Hematopoietic Markers CD34 Research Articles

The biological characteristics of chicken embryo mesenchymal stem cells isolated from chorioallantoic membrane.

Mesenchymal stem cells (MSCs) derived from fetal membranes (FMs) have the potential to exhibit immunosuppression, improve blood flow, and increase capillary density during transplantation. In the field of medicine, opening up new avenues for disease treatment. Chicken embryo chorioallantoic membrane (CAM), as an important component of avian species FM structure, has become a stable tissue engineering material in vivo angiogenesis, drug delivery, and toxicology studies. Although it has been confirmed that chorionic mesenchymal stem cells (Ch-MSCs) can be isolated from the outer chorionic layer of FM, little is known about the biological characteristics of MSCs derived from chorionic mesodermal matrix of chicken embryos. Therefore, we evaluated the characteristics of MSCs isolated from chorionic tissues of chicken embryos, including cell proliferation ability, stem cell surface antigen, genetic stability, and in vitro differentiation potential. Ch-MSCs exhibited a broad spindle shaped appearance and could stably maintain diploid karyotype proliferation to passage 15 in vitro. Spindle cells were positive for multifunctional markers of MSCs (CD29, CD44, CD73, CD90, CD105, CD166, OCT4, and NANOG), while hematopoietic cell surface marker CD34, panleukocyte marker CD45, and epithelial cell marker CK19 were negative. In addition, chicken Ch-MSC was induced to differentiate into four types of mesodermal cells in vitro, including osteoblasts, chondrocytes, adipocytes, and myoblasts. Therefore, the differentiation potential of chicken Ch-MSC in vitro may have great potential in tissue engineering. In conclusion, chicken Ch-MSCs may be an excellent model cell for stem cell regenerative medicine and chorionic tissue engineering.

A Simple High Yield Technique for Isolation of Wharton's Jelly-derived Mesenchymal Stem Cell.

The isolation of Mesenchymal Stem Cells (MSCs) from various tissues is possible, with the umbilical cord emerging as a competitive alternative to bone marrow. In order to fulfill the demands of cell therapy, it is essential to generate stem cells on a clinical scale while minimizing time, cost, and contamination. Here is a simple and effective protocol for isolating MSC from Wharton's Jelly (WJ-MSC) using the explant method with various supplements. Utilizing the explant method, small fragments of Wharton's jelly from the human umbilical cord were cultured in a flask. The multipotency of the isolated cells, were confirmed by their differentiation ability to osteocyte and adipocyte. Additionally, the immunophenotyping of WJ-MSCs showed positive expression of CD73, CD90, and CD105, while remaining negative for hematopoietic markers CD34 and CD45, meeting the criteria for WJ-MSC identification. Following that, to evaluate cells' proliferative capacity, various supplements, including basic Fibroblast Growth Factor (bFGF), Non-Essential amino acids (NEA), and L-Glutamine (L-Gln) were added to either alpha-Minimal Essential Medium (α-MEM) or Dulbecco's Modified Eagle's Medium-F12 (DMEM-F12), as the basic culture media. WJ-MSCs isolated by the explant method were removed from the tissue after seven days and transferred to the culture medium. These cells differentiated into adipocyte and osteocyte lineages, expressing CD73, CD90, and CD105 positively and CD34 and CD45 negatively. The results revealed that addition of bFGF to α-MEM or DMEMF12 media significantly increased the proliferation of MSCs when compared to the control group. However, there were no significant differences observed when NEA or LGln were added. Although bFGF considerably enhances cell proliferation, our study demonstrates that MSCs can grow and expand when properly prepared Wharton's jelly tissues of the human umbilical cord.

A Nonenzymatic Procedure to Obtain Human Mesenchymal Stromal Cells from the Dermis.

