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1.
Pharm Biol ; 60(1): 1160-1168, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-35695011

RESUMO

CONTEXT: The effects of Rhodiola rosea L. (Crassulaceae) polysaccharides (RRPs) on haematopoiesis are poorly understood. OBJECTIVE: To determine the effects of RRPs on haematopoiesis in mice with aplastic anaemia. MATERIALS AND METHODS: Aplastic anaemia was induced in Kunming mice by 60Coγ (2.0 Gy) irradiation and cyclophosphamide administration (50 mg/kg/day for 3 consecutive days; intraperitoneal injection). The in vivo effects of RRPs (10, 20, and 40 mg/kg; intraperitoneal injection) on haematopoiesis were analyzed using peripheral blood tests, histopathological examination of haematopoietic tissues, culture of haematopoietic progenitors and bone marrow stromal cells (BMSCs), and Western blotting of Fas and Fas ligand (FasL). The in vitro effects of RRPs on bone-marrow haematopoietic progenitors and BMSCs were also evaluated. RESULTS: Compared to anaemic controls, high-dose RRPs (40 mg/kg) significantly increased red blood cells (8.21 ± 0.57835 versus 6.13 ± 1.34623 × 1012/L), white blood cells (5.11 ± 1.6141 versus l.54 ± 1.1539 × 109/L), and BMSCs (10.33 ± 1.5542 versus 5.87 ± 3.1567 × 1012/L) in mice with aplastic anaemia (all p < 0.01). High-dose RRPs significantly increased the formation of colony-forming unit-granulocyte macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and colony-forming unit-erythroid (CFU-E; p < 0.01). Fas and FasL protein expression in BMSCs decreased after RRPs administration. Especially at the high dose, RRPs (150 µg/mL) significantly promoted in vitro CFUs-E, BFUs-E, and CFUs-GM formation. RRPs (150-300 µg/mL) also promoted BMSC proliferation. DISCUSSION AND CONCLUSIONS: RRPs helped to promote haematopoietic recovery in mice with aplastic anaemia, facilitating haematopoietic tissue recovery. This study indicated some mechanisms of the haematopoietic regulatory effects of RRPs. Our findings provide a laboratory basis for clinical research on RRPs.


Assuntos
Anemia Aplástica , Rhodiola , Anemia Aplástica/tratamento farmacológico , Anemia Aplástica/metabolismo , Animais , Medula Óssea , Células da Medula Óssea , Proliferação de Células , Ensaio de Unidades Formadoras de Colônias , Células-Tronco Hematopoéticas , Camundongos , Polissacarídeos/metabolismo , Polissacarídeos/farmacologia , Células Estromais
2.
Transfusion ; 62(8): 1595-1601, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35770742

RESUMO

BACKGROUND: The IL-3-pSTAT5 assay, a new, rapid, and standardized flow-cytometry-based assay may compensate for several limitations of the colony-forming unit (CFU) assay typically used for stem cell potency assessments of cord blood units (CBU). We performed an inter-laboratory evaluation of the performance of this new assay. STUDY DESIGN AND METHODS: This Biomedical Excellence for Safer Transfusion (BEST) Collaborative multicenter, international study included 15 participants from public cord blood banks (CBBs), CBB-supporting research laboratories, and stem cell laboratories. To perform the IL-3-pSTAT5 assay, participating centers received reagents, instructions, and 10 blind CBU samples, including eight normal samples and two samples exposed to a transient warming event. We measured inter-laboratory agreement qualitatively (proportion of correctly classified samples) and quantitatively (coefficient of variation [CV], correlation coefficients, receiver operating characteristics (ROC) curve, and intraclass correlation coefficient [ICC]). RESULTS: The qualitative agreement was 97.3% (i.e., 107/110; Fleiss' kappa = 0.835). The average CV on a per-sample basis was 11.57% among all samples, 8.99% among normal samples, and on a per-center basis was 9.42% among normal samples. In a correlation matrix that compared results across centers, the mean Pearson's correlation coefficient was 0.88 (standard deviation = 0.04). The ICC was 0.83 (95% confidence interval = 0.68-0.95). The area under the curve (AUC) from the ROC curve was 0.9974. DISCUSSION: Excellent qualitative and quantitative agreement was exhibited across laboratories. The IL-3-pSTAT5 assay may therefore be implemented in flow cytometry laboratories to rapidly and reliably provide standardized measures of stem cell potency in CBUs.


