In Utero Transplantation of Placenta-Derived Mesenchymal Stromal Cells for Potential Fetal Treatment of Hemophilia A.

Hemophilia A (HA) is an X-linked recessive disorder caused by mutations in the factor VIII ( FVIII) gene leading to deficient blood coagulation. The current standard of care is frequent infusions of plasma-derived FVIII or recombinant B-domain-deleted FVIII (BDD-FVIII). While this treatment is effective, many patients eventually develop FVIII inhibitors that limit the effectiveness of the infused FVIII. As a monogenic disorder, HA is an ideal target for gene or cell-based therapy. Several studies have investigated allogeneic stem cell therapy targeting in utero or postnatal treatment of HA but have not been successful in completely correcting HA. Autologous in utero transplantation of mesenchymal stem cells is promising for treatment of HA due to the naive immune status of the fetal environment as well as its potential to prevent transplant rejection and long-term FVIII inhibitor formation. HA can be diagnosed by chorionic villus sampling performed during the first trimester (10 to 13 wk) of gestation. In this study, we used an established protocol and isolated placenta-derived mesenchymal stromal cells (PMSCs) from first trimester chorionic villus tissue and transduced them with lentiviral vector encoding the BDD-FVIII gene. We show that gene-modified PMSCs maintain their immunophenotype and multipotency, express, and secrete high levels of active FVIII. PMSCs were then transplanted at embryonic day 14.5 (E14.5) into wild-type fetuses from time-mated pregnant mice. Four days after birth, pups were checked for engraftment, and varying levels of expression of human green fluorescent protein were found in the organs tested. This study shows feasibility of the approach to obtain PMSCs from first trimester chorionic villus tissue, genetically modify them with the FVIII gene, and transplant them in utero for cell-mediated gene therapy of HA. Future studies will involve evaluation of long-term engraftment, phenotypic correction in HA mice, and prevention of FVIII inhibitor development by this approach

Effect of a Rehabilitation Program After Mesenchymal Stromal Cell Transplantation for Advanced Osteonecrosis of the Femoral Head: A 10-Year Follow-Up Study

Objective: To assess the status of 10 patients with advanced osteonecrosis of the femoral head who underwent mesenchymal stromal cell transplants and a 12-week rehabilitation program 10 years earlier. Design: Retrospective study. Setting: University clinical research laboratory. Participants: Patients (N=10) who had undergone mesenchymal stromal cell transplantation and rehabilitation for a single hip osteonecrosis of the femoral head 10 years prior to the current study were recruited by telephone. The average age was 31.7 years and all participants were men; radiographic stages were 3A in 6 patients and 3B in 4 patients before treatment. Intervention: A 12-week rehabilitation program with follow-up once every 1 to 2 years was performed after mesenchymal stromal cell transplantation. Main Outcome Measures: Radiographic analysis, clinical score, timed Up and Go test, hip function (range of motion, muscle strength), and Short Form-36 scores were assessed before treatment and 1 and 10 years after treatment. Results: Upon imaging, 5 hips were found to be stable (stable group) and 5 had progressed (progressed group); 2 of the latter group required a total hip arthroplasty. The pretreatment radiographic stage of the progressed group was more advanced than that of the stable group. Body mass index was higher in the progressed group than in the stable group. Hip function and clinical score at 1 and 10 years after treatment improved in the hips of 8 patients without total hip arthroplasty. There were no severe adverse events during the rehabilitation. Conclusions: The 12-week rehabilitation program and annual follow-up after mesenchymal stromal cell transplantation for osteonecrosis of the femoral head was associated with pain reduction, maintaining hip muscle strength, widening range of motion, and improving quality of life. The level and timing of weight-bearing and social activity should be planned according to the individual's lifestyle and body composition

Immunomodulatory properties of mesenchymal stem cells : a review based on an interdisciplinary meeting held at the Kennedy Institute of Rheumatology Division, London, UK, 31 October 2005

