy. Joined research efforts bring hope that effective therapies for MDs are on the horizon to fulfil the unmet clinical need in patients. Graphical abstract

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Human Multi-Chimeric Cell (HMCC) Therapy as a Novel Approach for Tolerance Induction in Transplantation

Siemionow,

Cwykiel,

Brodowska

et al. 2023

Stem Cell Rev and Rep

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Cellular therapies are regarded as the most promising approach for inducing transplant tolerance without life-long immunosuppression in solid organ and vascularized composite allotransplantation (VCA). Currently, no therapies are achieving this goal. This study introduces a novel Human Multi-Chimeric Cell (HMCC) line created by fusion of umbilical cord blood (UCB) cells, from three unrelated donors as an alternative therapeutic approach to bone marrow transplantation and tolerance induction in solid organ and VCA transplants. We performed eighteen ex vivo polyethylene glycol mediated fusions of human UCB cells from three unrelated donors to create HMCC. Mononuclear cells labeled with PKH26, PKH67, and eFluor™ 670 fluorescent dyes were fused and sorted creating a new population of triple-labeled (PKH26/PKH67/eFluor™ 670) HMCC. The creation of HMCC from three unrelated human UCB donors was confirmed by flow cytometry and confocal microscopy. Genotyping analyses determined the tri-chimeric state of HMCC by presence of parent alleles and selected loci specific for each of three UCB donors. Phenotype characterization confirmed hematopoietic markers distribution, comparable to UCB donors. HMCC maintained viability and displayed a low apoptosis level. The COMET assay revealed absence of genotoxicity, confirming fusion safety. Colony forming units assay showed clonogenic properties of HMCC. This study confirmed the feasibility of HMCC creation from three unrelated human UCB donors and characterized tri-chimeric state, hematopoietic phenotype, viability, safety, and clonogenic properties of HMCC. The created HMCC line, representing genotype characteristics of three unrelated human UCB donors, introduces a novel therapeutic approach for bone marrow, solid organ, and VCA transplants. Graphical Abstract

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A Novel Human Myoblast Chimeric Cells Therapy for Restoration of Muscle Function

Cited by 5 publications

References 0 publications

Novel Human Umbilical Di-Chimeric (HUDC) cell therapy for transplantation without life-long immunosuppression

Novel Human Umbilical Di-Chimeric (HUDC) cell therapy for transplantation without life-long immunosuppression

Systemic cell therapy for muscular dystrophies

Human Multi-Chimeric Cell (HMCC) Therapy as a Novel Approach for Tolerance Induction in Transplantation

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