Autologous Progenitor Cell Research Articles

599 Human Pyloric Sphincters Bioengineered Using Autologous Pyloric Smooth Muscle Cells and Neural Progenitor Cells

599 Human Pyloric Sphincters Bioengineered Using Autologous Pyloric Smooth Muscle Cells and Neural Progenitor Cells

Intra-carotid arterial administration of autologous peripheral blood-derived endothelial progenitor cells improves acute ischemic stroke neurological outcomes in rats

ObjectiveWe tested the hypothesis that transfusion of autologous peripheral blood-derived endothelial progenitor cells (PBDEPC) via the internal carotid artery could reduce brain-infarct zone (BIZ) and neurological deficit in rats following acute ischemic stroke (IS) induced by 50-min left middle cerebral artery occlusion. DesignAdult male Sprague–Dawley rats (n=60) were equally divided into group 1 [sham control (SC)], group 2 [SC-PBDEPC (5.7×106/kg)], group 3 (IS), group 4 [IS-low-dose PBDEPC (1.7×106/kg)], group 5 [IS-high-dose PBDEPC (5.7×106/kg)]. Groups 2 to 5 received G-CSF (35μg/kg subcutaneously) for 4days before drawing blood for PBDEPC culture. Measurements and main resultsBy day 90, BIZ determined by histopathology (area) and brain MRI (volume) were highest in group 3, lowest in groups 1 and 2, higher in group 4 than in group 5 (all p<0.0001), and not significantly different between groups 1 and 2. Sensorimotor functional results exhibited an opposite pattern of BIZ among groups 3 to 5 (p<0.005). Angiogenesis biomarkers (SDF-1α, CXCR4, VEGF, angiopoietin-1) significantly increased progressively from groups 1 and 2 to group 5 (all p<0.0001). Oxidative-stress (NOX-1, NOX-2, oxidized protein), apoptotic (cleaved caspase 3 and PARP, mitochondrial Bax), inflammatory (MMP-9, TNF-α, AQP-4, GFAP, iNOS), and brain-damaged (cytosolic cytochrome-C) biomarkers showed an identical pattern, whereas anti-inflammatory (Bcl-2), mitochondrial preservation (mitochondrial cytochrome-C, PGC-1α), and endothelial function (CD31+, vWF+, eNOS) biomarkers, and vessel density showed an opposite pattern of BIZ among these five groups (all p<0.001). ConclusionHigher-dose was superior to lower-dose EPC treatment for reducing BIZ and improving neurological functional outcome.

In vitro evaluation of endothelial progenitor cells from adipose tissue as potential angiogenic cell sources for bladder angiogenesis.

Autologous endothelial progenitor cells (EPCs) might be alternative angiogenic cell sources for vascularization of tissue-engineered bladder, while isolation and culture of EPCs from peripheral blood in adult are usually time-consuming and highly inefficient. Recent evidence has shown that EPCs also exist in the adipose tissue. As adipose tissue is plentiful in the human body and can be easily harvested through a minimally invasive method, the aim of this study was to culture and characterize EPCs from adipose tissue (ADEPCs) and investigate their potential for the neovascularization of tissue-engineered bladder. Adipose stromal vascular fraction (SVF) was isolated and used for the culture of ADEPCs and adipose derived stem cells (ADSCs). After SVF was cultured for one week, ADEPCs with typical cobblestone morphology emerged and could be isolated from ADSCs according to their different responses to trypsinization. Rat bladder smooth muscle cells (RBSMCs) were isolated and cultured from rat bladder. RBSMCs exhibited typical spindle-shaped morphology. ADEPCs had higher proliferative potential than ADSCs and RBSMCs. ADEPCs stained positive for CD34, Stro-1, VEGFR-2, eNOS and CD31 but negative for α-SMA, CD14 and CD45. ADSCs stained positive for CD34, Stro-1 and α-SMA but negative for VEGFR-2, eNOS, CD31, CD14 and CD45. RBSMCs stained only positive for α-SMA. ADEPCs could be expanded from a single cell at an early passage to a cell cluster containing more than 10,000 cells. ADEPCs were able to uptake DiI-Ac-LDL, bind UEA-1 and form capillary-like structures in three-dimensional scaffolds (Matrigel and bladder acellular matrix). ADEPCs were also able to enhance the human umbilical vein endothelial cells’ capability of capillary-like tube formation on Matrigel. Additionally, significantly higher levels of mRNA and protein of vascular endothelial growth factor were found in ADEPCs than in RBSMCs. These results suggest the potential use of ADEPCs as angiogenic cell sources for engineering bladder tissue.

