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1.
Adv Exp Med Biol ; 1169: 243-256, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31487028

RESUMO

Heterogeneity among different subpopulations of human umbilical cord mesenchymal stem cell (hUCMSCs) lines is an ubiquitous phenomenon, with such variability being related to several factors including the identity of the individual donor, tissue source (Wharton's jelly vs. umbilical cord blood), culture conditions, as well as random variations in the cloning expansion process. In this chapter, we provide a general overview on the sources as well as available experimental techniques for proper identification of heterogeneity in hUCMSCs. Finally, we provide a brief discussion on the current scientific evidence regarding the potential superiority of subpopulations of hUCMSCs for specific clinical applications. Taking into account the exponential growth on the available experimental data on hUCMSCs in the past few years, this chapter is not intended to be comprehensive in nature, but rather is intended to provide a general overview about the central role which the topic of heterogeneity has in both basic science and clinical research in umbilical cord stem cells.


Assuntos
Células-Tronco Mesenquimais , Cordão Umbilical , Diferenciação Celular , Células Cultivadas , Sangue Fetal/citologia , Humanos , Células-Tronco Mesenquimais/citologia , Cordão Umbilical/citologia , Geleia de Wharton
3.
Life Sci ; 232: 116598, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31247209

RESUMO

Hematopoietic stem cells (HSCs) are a rare cell population in adult bone marrow, mobilized peripheral blood, and umbilical cord blood possessing self-renewal and differentiation capability into a full spectrum of blood cells. Bone marrow HSC transplantation has been considered as an ideal option for certain disorders treatment including hematologic diseases, leukemia, immunodeficiency, bone marrow failure syndrome, genetic defects such as thalassemia, sickle cell anemia, autoimmune disease, and certain solid cancers. Ex vivo proliferation of these cells prior to transplantation has been proposed as a potential solution against limited number of stem cells. In such culture process, MSCs have also been shown to exhibit high capacity for secretion of soluble mediators contributing to the principle biological and therapeutic activities of HSCs. In addition, endothelial cells have been introduced to bridge the blood and sub tissues in the bone marrow, as well as, HSCs regeneration induction and survival. Cell culture in the laboratory environment requires cell growth strict control to protect against contamination, symmetrical cell division and optimal conditions for maximum yield. In this regard, microfluidic systems provide culture and analysis capabilities in micro volume scales. Moreover, two-dimensional cultures cannot fully demonstrate extracellular matrix found in different tissues and organs as an abstract representation of three dimensional cell structure. Microfluidic systems can also strongly describe the effects of physical factors such as temperature and pressure on cell behavior.


Assuntos
Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Animais , Células da Medula Óssea/citologia , Técnicas de Cultura de Células/métodos , Técnicas de Cocultura , Células Endoteliais/citologia , Sangue Fetal/citologia , Humanos , Células-Tronco Mesenquimais/citologia
4.
Bioelectromagnetics ; 40(6): 375-390, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31157927

RESUMO

In this paper, morphological effects of electric fields on avian erythrocytes (nucleated red blood cells) have been studied in detail. Morphological changes include rounding and cytoplasm transparency. It has been shown that the effect is non-thermal. Careful imaging and image analyses have been carried out to show that the degree of this effect is frequency-dependent, and has a higher conversion rate at higher temperatures. Furthermore, to better understand the mechanisms behind the morphological changes, we investigated the dedifferentiation hypothesis and performed a series of tests on avian erythrocytes including fluorescence spectroscopy for hemoglobin, and tests on human umbilical cord blood, mesenchymal stem cells, and bone marrow mesenchymal stem cells including flow-cytometry analysis for expression of certain markers and calcium staining. Bioelectromagnetics. 2019;40:375-390. © 2019 Bioelectromagnetics Society.