Assuntos
Sangue Fetal , Interleucina-3 , Bancos de Sangue/métodos , Ensaio de Unidades Formadoras de Colônias , Humanos , Células-Tronco
3.
Cytokine ; 153: 155863, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35339859

RESUMO

Earlier research from our laboratory demonstrated the presence of stimulatory activity of different growth factors in the fetal liver (FL) extracts when collected in a medium known as fetal liver conditioned medium (FLCM) using Enzyme-linked Immunosorbent Assay (ELISA). In the present study, we have assessed two other cytokines viz. IL-6 and FMS like tyrosine kinase-3 (Flt-3) with the help of bioneutralization assay. FLCM was prepared by incubating fetal liver cells with Iscove's Modified Dulbecco's Medium (IMDM) containing 10% fetal bovine serum (FBS) and 10% Phytohemagglutinin and collected after 24hrs, 48hrs, 72 hrs. and on the 7th day of incubation. Clonal cultures were established for 1 X 105 normal bone marrow (BM) mononuclear cells (NBM MNC) per plate with methylcellulose medium containing cytokines SCF and EPO. Mean Colony forming units-granulocytes, erythrocytes, macrophages, megakaryocytes (CFU-GEMM) were assessed with and without the addition of FLCM. It was found that FLCM enhanced the number of colonies made by NBM MNCs. Further, cytokines IL-6 and Flt-3, present in FLCM, were bioneutralized with respective anti-cytokine antibodies. Neutralized FLCM was evaluated for the colony-forming potential of CFU-GEMM colonies. The maximum reduction of 42% was seen with 20 ng/ml of anti-IL-6 antibody. Maximum suppression up to 20% was observed with 0.7 ng/ml of anti Flt-3 antibody for CFU-GEMM colonies. Presence of cytokines IL-6 and Flt-3 in FL extracts and their colony stimulatory activity suggests that fetal liver infusion (FLI) may be a valuable alternative for managing BM recovery in certain clinical conditions such as AA.


Assuntos
Eritropoetina , Interleucina-6 , Células da Medula Óssea , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Meios de Cultivo Condicionados/farmacologia , Citocinas/farmacologia , Humanos , Interleucina-3/farmacologia , Interleucina-6/farmacologia , Fígado , Megacariócitos , Extratos Vegetais/farmacologia , Tirosina Quinase 3 Semelhante a fms
5.
Viruses ; 14(2)2022 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-35215979

RESUMO

Virus-like particles resemble infectious virus particles in size, shape, and molecular composition; however, they fail to productively infect host cells. Historically, the presence of virus-like particles has been inferred from total particle counts by microscopy, and infectious particle counts or plaque-forming-units (PFUs) by plaque assay; the resulting ratio of particles-to-PFUs is often greater than one, easily 10 or 100, indicating that most particles are non-infectious. Despite their inability to hijack cells for their reproduction, virus-like particles and the defective genomes they carry can exhibit a broad range of behaviors: interference with normal virus growth during co-infections, cell killing, and activation or inhibition of innate immune signaling. In addition, some virus-like particles become productive as their multiplicities of infection increase, a sign of cooperation between particles. Here, we review established and emerging methods to count virus-like particles and characterize their biological functions. We take a critical look at evidence for defective interfering virus genomes in natural and clinical isolates, and we review their potential as antiviral therapeutics. In short, we highlight an urgent need to better understand how virus-like genomes and particles interact with intact functional viruses during co-infection of their hosts, and their impacts on the transmission, severity, and persistence of virus-associated diseases.


