Non-cultured Cells Research Articles

Fetal mosaicism, should conventional karyotype always be performed?

The application of classical cytogenetic and DNA-based molecular techniques to detect cell lineages of mosaicism derived from cultured or noncultured fetal cells may result in discordant results. This retrospective study aimed to assess the inconsistent diagnostic outcomes, technical availability, and limitations of chromosomal microarray analysis (CMA) and karyotyping for mosaicism. A total of 75 fetuses diagnosed with mosaicism by karyotype analysis or CMA were selected, and the results from both the methods were compared and further analyzed. A total of 42 (56%, 42/75) CMA results were consistent with karyotypes, consisting of 22 cases of mosaic sex chromosomal abnormalities, 8 routine autosomal aneuploidy cases, 8 other autosome aneuploidy cases, 3 large cryptic genomic rearrangements, and 1 small supernumerary marker chromosome. Discrepancy between karyotype analysis and CMA was observed in 33 (44%, 33/75) mosaicisms involving 15 sex chromosomal abnormalities, 1 routine autosomal aneuploidies, 5 other autosome aneuploidy cases, 8 large cryptic genomic rearrangements, and 4 small supernumerary marker chromosomes. Considering the disparities between methods as well as the cell populations analyzed, both CMA and karyotype analysis have their own advantages and disadvantages. Therefore, CMA should ideally be used in combination with karyotyping to detect more cases of mosaicism than using either test alone.

DLP printed hDPSC-loaded GelMA microsphere regenerates dental pulp and repairs spinal cord

Dental pulp regeneration is ideal for irreversible pulp or periapical lesions, and in situ stem cell therapy is one of the most effective therapies for pulp regeneration. In this study, we provided an atlas of the non-cultured and monolayer cultured dental pulp cells with single-cell RNA sequencing and analysis. Monolayer cultured dental pulp cells cluster more closely together than non-cultured dental pulp cells, suggesting a lower heterogeneous population with relatively consistent clusters and similar cellular composition. We successfully fabricated hDPSC-loaded microspheres by layer-by-layer photocuring with a digital light processing (DLP) printer. These hDPSC-loaded microspheres have improved stemness and higher multi-directional differentiation potential, including angiogenic, neurogenic, and odontogenic differentiation. The hDPSC-loaded microspheres could promote spinal cord regeneration in rat spinal cord injury models. Moreover, in heterotopic implantation tests on nude mice, CD31, MAP2, and DSPP immunofluorescence signals were observed, implying the formation of vascular, neural, and odontogenetic tissues. In situ experiments in minipigs demonstrated highly vascularized dental pulp and uniformly arranged odontoblast-like cells in root canals of incisors. In short, hDPSC-loaded microspheres can promote full-length dental pulp regeneration at the root canals' coronal, middle, and apical sections, particularly for blood vessels and nerve formation, which is a promising therapeutic strategy for necrotic pulp.

A Comparative Study between Autologous Noncultured Trypsinized Melanocytes-Keratinocytes Cell Suspension and Autologous Noncultured Nontrypsinized Epidermal Cells Grafting (Jodhpur Technique) in Stable Vitiligo.

Noncultured epidermal cell suspension (NCES) and Jodhpur technique (JT) are two treatment options in vitiligo, in which the basic principle is the transfer of melanocytes from uninvolved skin to stable vitiligo patch in the form of either tissue graft or cellular graft. The aim of this study was to evaluate effectiveness of two different treatment methods (NCES and JT) in stable vitiligo. This was a randomized comparative study, which included 45 patients with 153 stable vitiligo patches. Cases were randomly divided into two groups. Appropriate statistical analysis was done. A value of P < 0.05 was considered statistically significant. Of 45 cases, female-to-male ratio was 1.36:1. Mean duration of disease and stability were found to be 10.44 ± 5.24 and 3.83 ± 2.31 years, respectively. Mean size of treated area for NCES and JT was 10.11 ± 10.31 and 10.13 ± 10.29 cm2, respectively. Earliest repigmentation was found at 3 weeks in NCES and 6 weeks in JT. At 6 weeks, repigmentation was better in NCES, whereas later JT showed better pigmentation. Follow-up was done at 8 days and 6, 10, 14, and 24 weeks. With respect to color match at 6 weeks, JT was found superior; however, at 24 weeks both showed similar efficacy. Mean reduction in daily life quality index was significantly decreased post-surgery. This was a single-center, small sample size study. Melanocyte plus keratinocyte cell transfer is very effective in the treatment of stable vitiligo. On the basis of the results, JT may show significant contribution in vitiligo.

