Lyophilized Platelet-rich plasma for the management of thin endometrium and facilitation of in-vitro fertilization.

OBJECTIVE: Adequate endometrial thickness has been considered an important parameter for hormonal response and blastocyst implantation in assisted reproduction therapies. While there is no consensus on the exact thickness of the endometrium considered 'adequate,' a thin endometrium (<7mm) has been associated with compromised outcomes in assisted reproduction therapies. Platelet-rich plasma (PRP), which is a concentrate obtained from peripheral blood, is a rich source of growth factors that play important roles in various cellular processes. The objective is to utilize lyophilized PRP (LPRP) to increase the thickness of the endometrium and enhance the outcomes of embryo transfer in women with poor response to previous in-vitro fertilization procedures. METHODS: This study enrolled nine women between 23 and 42 years of age, with a thin endometrium, who had undergone multiple previous unsuccessful assisted reproduction procedures. All patients underwent intrauterine infusion of LPRP, followed by frozen-thawed embryo transfer after 2-3 days. RESULTS: Endometrial thickness was assessed by ultrasound 2 weeks after LPRP infusion, which showed improved thickness in all patients (range, 0.7-2.2mm). Clinical pregnancy occurred in all patients and eight out of nine patients are currently between 9 weeks and 27 weeks of gestation. Twin fetal heartbeats were not detected at the eighth week in one patient. CONCLUSION: Infusion of LPRP was found to be beneficial to increase endometrium thickness in all patients. This regenerative technique could be considered to enhance the outcomes of assisted reproduction techniques in a minimally-invasive manner, without any side effects.

Genome-wide DNA hypermethylation opposes healing in patients with chronic wounds by impairing epithelial-mesenchymal transition.

An extreme chronic wound tissue microenvironment causes epigenetic gene silencing. An unbiased whole-genome methylome was studied in the wound-edge tissue of patients with chronic wounds. A total of 4,689 differentially methylated regions (DMRs) were identified in chronic wound-edge skin compared with unwounded human skin. Hypermethylation was more frequently observed (3,661 DMRs) in the chronic wound-edge tissue compared with hypomethylation (1,028 DMRs). Twenty-six hypermethylated DMRs were involved in epithelial-mesenchymal transition (EMT). Bisulfite sequencing validated hypermethylation of a predicted specific upstream regulator TP53. RNA-Seq analysis was performed to qualify findings from methylome analysis. Analysis of the downregulated genes identified the TP53 signaling pathway as being significantly silenced. Direct comparison of hypermethylation and downregulated genes identified 4 genes, ADAM17, NOTCH, TWIST1, and SMURF1, that functionally represent the EMT pathway. Single-cell RNA-Seq studies revealed that these effects on gene expression were limited to the keratinocyte cell compartment. Experimental murine studies established that tissue ischemia potently induces wound-edge gene methylation and that 5'-azacytidine, inhibitor of methylation, improved wound closure. To specifically address the significance of TP53 methylation, keratinocyte-specific editing of TP53 methylation at the wound edge was achieved by a tissue nanotransfection-based CRISPR/dCas9 approach. This work identified that reversal of methylation-dependent keratinocyte gene silencing represents a productive therapeutic strategy to improve wound closure.

Endothelial Phospholipase Cγ2 Improves Outcomes of Diabetic Ischemic Limb Rescue Following VEGF Therapy.

Therapeutic vascular endothelial growth factor (VEGF) replenishment has met with limited success for the management of critical limb-threatening ischemia. To improve outcomes of VEGF therapy, we applied single-cell RNA sequencing (scRNA-seq) technology to study the endothelial cells of the human diabetic skin. Single-cell suspensions were generated from the human skin followed by cDNA preparation using the Chromium Next GEM Single-cell 3' Kit v3.1. Using appropriate quality control measures, 36,487 cells were chosen for downstream analysis. scRNA-seq studies identified that although VEGF signaling was not significantly altered in diabetic versus nondiabetic skin, phospholipase Cγ2 (PLCγ2) was downregulated. The significance of PLCγ2 in VEGF-mediated increase in endothelial cell metabolism and function was assessed in cultured human microvascular endothelial cells. In these cells, VEGF enhanced mitochondrial function, as indicated by elevation in oxygen consumption rate and extracellular acidification rate. The VEGF-dependent increase in cell metabolism was blunted in response to PLCγ2 inhibition. Follow-up rescue studies therefore focused on understanding the significance of VEGF therapy in presence or absence of endothelial PLCγ2 in type 1 (streptozotocin-injected) and type 2 (db/db) diabetic ischemic tissue. Nonviral topical tissue nanotransfection technology (TNT) delivery of CDH5 promoter-driven PLCγ2 open reading frame promoted the rescue of hindlimb ischemia in diabetic mice. Improvement of blood flow was also associated with higher abundance of VWF+/CD31+ and VWF+/SMA+ immunohistochemical staining. TNT-based gene delivery was not associated with tissue edema, a commonly noted complication associated with proangiogenic gene therapies. Taken together, our study demonstrates that TNT-mediated delivery of endothelial PLCγ2, as part of combination gene therapy, is effective in diabetic ischemic limb rescue.