Human mesenchymal stromal cells (MSCs) have gained significant interest as cell-based therapeutics for organ restoration in the field of regenerative medicine. More recently, substantial attention has been directed toward cell-free therapy, achieved through the utilization of soluble factors possessing trophic and immunomodulatory properties present in the MSC secretome. This collection of soluble factors can be found either freely in the secretome or packed within its vesicular fraction, known as extracellular vesicles (EVs). MSCs can be derived from various tissue sources, each involving different extraction methods and yielding varying cell amounts. In this study, we describe a nonenzymatic procedure for a straightforward isolation of MSCs from the fetal dermis and the adult dermis. The results demonstrate the isolation of a cell population with a uniform MSC immunophenotype from the earliest passages (approximately 90% positive for the classical MSC markers CD90, CD105, and CD73, while negative for the hematopoietic markers CD34 and CD45, as well as HLA-DR). Additionally, we describe the procedures for cell expansion, banking, and secretome collection.

CD304 + adipose tissue-derived mesenchymal stem cell abundance in autologous fat grafts highly correlates with improvement of localized pain syndromes.

Surgery, burns or surgery-free accident are leading causes of scars with altered tissue consistency, a reduced degree of motion and pain. Autologous fat grafting can dramatically improve tissue consistency and elasticity but less frequently results in the reduction of pain. Therefore, we analyzed different cell populations present within the adipose tissue to be engrafted and correlated them with the reduction of pain after surgery. Here, we identify a population of CD3 - CD4 - CD304 + cells present in grafted adipose tissue, whose abundance highly correlates with pain improvement shortly after surgery ( r2 = 0.7243****) as well as persistently over time (3 months later: r2 = 0.6277****, 1 year later: r2 = 0.5346***, and 4 years later: r2 = 0.5223***). These cells are characterized by the absence of the hematopoietic marker CD45, whereas they express CD90 and CD34, which characterize mesenchymal stem cells (MSCs); the concomitant presence of CD10 and CD73 in the plasma membrane supports a function of these cells in pain reduction. We deduce that the enrichment of this adipose tissue-derived MSC subset could enhance the therapeutic properties of adipose grafts and ameliorate localized pain syndromes.

Characterization of Epithelial Stem Cells and Mesenchymal Stem Cells from Human Hair Follicle

Hair follicle mesenchymal stem cells (HF-MSCs) and epithelial stem cells (HF-EpSCs) play a key role in hair growth and regeneration. The isolation, culture and characterization of HF-MSCs and HF-EpSCs will provide clues for in vitro tissue models and regenerative medicine applications. In this study, HF-MSCs and HF-EpSCs from the hair bulge were harvested using the tissue culture method. HF-MSCs exhibited a fibroblast shape and expressed highly the mesenchymal markers CD44, CD73, CD90 and CD105 and a lack of hematopoietic markers CD45, CD34, CD11b, CD19 and HLA-DR. They showed the potential for differentiation into adipogenic, osteogenic and chondrogenic lineages. HF-EpSCs possessed the keratinocyte shape and expressed typical markers for epithelial stem cells such as CD200, CK15 and CK19. The stemness marker CD49f was preferentially expressed in HF-EpSCs over HF-MSCs. Moreover, the expression of pluripotent factors including c-myc, oct-4, sox-2, klf-4 and nanog at the mRNA level was higher in HF-EpSCs compared to the expression in HF-MSCs. In conclusion, we successfully isolated mesenchymal and epithelial stem cells via culturing the hair follicle bulge tissues and preliminarily evaluated the stemness and plasticity of these two populations. This information will provide clues for further studies on hair regeneration in vitro and in vivo.

Establishment of Cerebral Organoid‐like Structures from Dental Pulp Stem Cells (DPSCs) to Model Neurodegenerative Diseases.