The clinical application of mesenchymal stem cells and cardiac stem cells as a therapy for cardiovascular disease.

Cardiovascular disease (CVD) can be separated into two broad etiological categories, based on the presence or absence of ischemia as a causative factor. In both ischemic and non-ischemic heart disease, myocardial dysfunction or damage frequently results in the development of heart failure, characterized by dyspnea, fatigue and reduced survival. As one of the least regenerative organs in the human body, current standards of care are limited to mitigating loss and preventing recurrence of damage, rather than stimulating actual regeneration of functional heart tissue. Cell based therapies using progenitor cells from bone marrow and the heart itself have been evaluated in preclinical models, and have demonstrated some promise. Accordingly, several clinical trials using autologous stem and progenitor cells have been performed, showing that these cells can be used safely in humans, and suggesting that they may improve relevant clinical parameters in patients with heart disease. Two specific cell populations that are particularly promising are the bone marrow derived mesenchymal stem cell (MSC) and the heart muscle derived cardiac stem cell (CSC). This review will summarize preclinical studies evaluating these stem cell populations and will discuss the clinical application of these cells in contemporary clinical trials, and potential future investigations.

Transplantation of autologous endothelial progenitor cells to treat high blood flow-induced pulmonary artery hypertension

Objective To stidy the effects of the transplantation of autologous endothelial progenitor cells(EPCs)on high blood flow-induced pulmonary artery hypertension(PAH). Methods Thirty piglets were divided into three groups randomly.The animals in sham group(n= 10)underwent thoracotomy only, those in control group(n= 10) were subjected to the descending aorta and the main pulmonary artery shunt via left posterolateral thoracotomy,and those in EPCs+ shunt group(n= 10)were given EPCs(2× 107)intravenously after descending aorta and the main pulmonary artery shunt procedure for two weeks. All animals are normally raised for one month.Then their pulmonary artery systolic pressure(PASP)and pulmonary vascular resistance(PVR) were measured, the levels of serum matrix metalloproteinase (MMP)-9, endothelin(ET)-1, interleukin(IL)-6, and IL-8 were tested, p38 mitogen-activated protein kinase(p38MAPK)activity in the lung tissue was detected, and pulmonary artery structures were observed. Results As compared with the Sham group, the levels of PASP, PVR, MMP-9, ET-1, IL-6 and IL-8, and activity of p38MAPK were significantly increased in control group[(6. 63± 1. 20) k Pa,(10. 44±2. 53) wood's U,(76. 63±10. 39)μg/L,(103. 66±17. 31)μg/L,(43. 00± 7. 33)ng/L,(67. 39±9. 65)ng/L,3. 63±0. 33]and EPCs+ shunt group[(4. 07±0. 77)kPa,(6. 27± 0. 65) wood's U,(64. 11±9. 73)μg/L,(90. 44±13. 11)μg/L,(30. 44±8. 17) ng/L,(53. 23± 7. 37)ng/L, 1. 73±0. 37](P< 0. 01),30 days postoperatively.These values were remarkablely decreased in EPCs+ shunt group as compared with those in control group(P< 0. 05).There were most obvious lung histologic changes in control group, mild changes in EPCs+ shunt group, and no obvious pathological changes in Sham group. Conclusion Transplantation of autologous EPCs attenuates dynamic pulmonary hypertension, which may be associated with the decreased levels of MMP-9 and ET-1 which cause the lung tissue reconstruction, and inhibition of the inflammatory injury which plays a leader role in the process of PAH. Key words: High blood-induced pulmonary artery hypertension; Piglet model; Endothelial progenitor cells

Cartilage resurfacing potential of PLGA scaffolds loaded with autologous cells from cartilage, fat, and bone marrow in an ovine model of osteochondral focal defect.