GAS6 Promotes Robust Expansion of Human Hematopoietic Stem and Progenitor Cells with High Serial Engraftment Activity

Hematopoietic stem cell transplantation is a curative therapy for over 30 hematopoietic illnesses. The inability to sufficiently expand functional HSCs in cultures has partly hampered their therapeutic potential and limited molecular understanding of HSCs. Stem cell agonists (SCA) such as UM171 and StemReginin1 (SR1) have been shown to promote HSC expansion. Recently, we discovered that Ascorbic acid-2-phosphate (AA2P) can act as a novel SCA. Furthermore, using mathematical modeling we formulated a series of stem cell agonist cocktails (SCACs) composed of varying concentrations of 4 SCAs. SCACs promote robust expansion of HSPCs and limited dilution transplant assay revealed that the lead cocktail X2A promoted 15- and 4-fold expansions of scid repopulating cells, when compared to non-cultured cells or cells expanded with the combination of UM171 and SR1 (Manesia et al., 2019). The objective of the present study was to characterize the molecular programs supporting the strong expansion of HSCs in X2A cultures. RNA-Seq data from SCACs expanded CD34+CD45RA- cells revealed the upregulation of several receptor tyrosine kinases (RTKs) including AXL, but also its ligand GAS6 preferentially with X2A. AXL was recently shown to be essential for the self-renewal of leukemia initiating cells. Single cell RNA-seq analyses of CD34+ cells was done to characterize the graft composition of SCACs expanded HSPCs. These analyses revealed that AXL expression is enriched within CD34+CD45RA-CD90+CD49f+ HSC-enriched clusters. Image-flowcytometry analyses confirmed that HSC-enriched cell populations respond to GAS6-activation, resulting in the activation of ERK1/2 downstream of AXL activation. Epigenetic analysis revealed that GAS6/AXL upregulation is regulated by AA2P through DNA-demethylation in a TET dependent manner. Using genetic knockdown, chemical inhibition and GAS6 neutralizing antibody approaches we confirmed that GAS6 activation of AXL drives human HSPC expansion within X2A. In addition, supplementation of SCAC or single SCA-based cultures with GAS6 significantly enhanced the expansion of HSPC subsets with little induction of differentiation (e.g., 640- Vs. 1975-fold for CD34+CD45RA- HSPC in X2A and X2A+GAS6 cultures, respectively (n=3, p<0.05)). Transplant assays with day-14 expanded HSPC (progeny of 850 CD34+ starting cells/mouse) revealed that GAS6 supplementation in X2A cultures increased ensuing engraftment by 10-fold in both primary and secondary NSG mice (n=2, p<0.0001). For instance, the % of human CD45+ BM cells in secondary mice were of 2.3±0.1% and 25.7±1.3% in X2A and X2A+GAS6 groups. Remarkably, the net numbers of human colony-forming unit and CD34+ BM cells were increased by 7- and 15-fold (p<0.0001) in primary, and by 20- and 26-fold (p<0.0001) in secondary transplants by GAS6, respectively. In contrast, GAS6 neutralization in cultures impaired ensuing serial engraftment by 8- and 30-fold respectively Vs. X2A control (p<0.01). Lastly, SCAC and GAS6 were also shown to improve the expansion of adult G-CSF mobilized peripheral blood stem cells. In conclusion, we demonstrate that AA2P-mediated HSPC expansion is driven through DNA demethylation leading to enhanced expression of AXL and its ligand GAS6. We also show for the first time that GAS6 is a potent growth factor that promote robust expansion of human HSPCs including serial engrafting HSCs resulting in unexpectedly high engraftment.