Peer reviewedPublisher PD

Multi-Parameter Analysis of Biobanked Human Bone Marrow Stromal Cells Shows Little Influence for Donor Age and Mild Comorbidities on Phenotypic and Functional Properties

Heterogeneous populations of human bone marrow-derived stromal cells (BMSC) are among the most frequently tested cellular therapeutics for treating degenerative and immune disorders, which occur predominantly in the aging population. Currently, it is unclear whether advanced donor age and commonly associated comorbidities affect the properties of ex vivo-expanded BMSCs. Thus, we stratified cells from adult and elderly donors from our biobank (n = 10 and n = 13, mean age 38 and 72 years, respectively) and compared their phenotypic and functional performance, using multiple assays typically employed as minimal criteria for defining multipotent mesenchymal stromal cells (MSCs). We found that BMSCs from both cohorts meet the standard criteria for MSC, exhibiting similar morphology, growth kinetics, gene expression profiles, and pro-angiogenic and immunosuppressive potential and the capacity to differentiate toward adipogenic, chondrogenic, and osteogenic lineages. We found no substantial differences between cells from the adult and elderly cohorts. As positive controls, we studied the impact of in vitro aging and inflammatory cytokine stimulation. Both conditions clearly affected the cellular properties, independent of donor age. We conclude that in vitro aging rather than in vivo donor aging influences BMSC characteristics

The geometrical shape of mesenchymal stromal cells measured by quantitative shape descriptors is determined by the stiffness of the biomaterial and by cyclic tensile forces

Controlling mesenchymal stromal cell (MSC) shape is a novel method for investigating and directing MSC behaviour in vitro. it was hypothesized that specifigc MSC shapes can be generated by using stiffnessâ defined biomaterial surfaces and by applying cyclic tensile forces. Biomaterials used were thin and thick silicone sheets, fibronectin coating, and compacted collagen type I sheets. The MSC morphology was quantified by shape descriptors describing dimensions and membrane protrusions. Nanoscale stiffness was measured by atomic force microscopy and the expression of smooth muscle cell (SMC) marker genes (ACTA2, TAGLN, CNN1) by quantitative reverseâ transcription polymerase chain reaction. Cyclic stretch was applied with 2.5% or 5% amplitudes. Attachment to biomaterials with a higher stiffness yielded more elongated MSCs with fewer membrane protrusions compared with biomaterials with a lower stiffness. For cyclic stretch, compacted collagen sheets were selected, which were associated with the most elongated MSC shape across all investigated biomaterials. As expected, cyclic stretch elongated MSCs during stretch. One hour after cessation of stretch, however, MSC shape was rounder again, suggesting loss of stretchâ induced shape. Different shape descriptor values obtained by different stretch regimes correlated significantly with the expression levels of SMC marker genes. Values of approximately 0.4 for roundness and 3.4 for aspect ratio were critical for the highest expression levels of ACTA2 and CNN1. Thus, specific shape descriptor values, which can be generated using biomaterialâ associated stiffness and tensile forces, can serve as a template for the induction of specific gene expression levels in MSC. Copyright © 2017 John Wiley & Sons, Ltd.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141253/1/term2263.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/141253/2/term2263_am.pd

Generation of osteoarthritic mesenchymal stromal cell lines.

ResumenXunta de Galicia; R2016/036Xunta de Galicia; R2014/050Xunta de Galicia; CN2012/142Xunta de Gaicia; GPC2014/048Instituto de salud Carlos III; PI17/0219