AbstractBackground3D‐cell cultures are closer to in vivo conditions than the monolayers. There are different 3D‐cell culture models, the most relevant is the Cerebral Organoids (CO) model (Lancaster et al. 2014), it can show complex processes and proved to show molecular and structural changes when modeling Alzheimer´s disease (AD), and other diseases. One of the main limitations of using this model is the requirement for Induced Pluripotent Stem Cells (iPSC) or Human Embryonic Stem Cells (hESC) for the CO generation; because iPSC could undergo epigenetic changes during the process of reprogramming; moreover, hESC are controversial because of their ethical implications. We proposed to use DPSCs instead of iPSC or hESC to generate organoid‐like structures due to their capability to differentiate into the neuronal lineage, lack of pluripotency, and its easy access.MethodThe DPSCs were obtained by dissociating the dental pulp of a healthy 46 years old female with a colagenase‐dispase mix at 37°C, isolated and cultured. The cells phenotype was characterized by Western‐Blot, RT‐PCR and immunofluorescence. We cultured the DPSC into low‐adherence wells for 6 days in a DMEM/F12 medium + thiazovivin 50 mM, bFGF 4ng/mL, BFS 20%, Glutamax® 200x, MEM‐NEAA 1:100, 2‐mercaptoethanol 100 µm and murine LIF 10 µg/mL until cell aggregation into embryoid‐like structures. After that, neural induction was carried out.ResultWe successfully characterized DPSC phenotype by detecting pluripotential markers OCT4, Actin, NanoG, mesenchymal markers CD105, CD73, and the neuronal marker β‐III Tubulin which is always present in the DPSCs lineage, as well as the lack of hematopoietic markers CD14, CD34, and CD45. We obtained Cerebral Organoids‐like structures from de DPSCs after 35 days of the differentiation protocol and detected NeuN, MAP2, β‐III Tubulin, and tau, all neural markers that claim a neuronal phenotype.ConclusionOur results suggest a potential use of this approach to the CO generation using DPSCs instead of iPSC or hESC with the benefits of a shorter time and a less expensive protocol likewise, DPSCs could resemble the molecular aging changes. Further studies are needed to validate the use of this CO to model neurodegenerative diseases such as AD.

Reprogramming Cancer Cells to Antigen-presenting Cells.

Cancer cells evade the immune system by downregulating antigen presentation. Although immune checkpoint inhibitors (ICI) and adoptive T-cell therapies revolutionized cancer treatment, their efficacy relies on the intrinsic immunogenicity of tumor cells and antigen presentation by dendritic cells. Here, we describe a protocol to directly reprogram murine and human cancer cells into tumor-antigen-presenting cells (tumor-APCs), using the type 1 conventional dendritic cell (cDC1) transcription factors PU.1, IRF8, and BATF3 delivered by a lentiviral vector. Tumor-APCs acquire a cDC1 cell-like phenotype, transcriptional and epigenetic programs, and function within nine days (Zimmermannova et al., 2023). Tumor-APCs express the hematopoietic marker CD45 and acquire the antigen presentation complexes MHC class I and II as well as co-stimulatory molecules required for antigen presentation to T cells, but do not express high levels of negative immune checkpoint regulators. Enriched tumor-APCs present antigens to Naïve CD8+ and CD4+ T cells, are targeted by activated cytotoxic T lymphocytes, and elicit anti-tumor responses in vivo. The tumor-APC reprogramming protocol described here provides a simple and robust method to revert tumor evasion mechanisms by increasing antigen presentation in cancer cells. This platform has the potential to prime antigen-specific T-cell expansion, which can be leveraged for developing new cancer vaccines, neoantigen discovery, and expansion of tumor-infiltrating lymphocytes. Key features • This protocol describes the generation of antigen-presenting cells from cancer cells by direct reprogramming using lineage-instructive transcription factors of conventional dendritic cells type I. • Verification of reprogramming efficiency by flow cytometry and functional assessment of tumor-APCs by antigen presentation assays.

A comparison study of dental pulp stem cells derived from healthy and orthodontically intruded human permanent teeth for mesenchymal stem cell characterisation.