Cell therapy in corneal endothelial disease.

Endothelial keratoplasty is the current gold standard for treating corneal endothelial diseases, achieving excellent visual outcomes and rapid rehabilitation. There are, however, severe limitations to donor tissue supply and uneven access to surgical teams and facilities across the globe. Cell therapy is an exciting approach that has shown promising early results. Herein, we review the latest developments in cell therapy for corneal endothelial disease. We highlight the work of several groups that have reported successful functional outcomes of cell therapy in animal models, with the utilization of human embryonic stem cells, human-induced pluripotent stem cells and cadaveric human corneal endothelial cells (CECs) to generate populations of CECs for intracameral injection. The use of corneal endothelial progenitors, viability of cryopreserved cells and efficacy of simple noncultured cells, in treating corneal decompensation is of particular interest. Further additions to the collective understanding of CEC physiology, and the process of cultivating and administering effective cell therapy are reviewed as well. The latest developments in cell therapy for corneal endothelial disease are presented. The continuous growth in this field gives rise to the hope that a viable solution to the large numbers of corneal blind around the world will one day be reality.

Autofluorescence in freshly isolated adult human liver sinusoidal cells.

Autofluorescent granules of various sizes were observed in primary human liver endothelial cells (LSECs) upon laser irradiation using a wide range of wavelengths. Autofluorescence was detected in LAMP-1 positive vesicles, suggesting lysosomal location. Confocal imaging of freshly prepared cultures and imaging flow cytometry of non-cultured cells revealed fluorescence in all channels used. Treatment with a lipofuscin autofluorescence quencher reduced autofluorescence, most efficiently in the near UV-area. These results, combined with the knowledge of the very active blood clearance function of LSECs support the notion that lysosomally located autofluorescent material reflected accumulation of lipofuscin in the intact liver. These results illustrate the importance of careful selection of exogenous fluorophores, especially when labelling of live cells where the quencher is not compatible.

Non-cultured epidermal cells suspended in either platelet-rich plasma or ringer lactate for stable vitiligo: A prospective comparative study.

Noncultured epidermal cell suspension (NCES) transplantation is a commonly used surgical treatment for resistant stable vitiligo. The combination of platelet-rich plasma (PRP) with different therapeutic modalities for vitiligo yielded higher repigmentation response, probably due to platelet-derived growth factors. To evaluate the efficacy of PRP-suspended NCES compared to NCES suspended in Ringer's lactate (RL) solution in the treatment of stable vitiligo. A prospective comparative study was conducted on 40 patients with stable vitiligo. They were divided into two equal groups: group A (treated with RL-suspended NCES) and group B (treated with PRP-suspended NCES). All patients were followed-up for 6months for assessment of their therapeutic response regarding clinical outcomes and immunohistochemical expression of HMB-45 in lesional skin. Patients treated with PRP-suspended NCES showed a significantly higher repigmentation response compared to those treated with RL-suspended NCES at 1, 3, and 6months after treatment (p=0.015, 0.023, 0.029, respectively). The expression of HMB-45significantly increased in both groups after therapy, but without a significant difference between the two groups. The repigmentation response of NCES can be enhanced by suspending the melanocytes in autologous PRP.

Attenuation of Knee Osteoarthritis Progression in Mice through Polarization of M2 Macrophages by Intra-Articular Transplantation of Non-Cultured Human Adipose-Derived Regenerative Cells.