Current developments in tissue engineering strategies for articular cartilage regeneration focus on the design of supportive three-dimensional scaffolds and their use in combination with cells from different sources. The challenge of translating initial successes in small laboratory animals into the clinics involves pilot studies in large animal models, where safety and efficacy should be investigated during prolonged follow-up periods. Here we present, in a single study, the long-term (up to 1year) effect of biocompatible porous scaffolds non-seeded and seeded with fresh ex vivo expanded autologous progenitor cells that were derived from three different cell sources [cartilage, fat and bone marrow (BM)] in order to evaluate their advantages as cartilage resurfacing agents. An ovine model of critical size osteochondral focal defect was used and the test items were implanted arthroscopically into the knees. Evidence of regeneration of hyaline quality tissue was observed at 6 and 12months post-treatment with variable success depending on the cell source. Cartilage and BM-derived mesenchymal stromal cells (MSC), but not those derived from fat, resulted in the best quality of new cartilage, as judged qualitatively by magnetic resonance imaging and macroscopic assessment, and by histological quantitative scores. Given the limitations in sourcing cartilage tissue and the risk of donor site morbidity, BM emerges as a preferential source of MSC for novel cartilage resurfacing therapies of osteochondral defects using copolymeric poly-D,L-lactide-co-glycolide scaffolds.

Translational research of adult stem cell therapy

Congestive heart failure (CHF) secondary to chronic coronary artery disease is a major cause of morbidity and mortality world-wide. Its prevalence is increasing despite advances in medical and device therapies. Cell based therapies generating new cardiomyocytes and vessels have emerged as a promising treatment to reverse functional deterioration and prevent the progression to CHF. Functional efficacy of progenitor cells isolated from the bone marrow and the heart have been evaluated in preclinical large animal models. Furthermore, several clinical trials using autologous and allogeneic stem cells and progenitor cells have demonstrated their safety in humans yet their clinical relevance is inconclusive. This review will discuss the clinical therapeutic applications of three specific adult stem cells that have shown particularly promising regenerative effects in preclinical studies, bone marrow derived mesenchymal stem cell, heart derived cardiosphere-derived cell and cardiac stem cell. We will also discuss future therapeutic approaches.

One-staged aptamer-based isolation and application of endothelial progenitor cells in a porcine myocardial infarction model.

A multitude of stem cell types has been extensively studied and used for myocardial regenerative therapy. Amongst these endothelial progenitor cells form a promising source. In our present study, we investigated a one-staged approach for isolation and application of autologous endothelial progenitor cells in a pig model of myocardial infarction. Endothelial progenitor cell isolation was performed by immediately preprocedural bone marrow aspiration and consecutive positive selection by aptamer-based magnetic cell sorting. Animals were divided in three groups receiving endothelial progenitor cells, saline, or no intramyocardial injection respectively. Postprocedural follow-up consisted of weekly echocardiographic evaluations. Postmortem histological analysis after four weeks focused on detection of transplanted PKH26-positive endothelial progenitor cells and neovascularization within the infarcted myocardium. A significant difference in left ventricular ejection fraction could not be shown between the three groups. PKH26-stained endothelial progenitor cells could be found in the endothelial progenitor cells transplanted group, although detection was scarce. Large-sized capillaries were found to be significantly more in endothelial progenitor cells treated myocardium. The one-stage approach of endothelial progenitor cells isolation and application presented herein offers a new therapeutic concept. Even though a beneficial impact on myocardial function could not be assessed, increased neovascularization may indicate positive effects on remodeling processes. Being able to harvest endothelial progenitor cells right before application provides a wider scope of action in urgent cases.

The Stromal Vascular Fraction of Autologous Fat Graft Induces Proliferation of Epithelial Progenitor Cells in Healthy and Cancer-Containing Breast Tissue In Vitro