The objective of this study was to compare the characteristics of Dental Pulp Stem Cells (DPSCs) derived from healthy human permanent teeth with those that were orthodontically-intruded to serve as potential Mesenchymal Stem Cells (MSC). Recruited subjects were treated with orthodontic intrusion on one side of the maxillary first premolar while the opposite side served as the control for a period of six weeks before the dental pulp was extracted. Isolated DPSCs from both the control and intruded samples were analyzed, looking at the morphology, growth kinetics, cell surface marker profile, and multilineage differentiation for MSC characterisation. Our study showed that cells isolated from both groups were able to attach to the cell culture flask, exhibited fibroblast-like morphology under light microscopy, able to differentiate into osteogenic, adipogenic and chondrogenic lineages as well as tested positive for MSCs cell surface markers CD90 and CD105 but negative for haematopoietic cell surface markers CD34 and HLA-DR. Both groups displayed a trend of gradually increasing population doubling time from passage 1 to passage 5. Viable DPSCs from both groups were successfully recovered from their cryopreserved state. In conclusion, DPSCs in the dental pulp of upper premolar not only remained viable after 6 weeks of orthodontic intrusion using fixed appliances but also able to develop into MSCs.

Mesenchymal Stem Cell Identification After Delayed Cord Clamping.

We sought to determine the feasibility of identifying and quantifying mesenchymal stem cells (MSCs) from umbilical cord blood (UCB) after delayed cord clamping in preterm and term births. We obtained 3mL of UCB at various gestational ages after delayed cord clamping. UCB separated by density gradient centrifugation within 4h of delivery was passed through magnetic bead micro-columns to exclude the CD34 + cell population. The samples were incubated with fluorescent-tagged mesenchymal cell marker antibodies CD 29, CD44, CD73, CD105, and hematopoietic cell marker CD45. The cell populations were analyzed by flow cytometry. Viable cells were assessed with 7-aminoactinomycin-D. The results were expressed in median (minimum to maximum) MSCs and compared between preterm and term samples. A total of 12 UCB samples (32-40weeks) were obtained, 10 of which demonstrated MSCs, accounting for 0.0174% (0-14.7%) of the viable UCB mononuclear cells. MSCs comprised 0.148% (0.0006-1.59%) and 0.116% (0-14.7%) of the viable UCB mononuclear cells in the term (n = 5), 38.4 ± 1.3weeks, and preterm (n = 7) samples, 34.6 ± 1.1, respectively, p = 0.17. There was an overall median of 96 (0-39,574) MSCs. There was no difference in the median numbers of MSCs identified between term and preterm UCB samples, 3384 (23-6042) and 36 (0-39,574), respectively, p = 0.12. Mesenchymal stem cells were identified and quantified in 5 of 7 preterm and all 5 term UCB 3-mL samples obtained after delayed cord clamping.

Human Non-Hypertrophic Nonunion Tissue Contains Osteoblast Lineage Cells and E-BMP-2 Activates Osteogenic and Chondrogenic Differentiation.

In this study, we examined the proliferation capability and osteogenic and chondrogenic differentiation potential of non-hypertrophic nonunion cells (NHNCs), and the effect of Escherichia coli-derived BMP-2 (E-BMP-2) on them. We enrolled five patients with non-hypertrophic nonunion. NHNCs isolated from nonunion tissue sampled during surgery were cultured, passaged, counted every 14 days, and analyzed. NHNCs were homogenous fibroblastic adherent cells and long-lived through at least 10 passages, with a slight decline. The cells were consistently positive for mesenchymal stem cell-related markers CD73 and CD105, and negative for the hematopoietic markers CD14 and CD45. NHNCs could differentiate into osteoblast lineage cells; however, they did not have strong calcification or sufficient chondrogenic differentiation capability. E-BMP-2 did not affect the proliferative capability of the cells but improved their osteogenic differentiation capability by increasing alkaline phosphatase activity and upregulating the gene expression of osterix, bone sialoprotein, and osteocalcin. E-BMP-2 enhanced their chondrogenic differentiation capability by upregulating the gene expression of aggrecan and collagen type II. We showed, for the first time, that NHNCs have the capacity to differentiate into osteoblast-lineage cells, although the chondrogenic differentiation potential was poor. Local application of E-BMP-2 with preservation of nonunion tissue is a potential treatment option for non-hypertrophic nonunion.