Adipose-derived regenerative cells (ADRCs) are non-cultured heterogeneous or mixed populations of cells obtained from adipose tissue by collagenase digestion. The injection of ADRCs have been tried clinically for the treatment of osteoarthritis (OA). The purpose of this study was to evaluate the effect of intra-articular transplantation of human ADRCs on OA progression in mice and the effect of ADRCs on macrophage polarization. In in vivo experiments, BALB/c-nu mice with knee OA received intra-articular transplantation of either phosphate buffered-saline or human ADRCs. OA progression was evaluated histologically and significantly attenuated in the ADRC group at both four and eight weeks postoperatively. The expression of OA-related proteins in the cartilage and macrophage-associated markers in the synovium were examined by immunohistochemistry. The numbers of MMP-13-, ADAMTS-5-, IL-1β-, IL-6- and iNOS-positive cells significantly decreased, and type II collagen- and CD206-positive cells were more frequently detected in the ADRC group compared with that in the control group. In vitro co-culture experiments showed that ADRCs induced macrophage polarization toward M2. The results of this study suggest that the intra-articular transplantation of human ADRCs could attenuate OA progression possibly by reducing catabolic factors in chondrocytes and modulating macrophage polarization.

Tissue Harvesting Site Effect on the Canine Adipose Stromal Vascular Fraction Quantity and Quality.

Simple SummaryAdipose stromal vascular fraction (SVF) cells are freshly isolated non-cultured mesenchymal stem cells, which have been recently applied in the treatment of several musculoskeletal inflammatory conditions in dogs. However, the best adipose tissue (AT) sampling site is still challenging. This study first addressed the ideal AT harvesting site in canines ranging between middle and old age, the most susceptible age to chronic musculoskeletal problems. Our results showed that the peri-ovarian region is the best AT harvesting site, which yields high amounts of SVF cells with enough adipose-derived stem cells. These data may help the further set-up of cell-based regenerative therapies at the preclinical and experimental level in canines.Mesenchymal stem cells (MSCs) constitute a great promise for regenerative therapy, but these cells are difficultly recovered in large amounts. A potent alternative is the stromal vascular fraction (SVF), non-cultured MSCs, separated from adipose tissue (AT). We aim to evaluate AT harvesting site effect on the SVF cells’ quantity and quality in dogs. Subcutaneous abdominal fat, falciform ligament and peri-ovarian fat were sampled. After SVF isolation, the trypan blue exclusion test and a hemocytometer were used to assess the cell viability and cellular yield. SVF cells were labeled for four surface antigenic markers, clusters of differentiation CD90, CD44, CD29, and CD45, and then examined by flow cytometry. Semi-quantitative RT-PCR was used to evaluate the gene expression of the former markers in addition to OCT-4 and CD34. SVF cells in the peri-ovarian AT recorded the highest viability% (99.63 ± 0.2%), as well as a significantly higher cellular yield (36.87 ± 19.6 × 106 viable cells/gm fat, p < 0.001) and a higher expression of adipose-derived mesenchymal stem cells AD-MSCs surface markers than that of other sites. SVF cells from the peri-ovarian site revealed a higher expression of MSC markers (CD90, CD44, and CD29) and OCT-4 compared to the other sites, with weak CD45 and CD34 expressions. The positive OCT-4 expression demonstrated the pluripotency of SVF cells isolated from different sites. To conclude, the harvesting site is a strong determinant of SVF cells’ quantity and quality, and the peri-ovarian site could be the best AT sampling site in dogs.

Age-associated changes in the transcriptomes of non-cultured adipose-derived stem cells from young and old mice assessed via single-cell transcriptome analysis.

Adipose-derived stem cells (ASCs) exhibit self-renewal and pluripotency. The differentiation potency of ASCs has been reported to deteriorate with aging; however, relevant studies used ASCs that were isolated and subcultured several times. It is still unclear whether subcultured ASCs accurately reflect the in vivo state. To address this question, we used freshly isolated stromal vascular fractions (SVFs) and performed comprehensive single-cell transcriptome analysis. In this study, we identified three cell populations as putative ASC candidates in SVFs and three novel ASC-related genes: Adamts7, Snai2, and Tgfbr1, that are reported to be negative regulators of cell differentiation. Moreover, we identified age-associated high gene expression levels of Adamts7, Egfr, and Igfbp4 in the earliest differentiation stage of ASCs. These results suggest that aging may make it impossible to maintain the stringency of the regulation of the expression of some genes related to ASC differentiation.