Assuntos
Campos Eletromagnéticos , Eritrócitos/citologia , Sangue Fetal/citologia , Células-Tronco Mesenquimais/citologia , Animais , Aves , Diferenciação Celular , Linhagem Celular , Estimulação Elétrica , Humanos , Concentração de Íons de Hidrogênio , Temperatura Ambiente
5.
Nat Commun ; 10(1): 1634, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30967552

RESUMO

Gene correction in human long-term hematopoietic stem cells (LT-HSCs) could be an effective therapy for monogenic diseases of the blood and immune system. Here we describe an approach for X-linked sSevere cCombined iImmunodeficiency (SCID-X1) using targeted integration of a cDNA into the endogenous start codon to functionally correct disease-causing mutations throughout the gene. Using a CRISPR-Cas9/AAV6 based strategy, we achieve up to 20% targeted integration frequencies in LT-HSCs. As measures of the lack of toxicity we observe no evidence of abnormal hematopoiesis following transplantation and no evidence of off-target mutations using a high-fidelity Cas9 as a ribonucleoprotein complex. We achieve high levels of targeting frequencies (median 45%) in CD34+ HSPCs from six SCID-X1 patients and demonstrate rescue of lymphopoietic defect in a patient derived HSPC population in vitro and in vivo. In sum, our study provides specificity, toxicity and efficacy data supportive of clinical development of genome editing to treat SCID-Xl.


Assuntos
DNA Complementar/genética , Edição de Genes/métodos , Transplante de Células-Tronco Hematopoéticas , Subunidade gama Comum de Receptores de Interleucina/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapia , Animais , Antígenos CD34/metabolismo , Sistemas CRISPR-Cas/genética , Linhagem Celular , Códon de Iniciação/genética , Éxons/genética , Sangue Fetal/citologia , Vetores Genéticos/genética , Voluntários Saudáveis , Células-Tronco Hematopoéticas/metabolismo , Humanos , Masculino , Camundongos , Mutação , Parvovirinae/genética , Cultura Primária de Células , Fatores de Tempo , Transdução Genética/métodos , Quimeras de Transplante/genética , Transplante Heterólogo/métodos , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética
6.
Nat Commun ; 10(1): 1685, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30976008

RESUMO

Neonatal sepsis is characterized by hyperinflammation causing enhanced morbidity and mortality compared to adults. This suggests differences in the response towards invading threats. Here we investigate activated cord blood macrophages (CBMΦ) in comparison to adult macrophages (PBMΦ), indicating incomplete interferon gamma (IFN-γ) and interleukin 10 (IL-10)-induced activation of CBMΦ. CBMΦ show reduced expression of phagocytosis receptors and cytokine expression in addition to altered energy metabolism. In particular, IFN-γ as well as IL-10-activated CBMΦ completely fail to increase glycolysis and furthermore show reduced activation of the mTOR pathway, which is important for survival in sepsis. MTOR inhibition by rapamycin equalizes cytokine production in CBMΦ and PBMΦ. Finally, incubation of PBMΦ with cord blood serum or S100A8/A9, which is highly expressed in neonates, suppresses mTOR activation, prevents glycolysis and the expression of an PBMΦ phenotype. Thus, a metabolic alteration is apparent in CBMΦ, which might be dependent on S100A8/A9 expression.


Assuntos
Citotoxicidade Imunológica , Metabolismo Energético/imunologia , Macrófagos/metabolismo , Adulto , Fatores Etários , Calgranulina A/imunologia , Calgranulina A/metabolismo , Calgranulina B/imunologia , Calgranulina B/metabolismo , Diferenciação Celular/imunologia , Células Cultivadas , Sangue Fetal/citologia , Glicólise/imunologia , Voluntários Saudáveis , Humanos , Recém-Nascido , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-10/imunologia , Interleucina-10/metabolismo , Macrófagos/imunologia , Cultura Primária de Células , Sepse/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/imunologia , Serina-Treonina Quinases TOR/metabolismo
7.
Eur J Med Chem ; 174: 181-197, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31035239