BACKGROUND: Autologous fat grafting is widely used for managing contour asymmetries in breast reconstruction. Adipose tissue contains a rich population of both multipotent mesenchymal stem cells and vascular progenitor cells (collectively referred to as adipose-derived stem cells, or ASCs).1 When isolated, the cellular fraction of adipose tissue containing ASCs is referred to as stromal vascular fraction (SVF). Despite widespread clinical use, interactions between the SVF of lipoaspirate and breast parenchyma remain poorly understood. Our study investigated the effect of progenitor-rich SVF on the behavior of mammary epithelial progenitor cells derived from two sources: (i) healthy breast tissue and (ii) cancer adjacent breast tissue. METHODS: Healthy breast tissue and abdominal lipoaspirate were obtained from patients undergoing elective reduction mammoplasty and liposuction. Breast tissue from outside the tumor-free margins of mastectomy specimens (termed ‘cancer-adjacent breast tissue’) was obtained from patients undergoing primary free-flap reconstruction. Samples of SVF (from lipoaspirate) and breast parenchymal cells were then isolated using established cell digestion protocols. ASCs within the SVF were characterized using established differentiation, colony-forming unit-fibroblast (CFU-f), and cell surface marker assays.1 Finally, SVF cells were co-cultured with breast parenchymal cells from (i) healthy and (ii) cancer-adjacent sources were co-cultured using a three-dimensional matrix called Matrigel. Control cultures consisted of breast cells from (i) healthy and (ii) cancer-adjacent tissue in the absence of SVF. After 14 days, the total cell numbers and mammary epithelial progenitor cell populations from each culture group were quantified using colony forming cell (CFC) assays. RESULTS: Differentiation, CFU-f, and surface marker assays demonstrated the presence of ASCs in SVF samples. Co-cultures of cancer-adjacent breast parenchymal cells with SVF showed a 9-fold expansion of mammary epithelial progenitor cells (control = 3-fold) compared to a 5.5-fold expansion in co-cultures of healthy breast parenchymal cells (control = 2-fold) with SVF based on CFC assays. CONCLUSIONS: SVF is capable of increasing the proliferation of mammary epithelial progenitor cells in both healthy and cancer-adjacent breast tissue. As a result, this study demonstrates the potential for interaction between the SVF within autologous fat graft and progenitor cells contained within breast parenchymal tissue.

A PiggyBac-mediated approach for muscle gene transfer or cell therapy

An emerging therapeutic approach for Duchenne muscular dystrophy is the transplantation of autologous myogenic progenitor cells genetically modified to express dystrophin. The use of this approach is challenged by the difficulty in maintaining these cells ex vivo while keeping their myogenic potential, and ensuring sufficient transgene expression following their transplantation and myogenic differentiation in vivo. We investigated the use of the piggyBac transposon system to achieve stable gene expression when transferred to cultured mesoangioblasts and into murine muscles. Without selection, up to 8% of the mesoangioblasts expressed the transgene from 1 to 2 genomic copies of the piggyBac vector. Integration occurred mostly in intergenic genomic DNA and transgene expression was stable in vitro. Intramuscular transplantation of mouse Tibialis anterior muscles with mesoangioblasts containing the transposon led to sustained myofiber GFP expression in vivo. In contrast, the direct electroporation of the transposon-donor plasmids in the mouse Tibialis muscles in vivo did not lead to sustained transgene expression despite molecular evidence of piggyBac transposition in vivo. Together these findings provide a proof-of-principle that piggyBac transposon may be considered for mesoangioblast cell-based therapies of muscular dystrophies.

Accumulation of 4-1BBL+ B cells in the elderly induces the generation of granzyme-B+ CD8+ T cells with potential antitumor activity

AbstractAlthough the accumulation of highly-differentiated and granzyme B (GrB)-expressing CD8+CD28– T cells has been associated with aging, the mechanism for their enrichment and contribution to immune function remains poorly understood. Here we report a novel B-cell subset expressing 4-1BBL, which increases with age in humans, rhesus macaques, and mice, and with immune reconstitution after chemotherapy and autologous progenitor cell transplantation. These cells (termed 4BL cells) induce GrB+CD8+ T cells by presenting endogenous antigens and using the 4-1BBL/4-1BB axis. We found that the 4BL cells increase antitumor responses in old mice, which may explain in part the paradox of retarded tumor growth in the elderly. 4BL cell accumulation and its capacity to evoke the generation of GrB+CD8+ T cells can be eliminated by inducing reconstitution of B cells in old mice, suggesting that the age-associated skewed cellular immune responses are reversible. We propose that 4BL cells and the 4-1BBL signaling pathway are useful targets for improved effectiveness of natural antitumor defenses and therapeutic immune manipulations in the elderly.