A Novel Approach of Harvesting Viable Single Cells from Donor Corneal Endothelium for Cell-Injection Therapy.

Donor corneas with low endothelial cell densities (ECD) are deemed unsuitable for corneal endothelial transplantation. This study evaluated a two-step incubation and dissociation harvesting approach to isolate single corneal endothelial cells (CECs) from donor corneas for corneal endothelial cell-injection (CE-CI) therapy. To isolate CECs directly from donor corneas, optimization studies were performed where donor Descemet’s membrane/corneal endothelium (DM/CE) were peeled and incubated in either M4-F99 or M5-Endo media before enzymatic digestion. Morphometric analyses were performed on the isolated single cells. The functional capacities of these cells, isolated using the optimized simple non-cultured endothelial cells (SNEC) harvesting technique, for CE-CI therapy were investigated using a rabbit bullous keratopathy model. The two control groups were the positive controls, where rabbits received cultured CECs, and the negative controls, where rabbits received no CECs. Whilst it took longer for CECs to dislodge as single cells following donor DM/CE incubation in M5-Endo medium, CECs harvested were morphologically more homogenous and smaller compared to CECs obtained from DM/CE incubated in M4-F99 medium (p < 0.05). M5-Endo medium was hence selected as the DM/CE incubation medium prior to enzymatic digestion to harvest CECs for the in vivo cell-injection studies. Following SNEC injection, mean central corneal thickness (CCT) of rabbits increased to 802.9 ± 147.8 μm on day 1, gradually thinned, and remained clear with a CCT of 385.5 ± 38.6 μm at week 3. Recovery of corneas was comparable to rabbits receiving cultured CE-CI (p = 0.40, p = 0.17, and p = 0.08 at weeks 1, 2, and 3, respectively). Corneas that did not receive any cells remained significantly thicker compared to both SNEC injection and cultured CE-CI groups (p < 0.05). This study concluded that direct harvesting of single CECs from donor corneas for SNEC injection allows the utilization of donor corneas unsuitable for conventional endothelial transplantation.

The synovial fluid from patients with focal cartilage defects contains mesenchymal stem/stromal cells and macrophages with pro- and anti-inflammatory phenotypes

ObjectiveThe synovial fluid (SF) of patients with focal cartilage defects contains a population of poorly characterised cells that could have pathophysiological implications in early osteoarthritis and joint tissue repair. We have examined the cells within SF of such joints by determining their chondrogenic capacity following culture expansion and establishing the phenotypes of the macrophage subsets in non-cultured cells. DesignKnee SF cells were obtained from 21 patients receiving cell therapy to treat a focal cartilage defect. Cell surface immunoprofiling for stem cell and putative chondrogenic markers, and the expression analysis of key chondrogenic and hypertrophic genes were conducted on culture-expanded SF cells prior to chondrogenesis. Flow cytometry was also used to determine the macrophage subsets in freshly isolated SF cells. ResultsImmunoprofiling revealed positivity for the monocyte/macrophage marker (CD14), the haematopoietic/endothelial cell marker (CD34) and mesenchymal stem/stromal cell markers (CD73, CD90, CD105) on culture expanded cells. We found strong correlations between the presence of CD14 and the vascular cell adhesion marker, CD106 (r = 0.81, p = 0.003). Collagen type II expression after culture expansion positively correlated with GAG production (r = 0.73, p = 0.006), whereas CD90 (r = −0.6, p = 0.03) and CD105 (r = −0.55, p = 0.04) immunopositivity were inversely related to GAG production. Freshly isolated SF cells were positive for both pro- (CD86) and anti-inflammatory markers (CD163 and CD206). ConclusionsThe cellular content of the SF from patients with focal cartilage injuries is comprised of a heterogeneous population of reparative and inflammatory cells. Additional investigations are needed to understand the role played by these cells in the attempted repair and inflammatory process in diseased joints.