A novel type of mesenchymal stem cells derived from bovine metanephric mesenchyme

Mesenchymal stem cells (MSCs) are widely present in the interstitial tissue of embryos. Although the existence of the metanephros stromal stem cell population has been demonstrated, the focus has been on understanding the process of nephrogenesis, but the biological characterization of stromal stem cell population is less precise. Metanephric mesenchymal stem cells (MMSCs) have a vast potential in kidney tissue engineering and represent excellent candidates in cellular replacement therapy for human disease and veterinary research. Here, we aimed to isolate, culture, and characterize bovine MMSCs. We have successfully obtained a new population of stem cells using renal tissue isolated from three-month-old bovine embryo. MMSCs were isolated by collagenase digestion. The spindle-shaped cells adhered to plastic and exhibited extensive proliferation for more than 26 passages and good clonogenic ability in vitro. Moreover, the metanephric mesenchymal stem cells could be induced to differentiate into mesoderm-derived cells (such as osteoblasts, chondrocytes, and adipocytes) and endoderm-derived hepatocyte-like cells in vitro. These results indicated the multiple differentiations potential of MMSCs. Aside from colony-forming, self-renewal, and multilineage differentiation capabilities, the experiments of immunofluorescence and RT-PCR showed that spindle-shaped cells were positive for MSCs-related markers (CD29, CD44, CD73, CD90, CD106), nestin and vimentin, while the hematopoietic cell surface markers CD34 and pan-leukocyte marker CD45 were undetectable. This study provides a technical platform for the preservation of valuable bovine genetic resources and offers a new source for tissue engineering and cell transplantation therapy.

Tm4sf1-marked Endothelial Subpopulation Is Dysregulated in Pulmonary Arterial Hypertension.

The identification and role of endothelial progenitor cells in pulmonary arterial hypertension (PAH) remain controversial. Single-cell omics analysis can shed light on endothelial progenitor cells and their potential contribution to PAH pathobiology. We aim to identify endothelial cells that may have stem/progenitor potential in rat lungs and assess their relevance to PAH. Differential expression, gene set enrichment, cell-cell communication, and trajectory reconstruction analyses were performed on lung endothelial cells from single-cell RNA sequencing of Sugen-hypoxia, monocrotaline, and control rats. Relevance to human PAH was assessed in multiple independent blood and lung transcriptomic data sets. Rat lung endothelial cells were visualized by immunofluorescence in situ, analyzed by flow cytometry, and assessed for tubulogenesis invitro. A subpopulation of endothelial cells (endothelial arterial type 2 [EA2]) marked by Tm4sf1 (transmembrane 4 L six family member 1), a gene strongly implicated in cancer, harbored a distinct transcriptomic signature enriched for angiogenesis and CXCL12 signaling. Trajectory analysis predicted that EA2 has a less differentiated state compared with other endothelial subpopulations. Analysis of independent data sets revealed that TM4SF1 is downregulated in lungs and endothelial cells from patients and PAH models, is a marker for hematopoietic stem cells, and is upregulated in PAH circulation. TM4SF1+CD31+ rat lung endothelial cells were visualized in distal pulmonary arteries, expressed hematopoietic marker CD45, and formed tubules in coculture with lung fibroblasts. Our study uncovered a novel Tm4sf1-marked subpopulation of rat lung endothelial cells that may have stem/progenitor potential and demonstrated its relevance to PAH. Future studies are warranted to further elucidate the role of EA2 and Tm4sf1 in PAH.

Established Yazd human foreskin fibroblast lines (#8, #17, and #18) displaying similar characteristics to mesenchymal stromal cells: A lab resources report.