Myo-Inositol in Fermented Sugar Matrix Improves Human Macrophage Function.

SCOPE: Reactive oxygen species production by innate immune cells plays a central role in host defense against invading pathogens at wound-site. A weakened host-defense results in persistent infection leading to wound chronicity. Fermented Papaya Preparation (FPP), a complex sugar matrix, bolsters respiratory burst activity and improves wound healing outcomes in chronic wound patients. The objective of the current study was to identify underlying molecular factor/s responsible for augmenting macrophage host defense mechanisms following FPP supplementation. METHODS AND RESULTS: In depth LC-MS/MS analysis of cells supplemented with FPP led to identification of myo-inositol as a key determinant of FPP activity towards improving macrophage function. Myo-inositol, in quantities that is present in FPP, significantly improved macrophage respiratory burst and phagocytosis via de novo synthesis pathway of ISYNA1. In addition, myo-inositol transporters, HMIT and SMIT1, played a significant role in such activity. Blocking these pathways using siRNA attenuated FPP-induced improved macrophage host defense activities. FPP supplementation emerged as a novel approach to increase intracellular myo-inositol levels. Such supplementation also modified wound microenvironment in chronic wound patients to augment myo-inositol levels in wound fluid. CONCLUSION: These observations indicate that myo-inositol in FPP influences multiple aspects of macrophage function critical for host defense against invading pathogens.

Oncostatin M Improves Cutaneous Wound Re-Epithelialization and Is Deficient under Diabetic Conditions.

Impaired re-epithelialization characterized by hyperkeratotic nonmigratory wound epithelium is a hallmark of nonhealing diabetic wounds. In chronic wounds, the copious release of oncostatin M (OSM) from wound macrophages is evident. OSM is a potent keratinocyte (KC) activator. This work sought to understand the signal transduction pathway responsible for wound re-epithelialization, the primary mechanism underlying wound closure. Daily topical treatment of full-thickness excisional wounds of C57BL/6 mice with recombinant murine OSM improved wound re-epithelialization and accelerated wound closure by bolstering KC proliferation and migration. OSM activated the Jak-signal transducer and activator of transcription pathway as manifested by signal transducer and activator of transcription 3 phosphorylation. Such signal transduction in the human KC induced TP63, the master regulator of KC function. Elevated TP63 induced ITGB1, a known effector of KC migration. In diabetic wounds, OSM was more abundant than the level in nondiabetic wounds. However, in diabetic wounds, OSM activity was compromised by glycation. Aminoguanidine, a deglycation agent, rescued the compromised KC migration caused by glycated OSM. Finally, topical application of recombinant OSM improved KC migration and accelerated wound closure in db/db mice. This work recognizes that despite its abundance at the wound site, OSM is inactivated by glycation, and topical delivery of exogenous OSM is likely to be productive in accelerating diabetic wound closure.

Autologous Minimally Invasive Cell-Based Therapy for Meniscal and Anterior Cruciate Ligament Regeneration.

The meniscus is a fibrocartilaginous tissue that acts as a "shock absorber," along with performing functions such as stabilization and lubrication of the joint, proprioception, and load distribution. Sudden twisting movements during weight bearing or trauma can cause injury to the menisci, which leads to symptoms such as pain, swelling, and difficulty in performing movements, among others. Conventional pharmacological and surgical treatments are effective in treating the condition; however, do not result in regeneration of healthy tissues. In this report, we highlight the role of cell-based therapy in the management of medial and lateral meniscal and anterior cruciate ligament tears in a patient who was unwilling to undergo surgical treatment. We injected autologous mesenchymal stem cells obtained from the bone marrow and adipose tissue and platelet-rich plasma into the joint of the patient at the area of injury, as well as intravenously. The results of our study corroborate with those previously reported in the literature regarding the improvement in clinical parameters and regeneration of meniscal tissue and ligament. Thus, based on previous literature and improvements noticed in our patient, cell-based therapy can be considered a safe and effective therapeutic modality in the treatment of meniscal tears and cruciate ligament injury.

Cell-based Therapy Approach for Drug-resistant Epilepsy.

Drug-resistant epilepsy (DRE) is a global public health problem. This category includes patients who continue to experience seizures despite long-term anti-epileptic medications. DRE can lead to severe disability and morbidity in older children and adults and is associated with increased risk of mortality than the general population. This report describes the case of a 15-year-old male patient with DRE successfully managed with autologous cell-based and hyperbaric oxygen therapy. The patient underwent two sessions of cell-based therapy consisting of cells derived from the bone marrow, adipose tissue, and peripheral blood followed by neuro-physiotherapy and oxygen therapy. Post-treatment, the patient experienced decrease in the frequency of seizures and reduction in the dosage of anti-epileptic medications. Electroencephalogram taken one year after the therapy revealed improvement in seizure activity. The outcomes in this case may be considered a preliminary finding in formulating more robust treatment strategies using cell-based therapy for DRE.