A survey on cellular and engineered tissue therapies in Europe in 2008

Cellular therapy is an evolving investigational treatment modality in regenerative medicine, but little published information is available on its current use. Starting from the established European group for Blood and Marrow Transplantation activity survey on hematopoietic stem cell transplantation, a joint committee of four major scientific organizations made a coordinated attempt to collect detailed information in Europe for the year 2008. Thirty-three teams from 16 countries reported data on 656 patients to a "novel cellular therapy" survey, which were combined to additional 384 records reported to the standard European group for Blood and Marrow Transplantation survey. Indications were cardiovascular (29%; 100% autologous), musculoskeletal (18%; 97% autologous), neurological (9%; 39% autologous), epithelial/parenchymal (9%; 18% autologous), autoimmune diseases (12%; 77% autologous), or graft-versus-host disease (23%; 13% autologous). Reported cell types were hematopoietic stem cells (39%), mesenchymal stromal cells (47%), chondrocytes (5%), keratinocytes (7%), myoblasts (2%), and others (1%). In 51% of the grafts, cells were delivered after expansion; in 4% of the cases, cells were transduced. Cells were delivered intravenously (31%), intraorgan (45%), on a membrane or gel (14%), or using three-dimensional scaffolds (10%). This data collection platform is expected to capture and foresee trends for novel cellular therapies in Europe, and warrants further consolidation and extension