BackgroundFibroblasts from different parts of the human body have been used in cell biology, drug discovery and cell therapy studies. One of the most available sources of human fibroblasts is neonatal foreskin. Not only do these cells have wound-healing applications, but they are also the most popular source for pluripotent stem cell biotechnology. Moreover, several studies have indicated that different sources of fibroblasts display similar features to mesenchymal stem cells.ObjectiveGeneration and establishment of new human foreskin fibroblast cell lines called Yazd human foreskin fibroblasts (YhFFs).Materials and MethodsIn this lab resources study, the production of 3 YhFF cell lines (YhFF#8, YhFF#17, and YhFF#18) is reported. Their biological features were characterized using immunofluorescence, polymerase chain reaction, and flow-cytometry for mesenchymal markers such as fibronectin, vimentin, CD44, CD73, CD90, CD105, and hematopoietic markers CD34 and CD45.ResultsThe YhFF cell lines were passaged more than 40 times and their normal karyotype was checked using G-binding. Similarly to previous reports, the flow cytometry analysis revealed that the YhFF cell lines displayed mesenchymal stromal cell characteristics.ConclusionThis study will contribute to the development of clinical-grade cell-based products such as micro-vesicles and exosomes for future therapeutic applications in regenerative medicine.

Poster 104: The Role of Patient Demographic Factors in the Composition of Bone Marrow Aspirate Concentrate: A Flow Cytometry Analysis

Objectives:Bone marrow aspirate concentrate (BMAC) is one type of orthobiologic that has gained increased popularity over the last decade for a variety of orthopedic procedures. Multiple factors have been demonstrated to influence the MSCs concentration and chondrogenic potential in BMA and thus BMAC, ranging from aspiration location to amount extracted to peripheral blood platelet count. Some studies have suggested that age may affect chondrogenic potential of BMAC derived MSCs but may not affect initial MSC concentration. Therefore, the purpose of this study was to 1) investigate the concentration value and variance BMAC content and 2) investigate the effects of donor age, sex, and other demographic factors on the amount of MSCs as measured by flow cytometry in bone marrow aspirate concentrate (BMAC).Methods:A review of patients who underwent BMAC treatment as part of a clinical trial were enrolled and complete BMAC flow cytometry data were included in analysis. Chart review was performed to identify patient demographic factors to determine their relationship to flow cytometry cell count. Flow cytometry was performed on both patient bone marrow aspirate (BMA) and BMAC samples to identify multipotent MSC phenotype defined as cell-surface co-expression of the antigens CD105, CD73, and CD90 (≥95% positive) and the absence of hematopoietic lineage markers CD45, CD34, CD14 or CD11b, CD79a or CD19, and HLA-DR (≤2% positive). The ratio of cells in BMA: BMAC samples was calculated, and Spearman correlations and Kruskall-Wallis tests were used to determine the relationship of cell concentration to demographic factors.Results:A total of 80 patients were included in analysis (49% male, mean age: 49.93±12.17 years). Mean concentration of BMA and BMAC was 2,048.13±2,004.14 MSCs/mL and 5618.87±7568.54 MSC/mL, respectively, with a mean BMAC:BMA ratio of 4.35 ± 2.09 (Figure 1). A significantly higher MSC concentration was observed in the BMAC samples (P=0.005). No patient demographic factors (age, sex, height, weight, BMI, smoking status) significantly impacted MSC concentration in the BMAC samples.Conclusions:This study validates that BMAC concentrates and increases MSCs from BMA approximately four-fold. In addition, in opposition to our hypothesis, no demographic factors affected the final number of MSCs observed with flow cytometry analysis. These preliminary results suggest that BMAC successfully concentrates BMA and provides a higher number of MSCs. Baseline patient characteristics likely have minimal effects on the number and concentration of MSCs in BMAC, and therefore may not limit patient selection for BMAC for clinical use.Figure 1.Distribution of the ratio of BMAC to BMA

Bone Marrow-Derived Cells Contribute to the Maintenance of Thymic Stroma including TECs.