Safety and Efficacy of Targeted-Dose Busulfan and Bortezomib as a Conditioning Regimen for Patients with Relapsed Multiple Myeloma Undergoing a Second Autologous Blood Progenitor Cell Transplantation

Patients with multiple myeloma (MM) who relapse after autologous transplantation have limited therapeutic options. We conducted a prospective, multicenter, phase IIa study to investigate the safety and efficacy of i.v. busulfan (Bu) in combination with bortezomib as a conditioning regimen for a second autotransplantation. Because a safe Bu exposure was unknown in patients receiving this combination, Bu was initially targeted to a total area under the concentration–time curve (AUC) of 20,000 μM × minute. As no concentration-limiting toxicity was observed in 6 patients, this Bu exposure was utilized in the following treatment cohort (n = 24). Individualized Bu dose, based on test dose .8 mg/kg pharmacokinetics (PK), was administered daily for 4 consecutive days starting 5 days before transplantation, followed by a single dose of bortezomib (1.3 mg/m2) 1 day before transplantation. The total mean dose of i.v. Bu (including the test dose and 4-day administration) was 14.2 mg/kg (standard deviation = 2.48; range, 8.7 to 19.2). Confirmatory PK demonstrated that only 2 of 30 patients who underwent transplantation were dosed outside the Bu AUC target and dose adjustments were made for the last 2 doses of i.v. Bu. The median age was 59 years (range, 48 to 73). Median time from first to second transplantation was 28.0 months (range, 12 to 119). Of 26 evaluable patients, 10 patients attained a partial response (PR) or better at 3 months after transplantation, with 2 patients attaining a complete response. At 6 months after transplantation, 5 of 12 evaluable patients had maintained or improved their disease status. Median progression-free survival was 191 days, whereas median overall survival was not reached during the study period. The most common grade 3 and 4 toxicities were febrile neutropenia (50.0%) and stomatitis (43.3%). One transplantation-related death was observed. A combination of dose-targeted i.v. Bu and bortezomib induced PR or better in one third of patients with MM who underwent a second autotransplantation, with acceptable toxicity.

EACPT-0036 - The Beneficial Effect of Ultrasonic Microbubble Transfection on Aged Endothelial Progenitor Cells

Cell therapy for ischemic diseases by using autologous progenitor cells has been limited by the functional impairment of the cells, such as insufficient paracrine and angiogenic effects, owing to the presence of risk factors, including old age. Optimization of ultrasonic microbubble transfection (UMT) to increase the product of transferred gene in the progenitor cells is a potential strategy to improve the function of progenitor cells for clinical application. Therefore, we explored whether UMT has a beneficial effect on the angiogenic activity of aged endothelial progenitor cells (EPCs).

Abstract 7: Intracoronary Autologous Cardiac Progenitor Cell Transfer In Children With Hypoplastic Left Heart Syndrome: 2-year Results Of The Ticap Trial

Backgrounds: Hypoplastic left heart syndrome (HLHS) is a severe congenital heart malformation. Objective: The aim of this study is to determine whether intracoronary delivery of autologous cardiosphere-derived cells (CDCs) was feasible and safe to treat the children with HLHS. Methods: Four-teen patients with HLHS undergoing staged palliations were prospectively enrolled in this trial between January, 2011, and January, 2012. Seven patients constitutively assigned to receive intracoronary CDCs injection followed by 7 patients allocated to a control group with standard care alone. The primary endpoint was to assess the safety and the secondary endpoint was the preliminary efficacy by assessing the improvements of the right ventricular function during the follow-up. Results: No major complications were reported within 24 months of CDC infusion. Echocardiography showed that improvement of right ventricular ejection fraction (RVEF) was greater in the CDC-treated group (+5.3±3.2%) than in controls (+0.1±3.4%, P=0.01) at 3-month follow up. This cardiac function enhancement was manifested even in long-term observation (+7.8±4.9% vs. +2.2±3.1% at 1 year, P=0.03; +8.8±3.7% vs. +3.4±6.4% at 2 years, P=0.04). The absolute improvements in RVEF between 2 groups was confirmed by using right ventriculogram (RVG: +8.9±7.6% vs. +2.0±2.8% at 1 year, P=0.02; +8.1±6.0% vs. +2.9±3.9% at 2 years, P=0.04). In addition, RVEF on cMRI was also markedly improved in CDC-treated patients from 36.1±7.5% at baseline to 42.7±8.7% at 1 year (P=0.04) and to 42.4±7.6% at 2 years (P=0.047). Heart failure status was reduced in CDC-treated group as shown by significant decrease in Ross Heart Failure Class (2.6±0.8 at baseline vs. 1.4±0.5 at 2 years, P=0.01). Moreover, Z scores for weight-for-age was significantly increased from −4.0±2.7 at baseline to −2.2±1.4 at 2 year (P=0.02), whereas all of these parameters did not change in control subjects. Conclusion: These results of 2-year follow-up of TICAP trial suggest that intracoronary infusion of autologous CDCs is feasible and safe to treat the children with HLHS. This novel therapeutic strategy may impact on cardiac function as well as clinical symptom of heart failure status and somatic growth in long-term outcome.