Assuntos
Vírus Defeituosos/fisiologia , Vírion/fisiologia , Animais , Ensaio de Unidades Formadoras de Colônias , Genoma Viral , Humanos , Microscopia Eletrônica de Transmissão , Ensaio de Placa Viral , Viroses/virologia , Replicação Viral
6.
Gene ; 809: 146005, 2022 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-34673210

RESUMO

Stem cells from umbilical cord blood (UCB) are able to proliferate and differentiate into various somatic cell types. Thereby, they are considered as one of the attractive stem cell sources in tissue engineering and regenerative medicine. However, the limited number of hematopoietic CD 133+ stem cells in UCB restricted the clinical application of such stem cells. This study was aimed to expand CD 133+ stem cells derived from UCB on a 3D silk scaffold. UCB133+ stem cells were extracted using Magnetic cell sorting (MACS) and characterized by flow cytometry. Isolated cells were seeded on a fabricated electrospun silk scaffold and cultured for 7 days. The real-time PCR, cell counting, colony-forming assay, and MTT assay were performed to evaluate the expansion and homing of stem cells. The results showed a higher expression of CXCR4 gene, the number of cultured stem cells, and colony-forming units in the 3D silk scaffold group after 7 days when compared to the tissue culture plate. Moreover, higher viability and proliferation of stem cells were seen in cells cultured on silk scaffold. It seems electrospun silk scaffold could be used as a suitable substrate for UCB CD 133+ stem cell expansion.


Assuntos
Antígeno AC133/metabolismo , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Nanofibras , Técnicas de Cultura de Células/métodos , Proliferação de Células , Separação Celular/métodos , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Células-Tronco Hematopoéticas/metabolismo , Humanos , Microscopia Eletrônica de Varredura , Nanofibras/química , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Seda/química , Tecidos Suporte/química
7.
Stem Cell Reports ; 17(1): 68-81, 2022 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-34919810

RESUMO

Human pluripotent stem cells (hPSCs) grow as colonies with epithelial-like features including cell polarity and position-dependent features that contribute to symmetry breaking during development. Our study provides evidence that hPSC colonies exhibit position-dependent differences in apical structures and functions. With this apical difference, edge cells were preferentially labeled with amphipathic dyes, which enabled separation of edge and center cells by fluorescence-activated cell sorting. Transcriptome comparison between center and edge cells showed differential expression of genes related to apicobasal polarization, cell migration, and endocytosis. Accordingly, different kinematics and mechanical dynamics were found between center and edge cells, and perturbed actin dynamics disrupted the position-dependent apical polarity. In addition, our dye-labeling approach could be utilized to sort out a certain cell population in differentiated micropatterned colonies. In summary, hPSC colonies have position-dependent differences in apical structures and properties, and actin dynamics appear to play an important role in the establishment of this position-dependent cell polarity.


Assuntos
Citoesqueleto de Actina/metabolismo , Diferenciação Celular , Polaridade Celular , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Citoesqueleto de Actina/genética , Técnicas de Cultura de Células , Diferenciação Celular/genética , Linhagem Celular , Ensaio de Unidades Formadoras de Colônias , Imunofluorescência , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Imunofenotipagem
8.
EMBO J ; 41(4): e108415, 2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-34957577

RESUMO

Leptin receptor (LepR)-positive cells are key components of the bone marrow hematopoietic microenvironment, and highly enrich skeletal stem and progenitor cells that maintain homeostasis of the adult skeleton. However, the heterogeneity and lineage hierarchy within this population has been elusive. Using genetic lineage tracing and single-cell RNA sequencing, we found that Lepr-Cre labels most bone marrow stromal cells and osteogenic lineage cells in adult long bones. Integrated analysis of Lepr-Cre-traced cells under homeostatic and stress conditions revealed dynamic changes of the adipogenic, osteogenic, and periosteal lineages. Importantly, we discovered a Notch3+ bone marrow sub-population that is slow-cycling and closely associated with the vasculatures, as well as key transcriptional networks promoting osteo-chondrogenic differentiation. We also identified a Sca-1+ periosteal sub-population with high clonogenic activity but limited osteo-chondrogenic potential. Together, we mapped the transcriptomic landscape of adult LepR+ stem and progenitor cells and uncovered cellular and molecular mechanisms underlying their maintenance and lineage specification.