개의 골절 모델에서 골분화를 유도한 중간엽 줄기세포 시트의 골재생 효과

학위논문 (박사) -- 서울대학교 대학원 : 수의과대학 수의학과, 2020. 8. 강병재.Nonunion and delayed union during fracture repair are critical in veterinary orthopedic surgery. Mesenchymal stromal cells (MSC) sheets have potential for clinical application in bone regeneration. The bone regeneration capacity of gelatin-induced osteogenic differentiated mesenchymal stromal cell sheets (GCS) was evaluated in this study. The effects were similar to those of osteogenic differentiated mesenchymal stromal cell sheets (OCS). The study comprised two parts. First, OCS and undifferentiated mesenchymal stromal cell sheets (UCS) were compared for bone regeneration. Second, the efficacy of frozen and thawed GCS (FT-GCS) on the osteogenic potential of fresh GCS was assessed. Both parts of the study evaluated the bone healing capacity in canine model. The first chapter compared the bone generation capacity of UCS and OCS in vitro and in vivo. Quantitative real-time polymerase chain reaction (rt-PCR) showed that runt-related transcription factor 2 (Runx2), bone morphogenetic protein 7 (BMP7) and hepatocyte growth factor (HGF) were upregulated in OCS compared to UCS. The expression levels of cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), interleukin-10 (IL-10) and tumor necrosis factor - α (TNF-α) in UCS were markedly upregulated compared to OCS. In vivo, each stem cell sheet was applied in the radial fracture model. The proportions of external callus in the total bone volume of OCS group was significantly lower than that in the control. The OCS group showed significantly increased mature bone compared to the control and UCS groups. Fibrous connective tissue was increased in the UCS group. In OCS, the fracture sites could be stabilized by early bone healing and callus formation was reduced, suggesting that UCSs and OCSs had different tissue healing effects and OCS could be used for bone healing. The use of gelatin in the production of OCS increased bone differentiation-related factors, producing more solid OCS. In addition, the pathway of bone differentiation was different for OCS and GCS. Since it took several days to cultivate the OCS, the bone regeneration efficacy of the cell sheets was frozen and stored for easy evaluation of their clinical application. In the second chapter, the bone regeneration effects of fresh GCS (F-GCS) and FT-GCS were evaluated. The experiment was performed in two parts. The first part involved in vitro evaluation of osteogenic potential of F-GCS and FT-GCS. The second part involved in vivo examination of the bone healing effects of both sheets in a canine model of fracture. In vitro, rt-PCR revealed no significant difference in the values of osteogenic-related factors between the two groups, and in vivo results, the external callus and the connectivity of fracture sites in both the F-GCS and FT-GCS groups were significantly increased compared to the control group. The amount of mature bones at fracture sites was increased in the F-GCS and FT-GCS groups compared to the control group, with no difference between the F-GCS and FT-GCS groups. Thus, bone regeneration using fresh and frozen-thawed GCS was similarly effective. In conclusion, OCS can be clinically applied to promote early bone regeneration. In particular, OCS cultured with gelatin are more solid and osteogenic differentiated than OCS, even when frozen and stored, the bone regeneration effects of GCS could be expected.골절 회복 과정에서의 불유합과 지연유합의 회복반응은 수의정형외과학에서 중요한 주제이다. 중간엽 유래의 줄기세포를 이용하여 제작한 세포시트가 골재생에 효과가 있다고 연구되어 있다. 또한 젤라틴을 이용하여 골분화 유도 줄기세포 시트 (GCS)를 제작하였을 때 골분화 유도 줄기세포 시트 (OCS)와 골재생능에 있어 큰 차이가 없음이 연구되었다. 두 파트로 구성된 본 연구의 첫 번째 실험에서 OCS 와 미분화세포시트 (UCS)의 골재생능에 대하여 비교하였다. 두 번째 실험에서는 젤라틴을 이용하여 배양한 골분화유도 줄기세포 시트의 신선한 상태와 동결시켰던 세포시트의 골재생 효과 차이를 확인하였다. 추가로 두 연구 모두 개 골절 모델에 적용하여 골 회복 반응을 평가하였다. 첫 번째 실험은 골절 회복에 있어서, 미분화 줄기세포 시트(UCS)와 OCS의 골 재생 효과를in vitro 와 in vivo 상에서 비교한 것이다. 정량적 중합효소 연쇄반응 실험을 통하여 OCS의 경우 UCS에 비하여 골형성단백질7 (BMP7), 형성전화증식인자 (TGF-β), Runx2, 그리고 HGF가 상승된 것을 확인할 수 있었다. 반면 UCS의 경우 OCS에 비하여 시클로옥시게나이제-2 (COX-2), 인터류킨-6 (IL-6), 인터류킨-10 (IL-10) 종양괴사인자-알파 (TNF-α), 가 상승되어 있는 것을 확인할 수 있었다. 요골 골절 모델에서 각 줄기세포시트를 적용하였을 때, OCS를 적용한 군에서 가골의 양이 유의적으로 적게 형성된 것을 확인할 수 있었다. 조직염색을 통하여 OCS 군에서 성숙한 골화세포들을 확인할 수 있었다. 반면 UCS를 적용한 군에서는 섬유조직들의 유의적인 상승을 확인할 수 있었다. 따라서 OCS의 경우 초기 골재생을 통하여 골절부의 안정성이 증가되었고, 그로 인해 가골의 형성이 줄어든 것으로 생각된다. 또한 UCS와 OCS의 경우 조직재생에 있어 다른 효과가 있는 것으로 보이며, OCS는 골 재생을 기대할 수 있다. OCS 제작 과정에 젤라틴을 이용하면 골분화 관련 인자들의 상승과 더 단단한 골분화 유도 줄기세포 시트를 제작할 수 있음이 연구되었다. 또한, OCS 와 GCS 의 경우 골분화 경로가 다름이 밝혀졌다. 첫 번째 실험 과정에서 골분화 줄기세포 시트를 배양하기 위하여 몇 일간의 시간이 소요되었기에, 이를 임상에 적용하기 용이한 방법으로 동결하여 보관했던 줄기세포 시트의 골재생 효능을 평가하였다. 두 번째 실험에서는 젤라틴을 이용하여 배양한 골분화 유도 줄기세포 시트 (F-GCS)와, 그것을 동결시켜 보관하였다가 녹인 골분화 유도 줄기세포 시트 (FT-GCS)의 골 재생 효과를 비교하였다. 실험은 크게 두 파트로, in vitro에서 F-GCS 와 FT-GCS 의 골분화 관련 평가, in vivo 에서는 요골 골절 모델의 비글견에게 적용하여 골재생 정도를 평가하였다. In vitro 실험,정량적 중합효소 연쇄 반응 결과 F-GCS 와 FT-GCS 에서 골분화 관련 인자들의 수치들도 유의미적인 차이가 없었다. In vivo 결과에서는 가골이 세포시트를 적용해준 군에서 증가된 것을 확인할 수 있었고, 골절부분의 연결성은 통제군에 비하여 세포시트를 적용해준 군에서 유의미적으로 상승되었다. 조직 염색상에서 골절부의 피질골 사이 부분에서 F-GCS 군과 FT-GCS 군에서 성숙한 뼈의 양이 통제군에 비하여 유의미적으로 상승되어 있는 것을 확인할 수 있었고, 두 군의 차이는 없었다. 따라서 젤라틴을 이용하여 배양한 골분화유도 줄기세포 시트의 경우, 신선한 상태에서 바로 사용하는 것과 동결 후 냉동 과정을 거쳐서 사용하여도 골재생에 비슷한 정도의 효과를 보인다. 결론적으로 골분화 유도 줄기세포시트의 경우, 빠른 골재생을 기대할 때 임상적으로 적용이 가능하다. 특히 젤라틴을 이용하여 배양한 골분화 유도 줄기세포 시트의 경우에는 골분화 유도 줄기세포시트에 비하여 단단하고 골분화가 더 많이 진행되며, 동결하여 보관하다가 사용하더라도 골분화 유도 줄기세포 시트의 골재생 효과를 기대할 수 있다.General introduction 1 CHAPTER I. Different Bone Healing Effects of Undifferentiated and Osteogenic Differentiated Mesenchymal Stromal Cell Sheets in Canine Radial Fracture Model 4 Abstract 4 Introduction 6 Materials and methods 9 Results 16 Discussion 19 CHAPTER II. Frozen-thawed gelatin-induced osteogenic cell sheets of canine adipose-derived mesenchymal stromal cells improved fracture healing in canine model 33 Abstract 33 Introduction 35 Materials and methods 38 Results 46 Discussion 49 Conclusion 61 References 63 Abstract in korean 79Docto