The effect of autologous endothelial progenitor cell transplantation combined with extracorporeal shock-wave therapy on ischemic skin flaps in rats

BackgroundEndothelial progenitor cells (EPCs) have been used to revascularize ischemic tissues, but only limited effect can be achieved. Extracorporeal shock-wave therapy (ESWT) is a promising angiogenic strategy. We hypothesized that EPC transplantation combined with ESWT would greatly benefit the survival of ischemic skin flaps. MethodsSixty-four male Sprague-Dawley rats were divided into 4 groups (n = 16 in each group): group 1 (serving as sham control), group 2 (treated with subcutaneous EPC implantation, 1.0 × 106 cells), group 3 (treated with ESWT, 300 impulses at 0.10 mJ/mm2) and group 4 (treated with EPCs implantation combined with ESWT). Ischemic skin flaps were made on the backs of rats and treated accordingly. Blood flow of skin flaps was measured periodically after operation, and flap survival rates were compared. Tissue samples were harvested at 2 weeks postoperatively from each group. ResultsThe survival rate of skin flaps in group 4 was 87.5 ± 10.23%, which was statistically significantly higher than other groups. Histologic examination showed that the capillary density was higher in the dual-treatment group than in the two single-treatment groups. Compared with groups 2 and 3, blood perfusion increased significantly in group 4. A drastic increase of vWF+ cells was observed in the ischemic skin flaps on immunofluorescence staining in group 4. The expressions of chemotactic factors and angiogenic factors were higher in group 4. ConclusionsCombined treatment with EPCs and ESWT is superior to either EPCs or ESWT alone in improving the survival of ischemic skin flaps in rats.

A novel aging-associate B-cell subset controls antitumor CD8+ T-cell response (IRC3P.469)

Abstract CD8+CD28- T cells are considered an immune risk of mortality and morbidity in elderly. The mechanism of their enrichment and their function in elderly is poorly understood. In the present study we report a novel and evolutionary-conserved B-cell subset expressing 4-1BBL, which accumulates with aging of mammals, such as humans, rhesus macaques and mice. These cells (termed 4BL cells) induce antitumor effector CD8+ T cells by presenting endogenous antigens and utilizing 4-1BBL/4-1BB axis. 4BL cells are also responsible for the expansion of auto-reactive CD8+ T cells in patients treated with high dose chemotherapy and autologous progenitor cell transplantation. Thus, accumulation of antitumor 4BL cells explains the paradox of retarded tumor growth in the elderly, and most likely autoimmune disease outcome in the aged population. However, 4BL cell accumulation and their effects on CD8+ T-cell compartment can be eliminated by inducing B-cell lymphopoiesis in old mice (after B-cell depletion), suggesting that the aging-associated skewed cellular immune responses are reversible. We propose that 4BL cells and their 4-1BBL signaling pathway are useful targets for improved effectiveness of natural antitumor defenses and therapeutic immune manipulations in the elderly. How 4BL are generated, and their impact in the B-cell effector functions homeostasis in elderly will be further discussed.