Assuntos
Osso e Ossos/citologia , Receptores para Leptina/metabolismo , Análise de Célula Única/métodos , Células-Tronco/fisiologia , Envelhecimento/fisiologia , Animais , Antígenos Ly/metabolismo , Diferenciação Celular , Linhagem da Célula , Ensaio de Unidades Formadoras de Colônias , Feminino , Fraturas Ósseas , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Rosiglitazona/farmacologia , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Estresse Fisiológico
9.
Bull Exp Biol Med ; 172(2): 228-235, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34855083

RESUMO

We studied the effect of neural stem cells (NSC) and mesenchymal stem cells (MSC) from mouse adipose tissue on survival, clonogenic activity, and senescence of NSC after exposure to γ-radiation. It was found that survival and clonogenic activity of NSC irradiated in doses of 1 and 2 Gy was enhanced when irradiated cells were co-cultured with non-irradiated NSC and MSC in permeable Transwell inserts. The proportion of senescent NSC (cells with high ß-galactosidase activity) increased with increasing irradiation dose. Co-culturing with non-irradiated NSC in 3 days after irradiation in a dose of 1 Gy led to a decrease in the proportion of senescent cells among irradiated NSC. Factors secreted by NSC and MSC can become the basis for the development of means for prevention and treatment of damage to brain cells resulting from radiation therapy of head and neck cancer.


Assuntos
Raios gama/efeitos adversos , Células-Tronco Mesenquimais/citologia , Células-Tronco Neurais/efeitos da radiação , Tecido Adiposo/citologia , Animais , Apoptose/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Senescência Celular/fisiologia , Senescência Celular/efeitos da radiação , Técnicas de Cocultura , Ensaio de Unidades Formadoras de Colônias , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/citologia , Células-Tronco Neurais/fisiologia
10.
Cells ; 10(12)2021 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-34944042

RESUMO

Cartilage stem/progenitor cells (CSPCs) are cartilage-specific, multipotent progenitor cells residing in articular cartilage. In this study, we investigated the characteristics and potential of human CSPCs combined with poly(lactic-co-glycolic acid) (PLGA) scaffolds to induce osteochondral regeneration in rabbit knees. We isolated CSPCs from human adult articular cartilage undergoing total knee replacement (TKR) surgery. We characterized CSPCs and compared them with infrapatellar fat pad-derived stem cells (IFPs) in a colony formation assay and by multilineage differentiation analysis in vitro. We further evaluated the osteochondral regeneration of the CSPC-loaded PLGA scaffold during osteochondral defect repair in rabbits. The characteristics of CSPCs were similar to those of mesenchymal stem cells (MSCs) and exhibited chondrogenic and osteogenic phenotypes without chemical induction. For in vivo analysis, CSPC-loaded PLGA scaffolds produced a hyaline-like cartilaginous tissue, which showed good integration with the host tissue and subchondral bone. Furthermore, CSPCs migrated in response to injury to promote subchondral bone regeneration. Overall, we demonstrated that CSPCs can promote osteochondral regeneration. A monophasic approach of using diseased CSPCs combined with a PLGA scaffold may be beneficial for repairing complex tissues, such as osteochondral tissue.


Assuntos
Cartilagem Articular/citologia , Diferenciação Celular , Condrogênese , Células-Tronco/citologia , Tecidos Suporte/química , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Regeneração Óssea , Linhagem da Célula , Forma Celular , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Humanos , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Porosidade , Coelhos , Microtomografia por Raio-X
11.
Cells ; 10(12)2021 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-34943818