RESUMO

The scarcity of hematopoietic stem cells (HSCs) significantly hindered their clinical potentials. Umbilical cord blood (UCB) has become the leading source of HSCs for both research and clinical applications. But the low content of HSCs in a single UCB unit limited its use only to pediatric patients. Various cytokines and small molecules have demonstrated strong abilities in promoting HSC ex vivo expansion, of which UM171 is the newest and by far the most potent HSC ex vivo expansion agent. In this study, we synthesized 37 pyrimidoindole analogs and identified 6 compounds to be potent in promoting HSC ex vivo expansion. In particular, analog 11 was found to be the most effective in stimulating ex vivo expansion of UCB CD34+ cells and CD34+CD38- cells. Initial data indicated that compound 11 promoted the absolute number of long term HSCs and inhibited their differentiation. UCB HSCs expanded with 11 retained adequate multi-lineage differentiation capacity. In addition, compound 11 is not cytotoxic at its test concentrations, suggesting that it merits further investigation for potential clinical applications.


Assuntos
Proliferação de Células/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Indóis/farmacologia , Pirimidinas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Humanos , Indóis/síntese química , Indóis/química , Indóis/toxicidade , Estrutura Molecular , Pirimidinas/síntese química , Pirimidinas/química , Pirimidinas/toxicidade , Relação Estrutura-Atividade
8.
Transfus Apher Sci ; 58(2): 169-173, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30890311

RESUMO

Bone marrow transplantation is a treatment used for hematologic and non-hematologic disorders. A theory suggests that proliferation of cells in non-body condition helps to increase the efficiency of bone marrow transplant. There are different ways for proliferation of stem cells, in which, most studies have focused on stem cell culture in body-like conditions. The use of amniotic fluid as a rich resource of growth factors is developing in repair of tissues cornea. With regards to this condition, we discuss about the influence of amniotic fluid in proliferation and implantation of blood stem cells. The aim of this study was investigation of human amnion fluid (HAF) in support of growth and proliferation of umbilical cord in order to transplant and long period erythropoiesis. First, separating of CD-34+ stem cells by MACS was performed and check in 5% and 8% concentration of amniotic fluid (AF) in comprise with FBS10% in culture environment. After 7, 14 days cell count, and checking gene expression level of cyclinD1, BCL2, CXCR4, SDF1 by real-time PCR. The result show that BCL2, CXCR4 and cyclinD1 gene expression level were increased in cells that are growth in 5% AF with 5% FBS than other groups. After statistical analysis, proliferation of umbilical cord blood stem cells in 5% AF with 5% FBS was more than 8% AF with 2% FBS and 10% FBS. Therefore, HAF can play an effective role in increasing hematopoietic stem cells in cell culture before bone marrow transplant.


Assuntos
Líquido Amniótico/metabolismo , Transplante de Medula Óssea/métodos , Sangue Fetal/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Humanos
9.
Mol Med Rep ; 19(5): 4195-4204, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30896872

RESUMO

Regulatory T cells (Tregs) maintain immune homeostasis and modulate tumor­induced neovascularization. However, the mechanisms underlying the role of Tregs in acute lymphoblastic leukemia (ALL) remain to be elucidated. Helios, combined with forkhead box P3, is considered a suitable marker for discriminating functional Tregs. In the present study, a microenvironment was created with a high proportion of Helios+ Tregs in T cell­deficient nude mice to determine the mechanism underlying Tregs expressing Helios in ALL. It was revealed that umbilical cord blood­derived Helios+ Tregs had proliferation and immunosuppression abilities similar to those of normal pediatric Tregs. The accumulation of Helios+ Tregs accelerated leukemogenesis and the infiltration of leukemic cells into the bone marrow. Importantly, a high expression of Helios in Tregs promoted angiogenesis in the bone marrow via the vascular endothelial growth factor (VEGF)A/VEGF receptor 2 (VEGFR2) pathway. Furthermore, the expression of chemokine CC­chemokine ligand 22 (CCL22) in the bone marrow and serum of ALL mice infused with Helioshigh Treg cells was increased. The results demonstrated that Helios promotes the secretion of chemokine CCL22, which may recruit more Tregs into the bone marrow. Increased Helios+ Treg cells promoted angiogenesis in the bone marrow of ALL mice via the VEGFA/VEGFR2 pathway. Therefore, Helios may be a target to manipulate Treg activity in clinical settings.