A Comparative Study between Follicular Unit Transplantation and Autologous Non-cultured Non-trypsinized Epidermal Cells Grafting (Jodhpur Technique) in Stable Vitiligo.

Introduction:Several modalities are currently available for the treatment of vitiligo, but generally they do not result in complete cure of the disease. Despite the limitations and some side effects, surgical modalities appear to be the method of choice in recalcitrant stable vitiligo. Both Jodhpur technique (JT) and follicular unit transplantation (FUT) are simpler and cheaper methods, requiring minimal infrastructure.Materials and Methods:Hundred patches in 30 patients were divided into Group A and Group B, that is, hair follicle transplantation and autologous non-cultured non-trypsinized epidermal cell suspension grafting (JT), respectively. Fifty patches were included in each group. Efficacy of both the techniques, extent of pigmentation, and color match assessment were the main objectives.Results:Excellent repigmentation (>75%), at the end of 20 weeks post surgery, was observed in 70% lesions in FUT group and 72% of lesions in JT group. Good response (extent of repigmentation of 50%–75%) was seen in 18% of lesions in FUT group as compared to 26% in JT group. JT and FUT technique were almost equal in producing excellent repigmentation. Color match was same for both the groups, that is, 49 of 50 patches showed same color as surrounding in both groups at the end of 20 weeks. Repigmentation of the depigmented hairs occurred in 11 of 46 patients with associated leukotrichia.Conclusion:Our study indicates that both JT and FUT are safe and effective techniques in terms of repigmentation but JT is somewhat superior to FUT in producing excellent and good repigmentation, side effects, patient satisfaction, and dermatology life quality index reduction, while the color match was almost same with both techniques.

The effect of BPIFA1/SPLUNC1 genetic variation on its expression and function in asthmatic airway epithelium

Bacterial permeability family member A1 (BPIFA1), also known as short palate, lung, and nasal epithelium clone 1 (SPLUNC1), is a protein involved in the antiinflammatory response. The goal of this study was to determine whether BPIFA1 expression in asthmatic airways is regulated by genetic variations, altering epithelial responses to type 2 cytokines (e.g., IL-13). Nasal epithelial cells from patients with mild to severe asthma were collected from the National Heart, Lung, and Blood Institute Severe Asthma Research Program centers, genotyped for rs750064, and measured for BPIFA1. To determine the function of rs750064, cells were cultured at air-liquid interface and treated with IL-13 with or without recombinant human BPIFA1 (rhBPIFA1). Noncultured nasal cells with the rs750064 CC genotype had significantly less BPIFA1 mRNA expression than the CT and TT genotypes. Cultured CC versus CT and TT cells without stimulation maintained less BPIFA1 expression. With IL-13 treatment, CC genotype cells secreted more eotaxin-3 than CT and TT genotype cells. Also, rhBPIFA1 reduced IL-13-mediated eotaxin-3. BPIFA1 mRNA levels negatively correlated with serum IgE and fractional exhaled nitric oxide. Baseline FEV1% levels were lower in the asthma patients with the CC genotype (n = 1,016). Our data suggest that less BPIFA1 in asthma patients with the CC allele may predispose them to greater eosinophilic inflammation, which could be attenuated by rhBPIFA1 protein therapy.

Rapid treatment of full‐thickness skin loss using ovine tendon collagen typeIscaffold with skin cells

