Direct implantation of hair-follicle-associated pluripotent (HAP) stem cells repairs intracerebral hemorrhage and reduces neuroinflammation in mouse model.

Intracerebral hemorrhage (ICH) is a leading cause of mortality with ineffective treatment. Hair-follicle-associated pluripotent (HAP) stem cells can differentiate into neurons, glial cells and many other types of cells. HAP stem cells have been shown to repair peripheral-nerve and spinal-cord injury in mouse models. In the present study, HAP stem cells from C57BL/6J mice were implanted into the injured brain of C57BL/6J or nude mice with induced ICH. After allo transplantation, HAP stem cells differentiated to neurons, astrocytes, oligodendrocytes, and microglia in the ICH site of nude mice. After autologous transplantation in C57BL/6J mice, HAP stem cells suppressed astrocyte and microglia infiltration in the injured brain. The mRNA expression levels of IL-10 and TGF-β1, measured by quantitative Real-Time RT-PCR, in the brain of C57BL/6J mice with ICH was increased by HAP-stem-cell implantation compared to the non-implanted mice. Quantitative sensorimotor function analysis, with modified limb-placing test and the cylinder test, demonstrated a significant functional improvement in the HAP-stem-cell-implanted C57BL/6J mice, compared to non-implanted mice. HAP stem cells have critical advantages over induced pluripotent stem cells, embryonic stem cells as they do not develop tumors, are autologous, and do not require genetic manipulation. The present study demonstrates future clinical potential of HAP-stem-cell repair of ICH, currently a recalcitrant disease

Superiority of [11C]methionine over [18F]deoxyglucose for PET Imaging of Multiple Cancer Types Due to the Methionine Addiction of Cancer

Positron emission tomography (PET) is widely used to detect cancers. The usual isotope for PET imaging of cancer is [18F]deoxyglucose. The premise of using [18F]deoxyglucose is that cancers are addicted to glucose (The Warburg effect). However, cancers are more severely addicted to methionine (The Hoffman effect). [11C]methionine PET (MET-PET) has been effectively used for the detection of glioblastoma and other cancers in the brain, and in comparison, MET-PET has been shown to be more sensitive and accurate than [18F]deoxyglucose PET (FDG-PET). However, MET-PET has been limited to cancers in the brain. The present report describes the first applications of MET-PET to cancers of multiple organs, including rectal, bladder, lung, and kidney. The results in each case show that MET-PET is superior to FDG-PET due to the methionine addiction of cancer and suggest that the broad application of MET-PET should be undertaken for cancer detection

Superiority of [¹¹C]methionine over [¹⁸F]deoxyglucose for PET Imaging of Multiple Cancer Types Due to the Methionine Addiction of Cancer

Positron emission tomography (PET) is widely used to detect cancers. The usual isotope for PET imaging of cancer is [18F]deoxyglucose. The premise of using [18F]deoxyglucose is that cancers are addicted to glucose (The Warburg effect). However, cancers are more severely addicted to methionine (The Hoffman effect). [11C]methionine PET (MET-PET) has been effectively used for the detection of glioblastoma and other cancers in the brain, and in comparison, MET-PET has been shown to be more sensitive and accurate than [18F]deoxyglucose PET (FDG-PET). However, MET-PET has been limited to cancers in the brain. The present report describes the first applications of MET-PET to cancers of multiple organs, including rectal, bladder, lung, and kidney. The results in each case show that MET-PET is superior to FDG-PET due to the methionine addiction of cancer and suggest that the broad application of MET-PET should be undertaken for cancer detection

Hair-follicle-associated pluripotent stem cells derived from cryopreserved intact human hair follicles sustain multilineage differentiation potential.

The bulge area of the hair follicle contains hair-follicle-associated pluripotent (HAP) stem cells. Here, we present effective cryopreservation procedures of the human hair follicle that preserve the differentiation potential of HAP stem cells. Whole hair follicles isolated from human scalp were cryopreserved by a slow-rate cooling medium and stored in liquid nitrogen. A careful thawing method was used to collect the upper parts of the human hair follicles which were cultured for four weeks in a Dulbecco's Modified Eagle's Medium with fetal bovine serum (FBS). Proliferating hair follicle cells were then shifted to DMEM/Ham's Nutrient Mixture F-12 medium without FBS and allowed to grow for one week. These proliferating cells were able to produce HAP stem cell colonies with multilineage differentiation capacity. They produced keratinocytes, smooth muscle cells, cardiac muscle cells, neurons and glial cells. Interestingly, these cryopreserved hair follicles produced pluripotent HAP stem cell colonies similar to fresh follicles. These findings suggest that the cryopreserved whole human hair follicle preserves the ability to produce HAP stem cells, which will enable any individual to preserve a bank of these stem cells for personalized regenerative medicine

Rat hair-follicle-associated pluripotent (HAP) stem cells can differentiate into atrial or ventricular cardiomyocytes in culture controlled by specific supplementation.