RESUMO

The present study explored the effects of ascorbic-acid (AA)/retinol and timed inflammation on the stemness, the regenerative potential, and the transcriptomics profile of gingival mesenchymal stem/progenitor cells' (G-MSCs). STRO-1 (mesenchymal stem cell marker) immuno-magnetically sorted G-MSCs were cultured in basic medium (control group), in basic medium with IL-1ß (1 ng/mL), TNF-α (10 ng/mL) and IFN-γ (100 ng/mL, inflammatory-medium), in basic medium with AA (250 µmol/L) and retinol (20 µmol/L) (AA/retinol group) or in inflammatory medium with AA/retinol (inflammatory/AA/retinol group; n = 5/group). The intracellular levels of phosphorylated and total ß-Catenin at 1 h, the expression of stemness genes over 7 days, the number of colony-forming units (CFUs) as well as the cellular proliferation aptitude over 14 days, and the G-MSCs' multilineage differentiation potential were assessed. Next-generation sequencing was undertaken to elaborate on up-/downregulated genes and altered intracellular pathways. G-MSCs demonstrated all mesenchymal stem/progenitor cells characteristics. Controlled inflammation with AA/retinol significantly elevated NANOG (p < 0.05). The AA/retinol-mediated reduction in intracellular phosphorylated ß-Catenin was restored through the effect of controlled inflammation (p < 0.05). Cellular proliferation was highest in the AA/retinol group (p < 0.05). AA/retinol counteracted the inflammation-mediated reduction in G-MSCs' clonogenic ability and CFUs. Amplified chondrogenic differentiation was observed in the inflammatory/AA/retinol group. At 1 and 3 days, the differentially expressed genes were associated with development, proliferation, and migration (FOS, EGR1, SGK1, CXCL5, SIPA1L2, TFPI2, KRATP1-5), survival (EGR1, SGK1, TMEM132A), differentiation and mineral absorption (FOS, EGR1, MT1E, KRTAP1-5, ASNS, PSAT1), inflammation and MHC-II antigen processing (PER1, CTSS, CD74) and intracellular pathway activation (FKBP5, ZNF404). Less as well as more genes were activated the longer the G-MSCs remained in the inflammatory medium or AA/retinol, respectively. Combined, current results point at possibly interesting interactions between controlled inflammation or AA/retinol affecting stemness, proliferation, and differentiation attributes of G-MSCs.


Assuntos
Ácido Ascórbico/farmacologia , Diferenciação Celular , Gengiva/patologia , Inflamação/patologia , Células-Tronco Mesenquimais/patologia , Transcriptoma/genética , Vitamina A/farmacologia , Adolescente , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Ensaio de Unidades Formadoras de Colônias , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/genética , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Doadores de Tecidos , Transcriptoma/efeitos dos fármacos , Adulto Jovem , beta Catenina/metabolismo
12.
Nat Commun ; 12(1): 6790, 2021 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-34815405

RESUMO

Lineage commitment and differentiation is driven by the concerted action of master transcriptional regulators at their target chromatin sites. Multiple efforts have characterized the key transcription factors (TFs) that determine the various hematopoietic lineages. However, the temporal interactions between individual TFs and their chromatin targets during differentiation and how these interactions dictate lineage commitment remains poorly understood. Here we perform dense, daily, temporal profiling of chromatin accessibility (DNase I-seq) and gene expression changes (total RNA-seq) along ex vivo human erythropoiesis to comprehensively define developmentally regulated DNase I hypersensitive sites (DHSs) and transcripts. We link both distal DHSs to their target gene promoters and individual TFs to their target DHSs, revealing that the regulatory landscape is organized in distinct sequential regulatory modules that regulate lineage restriction and maturation. Finally, direct comparison of transcriptional dynamics (bulk and single-cell) and lineage potential between erythropoiesis and megakaryopoiesis uncovers differential fate commitment dynamics between the two lineages as they exit the stem and progenitor stage. Collectively, these data provide insights into the temporally regulated synergy of the cis- and the trans-regulatory components underlying hematopoietic lineage commitment and differentiation.