The full-thickness skin wound is a common skin complication affecting millions of people worldwide. Delayed treatment of this condition causes the loss of skin function and integrity that could lead to the development of chronic wounds or even death. This study was aimed to develop a rapid wound treatment modality using ovine tendon collagen type I (OTC-I) bio-scaffold with or without noncultured skin cells. Genipin (GNP) and carbodiimide (EDC) were used to cross-link OTC-I scaffold to improve the mechanical strength of the bio-scaffold. The physicochemical, biomechanical, biodegradation, biocompatibility, and immunogenicity properties of OTC-I scaffolds were investigated. The efficacy of this treatment approach was evaluated in an in vivo skin wound model. The results demonstrated that GNP cross-linked OTC-I scaffold (OTC-I_GNP) had better physicochemical and mechanical properties compared with EDC cross-linked OTC-I scaffold (OTC-I_EDC) and noncross-link OTC-I scaffold (OTC-I_NC). OTC-I_GNP and OTC-I_NC demonstrated no toxic effect on cells as it promoted higher cell attachment and proliferation of both primary human epidermal keratinocytes and human dermal fibroblasts compared with OTC-I_EDC. Both OTC-I_GNP and OTC-I_NC exhibited spontaneous formation of bilayer structure in vitro. Immunogenic evaluation of OTC-I scaffolds, in vitro and in vivo, revealed no sign of immune response. Finally, implantation of OTC-I_NC and OTC-I_GNP scaffolds with noncultured skin cells demonstrated enhanced healing with superior skin maturity and microstructure features, resembling native skin in contrast to other treatment (without noncultured skin cells) and control group. The findings of this study, therefore, suggested that both OTC-I scaffolds with noncultured skin cells could be promising for the rapid treatment of full-thickness skin wound.

Inter-Cell and Inter-Chromosome Variability of 5-Hydroxymethylcytosine Patterns in Noncultured Human Embryonic and Extraembryonic Cells

5-hydroxymethylcytosine (5hmC) is an oxidative derivative of 5-methylcytosine (5mC). Recent studies have revealed a sharp difference in the levels of 5hmC in 2 opposite DNA strands of a given chromosome and a chromosome-wide cell-to-cell variability in mammalian cells. This asymmetric 5hmC distribution was found in cultured cells, which may not fully mimic in vivo epigenetic processes. We have checked whether inter-chromosome and inter-cell variability of 5hmC patterns is typical for noncultured human cells. Using indirect immunofluorescence, we analyzed the localization of 5hmC and its co-distribution with 5mC on direct preparations of mitotically active cells from human embryonic lung and chorionic cytotrophoblast samples. We demonstrated 3 types of chromosomes according to the 5hmC accumulation pattern: hydroxymethylated (5hmC in both sister chromatids), hemihydroxymethylated (5hmC in only 1 sister chromatid), and nonhydroxymethylated ones. Each accumulation type was not specific to any particular chromosome, resulting in different 5hmC patterns between homologous chromosomes, among chromosomes within each metaphase plate, among metaphases in one tissue, and between the tissues. The 5mC distribution was stable: chromosomes were methylated in R-bands and, especially in embryonic lung cells, in the heterochromatic regions 1q12, 9q12, and 16q11.2. Our results provide the first evidence of inter-cell and inter-chromosome variability of 5hmC patterns in human noncultured embryonic and extraembryonic cells.

Non-cultured epithelial cells in platelet-rich plasma for the resurfacing of massive and chronic fullthickness burns: A case series

Non-cultured epithelial cells in platelet-rich plasma for the resurfacing of massive and chronic fullthickness burns: A case series

Fresh Noncultured Endothelial Progenitor Cells Improve Neonatal Lung Hyperoxia-Induced Alveolar Injury

Treatment of preterm human infants with high oxygen can result in disrupted lung alveolar and vascular development. Local or systemic administration of endothelial progenitor cells (EPCs) is reported to remedy such disruption in animal models. In this study, the effects of both fresh (enriched for KDR) and cultured bone marrow (BM)‐derived cell populations with EPC characteristics were examined following hyperoxia in neonatal mouse lungs. Intraperitoneal injection of fresh EPCs into five‐day‐old mice treated with 90% oxygen resulted in full recovery of hyperoxia‐induced alveolar disruption by 56 days of age. Partial recovery in septal number following hyperoxia was observed following injection of short‐term cultured EPCs, yet aberrant tissue growths appeared following injection of long‐term cultured cells. Fresh and long‐term cultured cells had no impact on blood vessel development. Short‐term cultured cells increased blood vessel number in normoxic and hyperoxic mice by 28 days but had no impact on day 56. Injection of fresh EPCs into normoxic mice significantly reduced alveolarization compared with phosphate buffered saline‐injected normoxic controls. These results indicate that fresh BM EPCs have a higher and safer corrective profile in a hyperoxia‐induced lung injury model compared with cultured BM EPCs but may be detrimental to the normoxic lung. The appearance of aberrant tissue growths and other side effects following injection of cultured EPCs warrants further investigation. Stem Cells Translational Medicine 2017;6:2094–2105

Transplantation of Noncultured Stromal Vascular Fraction Cells of Adipose Tissue Ameliorates Osteonecrosis of the Jaw-Like Lesions in Mice.