There has been only limited success to differentiate adult stem cells into cardiomyocyte subtypes. In the present study, we have successfully induced beating atrial and ventricular cardiomyocytes from rat hair-follicle-associated pluripotent (HAP) stem cells, which are adult stem cells located in the bulge area. HAP stem cells differentiated into atrial cardiomyocytes in culture with the combination of isoproterenol, activin A, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF), and cyclosporine A (CSA). HAP stem cells differentiated into ventricular cardiomyocytes in culture with the combination of activin A, BMP4, bFGF, inhibitor of Wnt production-4 (IWP4), and vascular endothelial growth factor (VEGF). Differentiated atrial cardiomyocytes were specifically stained for anti-myosin light chain 2a (MLC2a) antibody. Ventricular cardiomyocytes were specially stained for anti-myosin light chain 2v (MLC2v) antibody. Quantitative Polymerase Chain Reaction (qPCR) showed significant expression of MLC2a in atrial cardiomyocytes and MLC2v in ventricular cardiomyocytes. Both differentiated atrial and ventricular cardiomyocytes showed characteristic waveforms in Ca2+ imaging. Differentiated atrial and ventricular cardiomyocytes formed long myocardial fibers and beat as a functional syncytium, having a structure similar to adult cardiomyocytes. The present results demonstrated that it is possible to induce cardiomyocyte subtypes, atrial and ventricular cardiomyocytes, from HAP stem cells

Reversion of methionine addiction of osteosarcoma cells to methionine independence results in loss of malignancy, modulation of the epithelial-mesenchymal phenotype and alteration of histone-H3 lysine-methylation

Methionine addiction, a fundamental and general hallmark of cancer, known as the Hoffman Effect, is due to altered use of methionine for increased and aberrant transmethylation reactions. However, the linkage of methionine addiction and malignancy of cancer cells is incompletely understood. An isogenic pair of methionine-addicted parental osteosarcoma cells and their rare methionine-independent revertant cells enabled us to compare them for malignancy, their epithelial-mesenchymal phenotype, and pattern of histone-H3 lysine-methylation. Methionine-independent revertant 143B osteosarcoma cells (143B-R) were selected from methionine-addicted parental cells (143B-P) by their chronic growth in low-methionine culture medium for 4 passages, which was depleted of methionine by recombinant methioninase (rMETase). Cell-migration capacity was compared with a wound-healing assay and invasion capability was compared with a transwell assay in 143B-P and 143B-R cells in vitro. Tumor growth and metastatic potential were compared after orthotopic cell-injection into the tibia bone of nude mice in vivo. Epithelial-mesenchymal phenotypic expression and the status of H3 lysine-methylation were determined with western immunoblotting. 143B-P cells had an IC50 of 0.20 U/ml and 143B-R cells had an IC50 of 0.68 U/ml for treatment with rMETase, demonstrating that 143B-R cells had regained the ability to grow in low methionine conditions. 143B-R cells had reduced cell migration and invasion capability in vitro, formed much smaller tumors than 143B-P cells and lost metastatic potential in vivo, indicating loss of malignancy in 143B-R cells. 143B-R cells showed gain of the epithelial marker, ZO-1 and loss of mesenchymal markers, vimentin, Snail, and Slug and, an increase of histone H3K9me3 and H3K27me3 methylation and a decrease of H3K4me3, H3K36me3, and H3K79me3 methylation, along with their loss of malignancy. These results suggest that shifting the balance in histone methylases might be a way to decrease the malignant potential of cells. The present results demonstrate the rationale to target methionine addiction for improved sarcoma therapy

Beating cardiomyocytes differentiated from rat HAP stem cells.

Beating cardiomyocytes differentiated from rat HAP stem cells.</p

Rat HAP stem cells differentiated into atrial or ventricular cardiomyocytes.

(a-c) Immunofluorescence staining of the upper parts of rat vibrissa hair follicles, which were cultured for 21 days, differentiated into cTnI (a-upper; red) and cTnT (a-lower; red)-positive non-supplemented cardiomyocytes; cTnI (b-upper; red), cTnT (b-lower; red)- and MLC2a (b; green)-positive atrial cardiomyocytes; cTnI (c-upper; red), cTnT (c-lower; red)- and MLC2v (c; green)-positive ventricular cardiomyocytes. Nuclear staining with DAPI (blue). Scale bars; 100 μm. (d, e) qPCR analyses of cardiomyocytes differentiated from HAP stem cells. n = 4 per group for non-supplemented, atrial, and ventricular cardiomyocytes. Data are presented as mean ± SD. * P < 0.05, ** P < 0.005, two-sided Student’s t-test.</p