In paradox to critical functions for T-cell selection and self-tolerance, the thymus undergoes profound age-associated atrophy and loss of T-cell function, further enhanced by cancer therapies. Identifying thymic epithelial progenitor populations capable of forming functional thymic tissue will be critical in understanding thymic epithelial cell (TEC) ontogeny and designing strategies to reverse involution. We identified a new population of progenitor cells, present in both the thymus and bone marrow (BM) of mice, that coexpress the hematopoietic marker CD45 and the definitive thymic epithelial marker EpCAM and maintain the capacity to form functional thymic tissue. Confocal analysis and qRT-PCR of sorted cells from both BM and thymus confirmed coexpression of CD45 and EpCAM. Grafting of C57BL/6 fetal thymi under the kidney capsule of H2BGFP transgenic mice revealed that peripheral CD45+ EpCAM+ GFP-expressing cells migrate into the developing thymus and contribute to both TECs and FSP1-expressing thymic stroma. Sorted BM-derived CD45+ EpCAM+ cells contribute to reaggregate thymic organ cultures (RTOCs) and differentiate into keratin and FoxN1-expressing TECs, demonstrating that BM cells can contribute to the maintenance of TEC microenvironments previously thought to be derived solely from endoderm. BM-derived CD45+ EpCAM+ cells represent a new source of progenitor cells that contribute to thymic homeostasis. Future studies will characterize the contribution of BM-derived CD45+ EpCAM+ TEC progenitors to distinct functional TEC microenvironments in both the steady-state thymus and under conditions of demand. Cell therapies utilizing this population may help counteract thymic involution in cancer patients.

Isolation of the side population from neurogenic niches enriches for endothelial cells.

In stem cell research, DNA-binding dyes offer the ability to purify live stem cells using flow cytometry as they form a low-fluorescence side population due to the activity of ABC transporters. Adult neural stem cells exist within the lateral ventricle and dentate gyrus of the adult brain yet the ability of DNA-binding dyes to identify these adult stem cells as side populations remains untested. The following experiments utilize the efflux of a DNA-binding dye, Vyrbant DyeCycle Violet (DCV), to isolate bona fide side populations in the mouse dentate gyrus and subventricular zone (SVZ), and test their sensitivity to ABC transporter inhibitors. A distinct side population was found in both the adult lateral ventricle and dentate gyrus using DCV fluorescence and forward scatter instead of the conventional dual fluorescence approach. These side populations responded strongly to inhibition with the ABC transporter antagonists, verapamil and fumitremorgin C. The majority of the cells residing in the side populations of dentate gyrus and SVZ were characterized by their expression of CD31. Additionally, at least 90% of all CD31+ cells found in the dentate gyrus and SVZ were negative for the hematopoietic marker CD45, leading to the hypothesis that the CD31+ cells in the side population were endothelial cells. These findings, therefore, suggest that the side population analysis provides an efficient method to purify CD31-expressing endothelial cells, but not adult neural stem cells.

Differentiation of cancer stem cells into erythroblasts in the presence of CoCl2

Cancer stem cells (CSCs) are subpopulations in the malignant tumors that show self-renewal and multilineage differentiation into tumor microenvironment components that drive tumor growth and heterogeneity. In previous studies, our group succeeded in producing a CSC model by treating mouse induced pluripotent stem cells. In the current study, we investigated the potential of CSC differentiation into blood cells under chemical hypoxic conditions using CoCl2. CSCs and miPS-LLCcm cells were cultured for 1 to 7 days in the presence of CoCl2, and the expression of VEGFR1/2, Runx1, c-kit, CD31, CD34, and TER-119 was assessed by RT-qPCR, Western blotting and flow cytometry together with Wright-Giemsa staining and immunocytochemistry. CoCl2 induced significant accumulation of HIF-1α changing the morphology of miPS-LLCcm cells while the morphological change was apparently not related to differentiation. The expression of VEGFR2 and CD31 was suppressed while Runx1 expression was upregulated. The population with hematopoietic markers CD34+ and c-kit+ was immunologically detected in the presence of CoCl2. Additionally, high expression of CD34 and, a marker for erythroblasts, TER-119, was observed. Therefore, CSCs were suggested to differentiate into erythroblasts and erythrocytes under hypoxia. This differentiation potential of CSCs could provide new insight into the tumor microenvironment elucidating tumor heterogenicity.