Assuntos
Linhagem da Célula/genética , Cromatina/genética , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese/genética , Células-Tronco Hematopoéticas/fisiologia , Linhagem Celular , Cromatina/metabolismo , Ensaio de Unidades Formadoras de Colônias , Desoxirribonuclease I/metabolismo , Humanos , Leucócitos Mononucleares , Cultura Primária de Células , Regiões Promotoras Genéticas , RNA-Seq , Análise de Célula Única , Fatores de Transcrição/metabolismo
13.
Stem Cells Dev ; 30(24): 1228-1240, 2021 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-34714129

RESUMO

Bone and muscle tissues are mostly susceptible to different kinds of hypodynamia, including real and simulated microgravity (sµg). To evaluate the effect of sµg on bone marrow (BM), male C57Bl/6N mice were divided into three groups: vivarium control (VC), 30-day hindlimb suspension (HS), and subsequent 12-h short-term support reloading (RL). The effects on BM total mononucleated cells (MNCs) as well as stromal and hematopoietic progenitors from murine tibia were studied. The number of BM MNCs, immunophenotype, proliferation, colony-forming units (CFUs), differentiation and secretory activity of hematopoietic and stromal BM cells were determined. HS led to a twofold decrease in MNCs, alteration of surface molecule expression profiles, suppression of proliferative activity of BM cells, and change of soluble mediators' levels. The stromal compartment was characterized by a decrease of CFU of fibroblasts and suppression of spontaneous osteo-commitment after HS. Among the hematopoietic precursors, a decrease in the total number of CFUs was found mainly at the expense of suppression of CFU-GM and CFU-GEMM. After RL, restoration of the stromal precursor's functional activity to control levels and overabundance of paracrine mediator's production were detected, whereas the complete recovery of hematopoietic precursor's activity did not occur. These data demonstrate the fast functional reaction of the stromal compartment on restoration of loading support.


Assuntos
Medula Óssea , Tíbia , Animais , Células da Medula Óssea , Diferenciação Celular/fisiologia , Ensaio de Unidades Formadoras de Colônias , Masculino , Camundongos , Células Estromais
14.
STAR Protoc ; 2(4): 100903, 2021 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-34632411

RESUMO

Determining how hematopoietic stem and progenitor cells (HSPCs) can be infected by viruses is necessary to understand and predict how the immune system will drive the host response. We present here a protocol to analyze the capacity of SARS-CoV-2 to infect different subsets of human HSPCs, inlcuding procedures for SARS-CoV-2 production and titration, isolation of human HSPCs from different sources (bone marrow, umbilical cord, or peripheral blood), and quantification of SARS-Cov-2 infection capacity by RT-qPCR and colony forming unit assay. For complete details on the use and execution of this protocol, please refer to Huerga Encabo et al. (2021).


Assuntos
Medula Óssea/virologia , Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/virologia , Ensaio de Unidades Formadoras de Colônias/métodos , Sangue Fetal/virologia , Células-Tronco Hematopoéticas/virologia , SARS-CoV-2/isolamento & purificação , COVID-19/patologia , Células-Tronco Hematopoéticas/patologia , Humanos
15.
STAR Protoc ; 2(4): 100846, 2021 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-34622219

RESUMO

Hematopoietic stem and progenitor cells (HSPCs) reside in the bone marrow and supply blood cells. Efficient methods for isolation of HSPCs are required. Here, we present protocols for the isolation of human and murine HSPCs using manual and FACS-assisted techniques. Isolated HSPCs can be used for downstream applications, including colony forming unit assays and DNA damage and repair assays. For complete details on the use and execution of this protocol, please refer to Rodríguez et al. (2021a) and (2021b).


Assuntos
Medula Óssea , Células-Tronco Hematopoéticas , Animais , Ensaio de Unidades Formadoras de Colônias , Dano ao DNA/genética , Reparo do DNA , Humanos , Camundongos
16.
Exp Hematol ; 104: 1-8, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34688837

RESUMO

Hematopoietic stem cells (HSCs) have been studied extensively since their initial functional description in 1961 when Dr. James Till and Dr. Ernest McCulloch developed the first in vivo clonal strategy, termed the spleen colony-forming unit (CFU-S) assay, to assess the functional capacity of bone marrow-derived hematopoietic progenitors at the single-cell level. Through transplantation of bone marrow cells and analysis of the resulting cellular nodules in the spleen, the CFU-S assay revealed both the self-renewal and clonal differentiation capacity of hematopoietic progenitors. Further development and use of this assay have identified highly proliferative, self-renewing, and differentiating HSCs that possess clonal, multilineage differentiation. The CFU-S strategy has also been adapted to interrogating single purified hematopoietic stem and progenitor cell populations, advancing our knowledge of the hematopoietic hierarchy. In this review, we explore the major discoveries made with the CFU-S assay, consider its modern use and recent improvements, and compare it with commonly used long-term transplantation assays to determine the continued value of the CFU-S assay for understanding HSC biology and hematopoiesis.