Assuntos
Quimiocina CCL22/metabolismo , Sangue Fetal/citologia , Neovascularização Patológica/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/etiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Linfócitos T Reguladores/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Medula Óssea/patologia , Humanos , Camundongos , Neovascularização Patológica/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/imunologia
10.
Mol Immunol ; 109: 99-107, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30921683

RESUMO

The relationship between breastfeeding and infant health has been well elucidated in past decades. Our previous study has shown that human ß-defensin 1 (hBD-1) in human breast milk plays a protective role in reducing the incidence of upper respiratory infection in infants younger than 6 months. In the present study, we aim to reveal the mechanism underlying the protective role of hBD-1 by focusing on its immunoregulatory function in neonates. Cord blood (CB) from newborns' umbilical cords, which can simulate many of the neonatal symptoms, was used to study the immunomodulatory role of hBD-1 in neonates in vitro. Our results showed that hBD-1 promotes the GM-CSF- and IL-4-driven differentiation of neonatal umbilical CB monocytes to immature dendritic cells (DCs) and the final maturation of CB monocyte-derived DCs (moDCs) induced by LPS but not inflammatory cytokine production. In addition, hBD-1 inhibits apoptosis in neonatal moDCs through CCR6, which might be a possible mechanism of the hBD-1-induced phenotypes in moDCs. Furthermore, we found that hBD-1 promotes the proliferation and activation, but not the maturation, of neonatal CB CD4 + T cells. These results extend the immunoregulatory effects of hBD-1 and provide a potential mechanism for the protective role of hBD-1 in early infants, which will inform the development of infant nutrition, novel vaccines and anti-infective strategies in the future.


Assuntos
Células Dendríticas/citologia , Sangue Fetal/citologia , Linfócitos T/citologia , beta-Defensinas/imunologia , Apoptose , Biomarcadores/metabolismo , Diferenciação Celular , Proliferação de Células , Citocinas/biossíntese , Células Dendríticas/metabolismo , Endocitose , Humanos , Recém-Nascido , Lipopolissacarídeos , Ativação Linfocitária/imunologia , Monócitos/citologia , Monócitos/metabolismo , Receptores CCR6/metabolismo , Linfócitos T/metabolismo
11.
Vox Sang ; 114(4): 330-339, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30900265

RESUMO

BACKGROUND AND OBJECTIVES: Several sources of haematopoietic stem cells have been used for static culture of megakaryocytes to produce platelets in vitro. This study compares and characterizes platelets produced in shear flow using precursor cells from either umbilical (UCB) or adult peripheral blood (PB). MATERIALS AND METHODS: The efficiency of platelet production of the cultured cells was studied after perfusion in custom-built von Willebrand factor-coated microfluidic flow chambers. Platelet receptor expression and morphology were investigated by flow cytometry and microscopy, respectively. RESULTS: Proliferation of stem cells isolated out of UCB was significantly higher (P < 0·0001) compared to PB. Differentiation of these cells towards megakaryocytes was significantly lower from PB compared to UCB where the fraction of CD42b/CD41 double positive events was 44 ± 9% versus 76 ± 11%, respectively (P < 0·0001). However, in vitro platelet production under hydrodynamic conditions was more efficient with 7·4 platelet-like particles per input cell from PB compared to 4·2 from UCB (P = 0·02). The percentage of events positive for CD42b, CD41 and CD61 was comparable between both stem cell sources. The mean number of receptors per platelet from UCB and PB was similar to that on blood bank platelets with on average 28 000 CD42b, 57 000 CD61 and 5500 CD49b receptors. Microscopy revealed platelets appearing similar to blood bank platelets in morphology, size and actin cytoskeleton, alongside smaller fragments and source megakaryocytes. CONCLUSION: This characterization study suggests that platelets produced in vitro under flow either from UCB or from PB share receptor expression and morphology with donor platelets stored in the blood bank.