Isolation and phenotyping of cardiac-derived progenitor cells from neonatal mice

Dysfunctions of resident progenitor cells play a significant role in the pathogenesis of decreased myocardial contractility in heart failure, so the most promising approaches for the treatment of heart disease are cardiac-derived stem/progenitor cells (CSCs). Materials and methods. Protocols for progenitor cell cultures from different parts of the heart of newborn FVB/N mice have been developed and their proliferative potential has been characterized. Comparative analysis of the expression of CD31, CD34, CD44, CD45, CD73, CD90, CD105, CD117, CD309 and troponin I by cells from native myocardial biopsies and in the obtained cultures was performed by flow cytometric immunophenotyping. Results. The expression of mesenchymal markers CD44 and CD90 in the absence of the hematopoietic marker CD45 was demonstrated in early passages in mouse myocardial progenitor cell cultures. Relatively high expression of CD34 and CD31 was found. The presence of a minor population of CD44+117+ cells which correspond to the phenotype of cardiac progenitor cells, was detected. Expression of troponin I as one of the key markers of cardiomyocytes as well as the vascular endothelial growth factor receptor has been confirmed in terminally differentiated cultures of cells with contractile activity. Conclusions. It was found that newborn mice in the myocardial tissue contain more cells with the expression of markers of cardiac progenitors than in adult animals. The relative content of such cells is higher in the atria than in the ventricles. Cardiac progenitor cells in neonatal mice derived from the atrial appendages have better proliferative potential than cell cultures isolated from the ventricles.

A flow cytometric method for measuring and isolating mammary epithelial cells from bovine milk

Sampling frequent time points of mammary signaling pathways is not possible with tissue biopsies. We have validated a flow cytometry and cell sorting procedure for isolating live bovine mammary epithelial cells from somatic cell populations in milk using butyrophilin 1A1 as a marker for mammary epithelial cells and CD45 as a marker for hematopoietic cells. Hoechst 33342 staining and propidium iodide exclusion were used to select for nucleated live cells. Positive selection of butyrophilin (BTN)-expressing cells was performed by fluorescence-activated cell sorting. Quantitative real-time PCR performed on mRNA isolated from these cells showed a 226-fold increase in κ-casein (CSN3) mRNA expression in BTN single-positive cells compared with unsorted cells, whereas CD45 single-positive cells showed a significant decrease. A negative selection strategy for cells not expressing the hematopoietic cell marker CD45 also resulted in a cell population with a 196-fold increase in CSN3 mRNA expression compared with unsorted cells. We found no enrichment of CSN3 mRNA expression after sorting cells using cytokeratin antibodies. The noninvasive assays described here allow for daily or more frequent sampling time points for measurement of mammary epithelial cells during the course of lactation.

Identification and characterization of pulmonary mesenchymal stem cells derived from rat fetal lung tissue

Pulmonary mesenchymal stem cells (PMSCs) have great potential in lung tissue repair and regeneration, which have been isolated from some mammalian species, including mice, bovine and pig. However, the isolation, characteristics and differentiation potential of rat PMSCs have not been reported. In this study, we successfully isolated PMSCs from Sprague-Dawley rat fetal lung tissue in vitro for the first time and attempted to evaluate its multilineage differentiation potentials. The cultured PMSCs showed typical spindle-shaped morphology and high proliferative potential, and could be passaged for at least 13 passages and maintained high hereditary stability with more than 93.6 % of cells were diploid (2n = 42) by G-banding analysis. Furthermore, the PMSCs could express mesenchymal markers Sca-1, CD29, CD44, CD73 and CD90, but not hematopoietic markers CD34 and CD45. Besides, the expression of cell markers of AT2 (SFTPC), AT1 (PDPN) and macrophage (CD11b) were also negative. Cell cycle examination revealed majority of the PMSCs were in G0/G1 phase, which are similar with previously reported pig PMSCs. In addition, the PMSCs were multipotent and could differentiated into osteocytes, adipocytes, hepatocytes and neurons in vitro. Together, the present study demonstrated the stemness and multi-differentiation potentials of rat PMSCs, which conferred a potential regenerative cell resource for cell regenerative therapy of lung injury.