Comparison of Mesenchymal Stromal Cells Isolated From Murine Adipose Tissue and Bone Marrow in the Treatment of Spinal Cord Injury

The use of mesenchymal stromal cell (MSC) transplantation to repair the injured spinal cord has shown consistent benefits in preclinical models. However, the low survival rate of grafted MSC is one of the most important problems. In the injured spinal cord, transplanted cells are exposed to hypoxic conditions and exposed to nutritional deficiency caused by poor vascular supply. Also, the transplanted MSCs face cytotoxic stressors that cause cell death. The aim of this study was to compare adipose-derived MSCs (AD-MSCs) and bone marrow-derived MSCs (BM-MSCs) isolated from individual C57BL6/J mice in relation to: (i) cellular characteristics, (ii) tolerance to hypoxia, oxidative stress and serum-free conditions, and (iii) cellular survival rates after transplantation. AD-MSCs and BM-MSCs exhibited a similar cell surface marker profile, but expressed different levels of growth factors and cytokines. To research their relative stress tolerance, both types of stromal cells were incubated at 20.5% O2 or 1.0% O2 for 7 days. Results showed that AD-MSCs were more proliferative with greater culture viability under these hypoxic conditions than BM-MSCs. The MSCs were also incubated under H2O2-induced oxidative stress and in serum-free culture medium to induce stress. AD-MSCs were better able to tolerate these stress conditions than BMMSCs; similarly when transplanted into the spinal cord injury region in vivo, AD-MSCs demonstrated a higher survival rate post transplantation Furthermore, this increased AD-MSC survival post transplantation was associated with preservation of axons and enhanced vascularization, as delineated by increases in anti-gamma isotype of protein kinase C and CD31 immunoreactivity, compared with the BM-MSC transplanted group. Hence, our results indicate that AD-MSCs are an attractive alternative to BM-MSCs for the treatment of severe spinal cord injury. However, it should be noted that the motor function was equally improved following moderate spinal cord injury in both groups, but with no significant improvement seen unfortunately following severe spinal cord injury in either grou