Assuntos
Hematopoiese Clonal , Ensaio de Unidades Formadoras de Colônias/métodos , Análise de Célula Única/métodos , Animais , Autorrenovação Celular , Células-Tronco Hematopoéticas/citologia , Humanos
17.
Tissue Cell ; 73: 101628, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34479072

RESUMO

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.


Assuntos
Feto/citologia , Pulmão/citologia , Pulmão/embriologia , Células-Tronco Mesenquimais/citologia , Adipócitos/citologia , Animais , Biomarcadores/metabolismo , Ciclo Celular , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Forma Celular , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Hepatócitos/citologia , Cariótipo , Neurônios/citologia , Osteócitos/citologia , Ratos Sprague-Dawley
18.
Am J Sports Med ; 49(12): 3404-3413, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34398643

RESUMO

BACKGROUND: Umbilical cord (UC) connective tissues contain plastic-adherent, colony forming unit-fibroblasts (CFU-Fs) amenable to culture expansion for potential therapeutic use. Recently, UC-derived allograft products have been made available to practitioners in orthopaedics and other specialties, by companies purporting "stem cell"-based healing. However, such marketing claims conflict with existing regulations for these human tissues, generating questions over the cellular and protein composition of current commercially available UC allograft products. PURPOSE: To evaluate commercial UC allograft products for viable cells, CFU-Fs, and protein makeup. STUDY DESIGN: Descriptive laboratory study. METHODS: Five commercial UC allograft products claiming to contain viable, undescribed "stem cells," 2 obtained from UC blood (UCB) and 3 from UC tissue (UCT), were analyzed. Image-based methods were used to measure cell concentration and viability, a traditional CFU-F assay was used to evaluate in vitro behavior indicative of a connective tissue progenitor cell phenotype often referred to as mesenchymal stem/stromal cells, and quantitative immunoassay arrays were used to measure a combination of cytokines and growth factors. Bone marrow concentrate (BMC) and plasma derived from the blood and bone marrow of middle-aged individuals served as comparative controls for cell culture and protein analyses, respectively. RESULTS: Viable cells were identified within all 5 UC allograft products, with those derived from UCB having greater percentages of living cells (40%-59%) than those from UCT (1%-22%). Compared with autologous BMC (>95% viability and >300 million living cells), no CFU-Fs were observed within any UC allograft product (<15 million living cells). Moreover, a substantial number of proteins, particularly those within UCB allograft products, were undetectable or present at lower concentrations compared with blood and bone marrow plasma controls. Interestingly, several important growth factors and cytokines, including basic fibroblast growth factor, hepatocyte growth factor, interleukin-1 receptor antagonist, and osteoprotegerin, were most prevalent in 1 or more UCT allograft products as compared with blood and bone marrow plasma. CONCLUSION: CFU-Fs, often referred to as stem cells, were not found within any of the commercial UC allograft products analyzed, and clinicians should remain wary of marketing claims stating otherwise. CLINICAL RELEVANCE: Any therapeutic benefit of current UC allograft products in orthopaedic medicine is more likely to be attributed to their protein composition (UCT > UCB) or inclusion of cells without colony forming potential (UCB > UCT).