Assuntos
Plaquetas/citologia , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Dispositivos Lab-On-A-Chip , Citoesqueleto de Actina/metabolismo , Antígenos CD34/metabolismo , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Citometria de Fluxo , Humanos , Integrina beta3/metabolismo , Megacariócitos/citologia , Microscopia , Fenótipo , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Refrigeração
12.
Cell Prolif ; 52(3): e12594, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30847992

RESUMO

OBJECTIVE: Ex vivo expansion is an effective way to produce cytokine-induced killer (CIK) cells needed for clinical trials. Here, ex vivo expansion and metabolism characters of CIK cells in static and dynamic cultures and the relationship between cell expansion and metabolism were investigated. MATERIALS AND METHODS: Oxygen transfer efficiency was assessed by computational fluid dynamics technique. Cell phenotype, apoptosis and of transporter expression were determined by flow cytometry and Western blotting. Metabolites and enzyme activities were assessed by biochemical methods. RESULTS: Dynamic cultures favoured better CIK cell expansion without impairing their phenotype and cytotoxicity, enhanced oxygen transfer efficiency. The glucose metabolism flux of cells in dynamic cultures was enhanced by upregulating surface glucose transporter 1 expression and phosphofructokinase activity. Moreover, pentose phosphate pathway (PPP) metabolic flux was enhanced through upregulating glucose-6-phosphate dehydrogenase activity. Glutaminolysis was also accelerated via boosting glutamine transporters expression, glutaminase (GLS) and glutamate dehydrogenase activities. Together with higher oxygen consumption rate and extracellular acidification rate, it was suggested that cells in dynamic cultures were in a more vigorous metabolic state for ATP production. CONCLUSION: Dynamic cultures accelerated glucose and glutamine metabolic flux to promote ATP production, elevated glucose metabolic flux through PPP to promote biosynthesis for better cell expansion. These findings may provide the basis for ex vivo CIK cell expansion process optimization.


Assuntos
Trifosfato de Adenosina/biossíntese , Células Matadoras Induzidas por Citocinas/metabolismo , Via de Pentose Fosfato , Técnicas de Cultura de Células , Proliferação de Células , Células Matadoras Induzidas por Citocinas/citologia , Células Matadoras Induzidas por Citocinas/imunologia , Sangue Fetal/citologia , Glucose/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Glutamina/metabolismo , Glicólise , Humanos , Imunoterapia Adotiva , Modelos Biológicos , Neoplasias/imunologia , Neoplasias/terapia , Consumo de Oxigênio
13.
Nature ; 566(7744): 398-402, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30760926

RESUMO

The human genome contains approximately 20 thousand protein-coding genes1, but the size of the collection of antigen receptors of the adaptive immune system that is generated by the recombination of gene segments with non-templated junctional additions (on B cells) is unknown-although it is certainly orders of magnitude larger. It has not been established whether individuals possess unique (or private) repertoires or substantial components of shared (or public) repertoires. Here we sequence recombined and expressed B cell receptor genes in several individuals to determine the size of their B cell receptor repertoires, and the extent to which these are shared between individuals. Our experiments revealed that the circulating repertoire of each individual contained between 9 and 17 million B cell clonotypes. The three individuals that we studied shared many clonotypes, including between 1 and 6% of B cell heavy-chain clonotypes shared between two subjects (0.3% of clonotypes shared by all three) and 20 to 34% of λ or κ light chains shared between two subjects (16 or 22% of λ or κ light chains, respectively, were shared by all three). Some of the B cell clonotypes had thousands of clones, or somatic variants, within the clonotype lineage. Although some of these shared lineages might be driven by exposure to common antigens, previous exposure to foreign antigens was not the only force that shaped the shared repertoires, as we also identified shared clonotypes in umbilical cord blood samples and all adult repertoires. The unexpectedly high prevalence of shared clonotypes in B cell repertoires, and identification of the sequences of these shared clonotypes, should enable better understanding of the role of B cell immune repertoires in health and disease.