Three-Dimensional Perfusion Cultivation of Human Cardiac-Derived Progenitors Facilitates Their Expansion While Maintaining Progenitor State

The therapeutic application of autologous cardiac-derived progenitor cells (CPCs) requires a large cell quantity generated under defined conditions. Herein, we investigated the applicability of a three-dimensional (3D) perfusion cultivation system to facilitate the expansion of CPCs harvested from human heart biopsies and characterized by a relatively high percentage of c-kit(+) cells. The cells were seeded in macroporous alginate scaffolds and after cultivation for 7 days under static conditions, some of the constructs were transferred into a perfusion bioreactor, which was operated for an additional 14 days. A robust and highly reproducible human CPC (hCPC) expansion of more than seven-fold was achieved under the 3D perfusion culture conditions, while under static conditions, the expansion of CPCs was limited only to the first 7 days, after which it leveled-off. On day 21 of perfusion cultivation, the expanded cells exhibited a higher expression level of the progenitor marker c-kit, suggesting that the c-kit-positive CPCs are the main cell population undergoing proliferation. The profile of the spontaneous differentiation in the perfused construct was different from that in the static cultivated constructs; genes typical for cardiac and endothelial cell lineages were more widely expressed in the perfused constructs. By contrast, the differentiation to osteogenic (Von Kossa staining and alkaline phosphatase activity) and adipogenic (Oil Red staining) lineages was reduced in the perfused constructs compared with static cultivated constructs. Collectively, our results indicate that 3D perfusion cultivation mode is an appropriate system for robust expansion of human CPCs while maintaining their progenitor state and differentiation potential into the cardiovascular cell lineages.

Tissue Engineering Bone Using Autologous Progenitor Cells in the Peritoneum

Despite intensive research efforts, there remains a need for novel methods to improve the ossification of scaffolds for bone tissue engineering. Based on a common phenomenon and known pathological conditions of peritoneal membrane ossification following peritoneal dialysis, we have explored the possibility of regenerating ossified tissue in the peritoneum. Interestingly, in addition to inflammatory cells, we discovered a large number of multipotent mesenchymal stem cells (MSCs) in the peritoneal lavage fluid from mice with peritoneal catheter implants. The osteogenic potential of these peritoneal progenitor cells was demonstrated by their ability to easily infiltrate decalcified bone implants, produce osteocalcin and form mineralized bone in 8 weeks. Additionally, when poly(l-lactic acid) scaffolds loaded with bone morphogenetic protein-2 (a known osteogenic differentiation agent) were implanted into the peritoneum, signs of osteogenesis were seen within 8 weeks of implantation. The results of this investigation support the concept that scaffolds containing BMP-2 can stimulate the formation of bone in the peritoneum via directed autologous stem and progenitor cell responses.

Production of endothelial no-synthase gene-enhanced autologous progenitor cell therapies for cardio-pulmonary diseases

Production of endothelial no-synthase gene-enhanced autologous progenitor cell therapies for cardio-pulmonary diseases

Anal Sphincter Repair With Muscle Progenitor Cell Transplantation: Serial Assessment With Iron Oxide–Enhanced MRI

The purpose of this study was to assess homing of ultrasmall superparamagnetic iron oxide (USPIO)-labeled muscle progenitor cells in an experimental rabbit model of anal sphincter repair using MRI. Twelve rabbits underwent external anal sphincterotomy and randomly received injection of either autologous muscle progenitor cells labeled with USPIO at a concentration of 4 mg/10(6) cells (experimental group) or saline (control group) at the site of sphincter damage. In vivo MRI, electromyography, and manometry were performed before, 1 hour after, and 1, 2, and 4 weeks after the injection. At the end time point, anal sphincter sections were obtained for histologic analysis. Semiquantitative analysis of fibrosis, desmin, iron, CD3, and CD68 was performed using two microscopic fields in two distinct regions of the sphincter according to either presence (zone I) or absence (zone II) of signal loss on the corresponding MR images. Labeling efficiency was 88.67% and did not influence cell viability. On follow-up images of the cell-transplanted rabbits, significant influence was reported at 1 hour, 1 and 2 weeks after transplantation. The maximum signal loss was detected at 1 hour (75.7%). Regenerating myofibers stained positively for desmin and mainly correlated to zone I on MR images. Clusters of iron-positive particles were detectable in the myofibers located mainly at the site of injection, which correlated well to zone I. Significant signal loss and Perls Prussian blue-positive area were not detected in the control group. Functional studies showed significant improvement in sphincter pressure and electrical activity in the experimental group after 4 weeks (p<0.001). Our results support the potential of iron oxide-enhanced MRI for serial monitoring of transplanted cells in an animal model of anal sphincter repair.