The precise pathoetiology and effective treatment strategies for bisphosphonate-related osteonecrosis of the jaw (BRONJ) remain unknown. Transplantation of noncultured stromal vascular fraction (SVF) cells has been shown to be a useful method for regenerative medicine in place of stem cell therapy. This study investigated the effects of noncultured SVF transplantation on tooth extraction socket healing in mice. Both chemotherapeutic/bisphosphonate combination therapy for 7 weeks and tooth extraction of maxillary first molars at 3 weeks after drug administration were performed using female C57BL/6J mice. Osseous and soft tissue wound healing were validated at 4 weeks postextraction using gross wound healing and histomorphometry. Here, we created a new animal model of high-prevalence ONJ-like lesions that mimic human progression, because human ONJ mainly occurs in female patients taking both chemotherapeutic and bisphosphonate following tooth extraction. Moreover, mice with chemotherapeutic and bisphosphonate combination therapy for 5 weeks received SVF transplantation just after tooth extraction at 3 weeks post-drug administration. Euthanasia was performed at 2 weeks postextraction to assess the transplantation effects on wound healing using gross wound healing, histomorphometry, immunohistomorphometry, quantitative real-time polymerase chain reaction, and microcomputed tomography. We showed that systemic transplantation of noncultured SVF cells ameliorates ONJ-like lesions by improving both osseous and soft tissue healing of tooth extraction sockets. SVF therapy significantly increased blood vessels and the ratio of M2/M1 macrophages. In addition, SVF transplantation reduced the increases in tartrate-resistant acid phosphatase-positive (TRAP+ ) mononuclear cells (MNCs) and nonattached osteoclasts from the bone surface, which were significantly detected in the connective tissue of tooth extraction sockets and bone marrow by chemotherapeutic/bisphosphonate combination therapy. Our findings suggest that transplantation of noncultured SVF cells is a suitable treatment for BRONJ. Abnormal TRAP+ MNCs and nonattached osteoclasts in systemic and local environments may contribute to the development of BRONJ. © 2017 American Society for Bone and Mineral Research.

Hair follicle growth by stromal vascular fraction-enhanced adipose transplantation in baldness.

Great interest remains in finding new and emerging therapies for the treatment of male and female pattern hair loss. The autologous fat grafting technique is >100 years old, with a recent and dramatic increase in clinical experience over the past 10–15 years. Recently, in 2001, Zuk et al published the presence of adipose-derived stem cells, and abundant research has shown that adipose is a complex, biological active, and important tissue. Festa et al, in 2011, reported that adipocyte lineage cells support the stem cell niche and help drive the complex hair growth cycle. Adipose-derived regenerative cells (also known as stromal vascular fraction [SVF]) is a heterogeneous group of noncultured cells that can be reliably extracted from adipose by using automated systems, and these cells work largely by paracrine mechanisms to support adipocyte viability. While, today, autologous fat is transplanted primarily for esthetic and reconstructive volume, surgeons have previously reported positive skin and hair changes posttransplantation. This follicular regenerative approach is intriguing and raises the possibility that one can drive or restore the hair cycle in male and female pattern baldness by stimulating the niche with autologous fat enriched with SVF. In this first of a kind patient series, the authors report on the safety, tolerability, and quantitative, as well as photographic changes, in a group of patients with early genetic alopecia treated with subcutaneous scalp injection of enriched adipose tissue. The findings suggest that scalp stem cell-enriched fat grafting may represent a promising alternative approach to treating baldness in men and women.