Assuntos
Sangue Fetal , Cordão Umbilical , Aloenxertos , Técnicas de Cultura de Células , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Humanos , Pessoa de Meia-Idade
19.
J Hand Surg Asian Pac Vol ; 26(3): 445-450, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34380405

RESUMO

Background: Numerous studies have indicated the presence of mesenchymal stem cells (MSCs) in the bone marrow aspirated from the vertebral body, distal femur, proximal tibia, humeral head, sternum, and iliac crest. However, their presence has not been reported in the radius thus far. In this study, we aimed to compare the number of MSCs in bone marrow aspirated from radius and iliac crest in patients with Kienböck's disease. Furthermore, we examined the association between the number of MSCs in the bone marrow and patient age. Methods: A total of 17 patients were recruited. Owing to difficulties in obtaining samples for 5 cases, only 12 cases were included. Hematological analyses and fibroblastic colony-forming unit (CFU-F) assay were performed using bone marrow samples aspirated from the radius (group R), the first sample aspirated from the iliac crest (group I-1), and the second sample aspirated from the iliac crest (group I-2). The CFU-F numbers among the three groups were compared using Mann-Whitney U-test. Pearson's correlation coefficient was calculated to evaluate the association between the CFU-F numbers and patient age. Results: The average numbers of CFU-Fs/ml in the bone marrow samples from the R, I-1, and I-2 groups were 3.4, 57.3, and 13.7, respectively. The CFU-F number in Group I-1 was significantly higher than that in the other two groups; the CFU-F number was lower in group R than in group I-2. The correlation coefficients were -0.168, 0.166, and 0.036 for samples from groups R, I-1, and I-2, respectively. No significant association between the CFU-F numbers and patient age was observed. Conclusions: The presence of MSCs in the radius was indicated by CFU-Fs in patients with Kienböck's disease. The number of CFU-Fs was lower in the radius than in the iliac crest; the CFU-F number was not associated with patient age.


Assuntos
Células-Tronco Mesenquimais , Ensaio de Unidades Formadoras de Colônias , Humanos , Ílio , Rádio (Anatomia) , Células-Tronco
20.
Stem Cell Res Ther ; 12(1): 444, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34365970

RESUMO

BACKGROUND: Age-associated changes attenuate human blood system functionality through the aging of hematopoietic stem and progenitor cells (HSPCs), manifested in human populations an increase in myeloproliferative disease and even leukemia; therefore, study on HSPC senescence bears great significance to treat hematopoietic-associated disease. Furthermore, the mechanism of HSPC aging is lacking, especially the cellular memory mechanism. Here, we not only reported a new HSPC senescence model in vitro, but also propose and verify the cellular memory mechanism of HSPC aging of the Polycomb/Trithorax system. METHODS: HSPCs (Lin-c-kit+ cells) were isolated and purified by magnetic cell sorting (MACS). The proportions and cell cycle distribution of cells were determined by flow cytometry; senescence-related ß-galactosidase assay, transmission electron microscope (TEM), and colony-forming unit (CFU)-mix assay were detected for identification of the old HSPC model. Proteomic tests and RNA-seq were applied to analyze differential pathways and genes in the model cells. qPCR, Western blot (WB), and chromatin immunoprecipitation PCR (CHIP-PCR) were used to detect the gene expression of cell memory-related proteins. Knockdown of cell memory-related key genes was performed with shRNA interference. RESULTS: In the model old HSPCs, ß-gal activity, cell cycle, colony-forming ability, aging-related cell morphology, and metabolic pathway were significantly changed compared to the young HSPCs. Furthermore, we found the model HSPCs have more obvious aging manifestations than those of natural mice, and IL3 is the major factor contributing to HSPC aging in the model. We also observed dramatic changes in the expression level of PRC/TrxG complexes. After further exploring the downstream molecules of PRC/TrxG complexes, we found that Uhrf1 and TopII played critical roles in HSPC aging based on the HSPC senescence model. CONCLUSIONS: These findings proposed a new HSPC senescence model in vitro which we forecasted could be used to preliminary screen the drugs of the HSPC aging-related hemopathy and suggested cellular memory mechanism of HSPC aging.


Assuntos
Células-Tronco Hematopoéticas , Proteômica , Animais , Senescência Celular , Ensaio de Unidades Formadoras de Colônias , Camundongos
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