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Human platelet lysate improves human cord blood derived ECFC survival and vasculogenesis in three dimensional (3D) collagen matrices.

ABSTRACT: Human cord blood (CB) is enriched in circulating endothelial colony forming cells (ECFCs) that display high proliferative potential and in vivo vessel forming ability. Since diminished ECFC survival is known to dampen the vasculogenic response in vivo, we tested how long implanted ECFC survive and generate vessels in three-dimensional (3D) type I collagen matrices in vitro and in vivo. We hypothesized that human platelet lysate (HPL) would promote cell survival and enhance vasculogenesis in the 3D collagen matrices. We report that the percentage of ECFC co-cultured with HPL that were alive was significantly enhanced on days 1 and 3 post-matrix formation, compared to ECFC alone containing matrices. Also, co-culture of ECFC with HPL displayed significantly more vasculogenic activity compared to ECFC alone and expressed significantly more pro-survival molecules (pAkt, p-Bad and Bcl-xL) in the 3D collagen matrices in vitro. Treatment with Akt1 inhibitor (A-674563), Akt2 inhibitor (CCT128930) and Bcl-xL inhibitor (ABT-263/Navitoclax) significantly decreased the cell survival and vasculogenesis of ECFC co-cultured with or without HPL and implicated activation of the Akt1 pathway as the critical mediator of the HPL effect on ECFC in vitro. A significantly greater average vessel number and total vascular area of human CD31(+) vessels were present in implants containing ECFC and HPL, compared to the ECFC alone implants in vivo. We conclude that implantation of ECFC with HPL in vivo promotes vasculogenesis and augments blood vessel formation via diminishing apoptosis of the implanted ECFC.

SUBMITTER: Kim H

PROVIDER: S-EPMC4537830 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Human platelet lysate improves human cord blood derived ECFC survival and vasculogenesis in three dimensional (3D) collagen matrices.

Kim Hyojin H Prasain Nutan N Vemula Sasidhar S Ferkowicz Michael J MJ Yoshimoto Momoko M Voytik-Harbin Sherry L SL Yoder Mervin C MC

Microvascular research 20150627

Human cord blood (CB) is enriched in circulating endothelial colony forming cells (ECFCs) that display high proliferative potential and in vivo vessel forming ability. Since diminished ECFC survival is known to dampen the vasculogenic response in vivo, we tested how long implanted ECFC survive and generate vessels in three-dimensional (3D) type I collagen matrices in vitro and in vivo. We hypothesized that human platelet lysate (HPL) would promote cell survival and enhance vasculogenesis in the ...[more]

PMID: 26122935

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Project description:BackgroundCurrent treatments for several corneal lesions show limited efficacy. Here we report the clinical evaluation of the efficacy of a novel eye drop preparation produced in a public cord blood (CB) bank.Material and methodsIn a multicentre, retrospective, consecutive case study we evaluated 33 patients (46 eyes) unresponsive to conventional treatments who required urgent intervention. The patients were given allogeneic eye drops obtained from cord blood platelet lysate (CBED) to treat severe ocular surface lesions under a compassionate use protocol. The CBED were prepared from CB units donated for haematopoietic stem cell transplantation that did not contain the minimum stem cell dose required for this use. Patients were grouped by acute conditions (neurotrophic ulcers: group I; other corneal ulcers: group II; corneal burns: group III), and chronic conditions (ocular graft-versus-host disease: group IV; severe dry eye syndrome: group V). The patients received one or two drops of the product to the affected eye four to six times per day for 19 days. A further 19-day cycle of treatment could be repeated according to the initial clinical response.ResultsPatients received a median of 19 CBED vials (interquartile range 19-57, range 19-442) to complete the therapy. Group I-II-III patients showed full and partial ulcer recovery in 25 (78%) and six (19%) eyes respectively. One eye (3%) did not respond to treatment. For groups IV-V improvement was reported for 12 (85%) eyes and lesions worsened on treatment in both eyes (15%) of one patient. No severe adverse events were directly attributed to CBED.DiscussionPromptly available CBED resulted in a well-tolerated allogeneic treatment that showed evidence of efficacy in this cohort of patients. These positive results support further studies on CBED from platelet lysate as a novel product of CB banks. A prospective clinical trial in neurotrophic keratitis (NCT03084861) is ongoing to confirm these preliminary data.

Project description:The mechanobiological behavior of mesenchymal stem cells (MSCs) in two- (2D) or three-dimensional (3D) cultures relies on the formation of actin filaments which occur as stress fibers and depends on mitochondrial dynamics involving vimentin intermediate filaments. Here we investigate whether human platelet lysate (HPL), that can potentially replace fetal bovine serum for clinical-scale expansion of functional cells, can modulate the stress fiber formation, alter mitochondrial morphology, change membrane elasticity and modulate immune regulatory molecules IDO and GARP in amnion derived MSCs. We can provide evidence that culture supplementation with HPL led to a reduction of stress fiber formation in 2D cultured MSCs compared to a conventional growth medium (MSCGM). 3D MSC cultures, in contrast, showed decreased actin concentrations independent of HPL supplementation. When stress fibers were further segregated by their binding to focal adhesions, a reduction in ventral stress fibers was observed in response to HPL in 2D cultured MSCs, while the length of the individual ventral stress fibers increased. Dorsal stress fibers or transverse arcs were not affected. Interestingly, ventral stress fiber formation did not correlate with membrane elasticity. 2D cultured MSCs did not show differences in the Young's modulus when propagated in the presence of HPL and further cultivation to passage 3 also had no effect on membrane elasticity. In addition, HPL reduced the mitochondrial mass of 2D cultured MSCs while the mitochondrial mass in 3D cultured MSCs was low initially. When mitochondria were segregated into punctuate, rods and networks, a cultivation-induced increase in punctuate and network mitochondria was observed in 2D cultured MSCs of passage 3. Finally, mRNA and protein expression of the immunomodulatory molecule IDO relied on stimulation of 2D culture MSCs with pro-inflammatory cytokines IFN-? and TNF-? with no effect upon HPL supplementation. GARP mRNA and surface expression was constitutively expressed and did not respond to HPL supplementation or stimulation with IFN-? and TNF-?. In conclusion, we can say that MSCs cultivated in 2D and 3D are sensitive to medium supplementation with HPL with changes in actin filament formation, mitochondrial dynamics and membrane elasticity that can have an impact on the immunomodulatory function of MSCs.

Dataset Information

Human platelet lysate improves human cord blood derived ECFC survival and vasculogenesis in three dimensional (3D) collagen matrices.

Publications

Human platelet lysate improves human cord blood derived ECFC survival and vasculogenesis in three dimensional (3D) collagen matrices.

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