Assuntos
Anticorpos/genética , Anticorpos/imunologia , Linfócitos B/imunologia , Células Clonais/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Adulto , Sequência de Aminoácidos , Anticorpos/química , Antígenos/imunologia , Linfócitos B/citologia , Linfócitos B/metabolismo , Sequência de Bases , Células Clonais/citologia , Células Clonais/metabolismo , Feminino , Sangue Fetal/citologia , Sangue Fetal/imunologia , Voluntários Saudáveis , Humanos , Recém-Nascido , Masculino , Receptores de Antígenos de Linfócitos B/química , Receptores de Antígenos de Linfócitos B/genética , Análise de Sequência de DNA
14.
Stem Cell Res ; 35: 101392, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30711803

RESUMO

Here we report the reprogramming of CD34+ cells obtained from UCB of a healthy donor female child belonging to the Indian ethnic population. These CD34+cells were subjected to nucleofection for delivery of episomal vectors expressing Oct4, Sox2, L-Myc, Lin28, Klf4 and p53DD (negative mutation in p53). The iPSC colonies expressed pluripotency markers as detected by PCR, immunofluorescence and flow-cytometry. The removal of plasmid was confirmed by its absence in cells at higher passages. Karyotype analysis revealed a stable genome. The property of in vitro differentiation to tri-lineage was confirmed by expression of markers by immunofluorescence.


Assuntos
Antígenos CD34/metabolismo , Linhagem Celular , Técnicas de Reprogramação Celular , Sangue Fetal , Células-Tronco Pluripotentes Induzidas , Grupo com Ancestrais do Continente Asiático , Reprogramação Celular , Feminino , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Recém-Nascido , Cariótipo
15.
Curr Stem Cell Res Ther ; 14(4): 367-372, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30806326

RESUMO

Cord Blood (CB) is a unique and readily available source of hematopoietic stem cells for transplantation. CB also contains other types of stem cells, including endothelial stem cells and mesenchymal stem cells, that may prove useful in non-traditional clinical uses. Genetic and molecular analyses have demonstrated that CB stem cells lie somewhere between mature stem cells like those found in Bone Marrow (BM), and fetal stem cells. After 25 years of clinical experience, CB is now used in the same fashion as BM for all typical malignant and genetic diseases treated by bone marrow transplant. Due to the establishment of CB banks in the US and abroad, more than 35,000 CB transplants have been performed over the past 25 years. An average of 700-800 CB transplants are performed annually. In addition, CB is now used more frequently for regenerative medicine and tissue engineering applications. At first glance, it seems that everything could not be better with the public cord blood banks and the use of their samples in the clinic. However, a recent report by the Rand Corp. reviewed the US national cord blood stem cell banking program and detailed many ongoing problems. However, some details were omitted from the report that would shed some light on the causes of many of the problems. This paper will summarize the status of the public cord blood stem cell banking program in the US, detail the problems associated with the program that could jeopardize its existence and suggest possible solutions to resolve these issues.


Assuntos
Células-Tronco Adultas/citologia , Bancos de Sangue/economia , Transplante de Células-Tronco de Sangue do Cordão Umbilical/economia , Sangue Fetal/citologia , Investimentos em Saúde , Células-Tronco Mesenquimais/citologia , Setor Público , Humanos , Imposto de Renda , Estados Unidos
16.
Gene ; 696: 10-20, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30769140

RESUMO

Human umbilical cord (UC) and cord blood (CB) provide attractive sources of mesenchymal stem cells (MSCs) for cell therapy. Both UCMSCs and CBMSCs have been demonstrated to play prominent roles in clinical therapy. However, little is known about their functional differences in clinical application. Our transcriptome analysis uncovered high activity of insulin secretion related signaling pathways for CBMSCs and cell adhesion related signaling pathways for UCMSCs. Expression of a large number of immune related signaling pathways also showed the difference in both cells, implying their distinct immune modulatory functions. As the therapeutic effects of MSCs mainly dependent on the cytokines and growth factors produced by transplanted MSCs, we further compared the cytokine profiles of UCMSCs and CBMSCs using antibody array. By evaluating the expression of 106 cytokines, we found both MSCs abundantly secreted TSP-1, TSG-14, TIMP-1, IL-8, IL-6, CXCL1, GIF and IGFBP3. However, the expression of CCL2 in UCMSCs showed significantly higher than CBMSCs. IGFBP1 and IGFBP2 were secreted by CBMSCs with higher abundance than UCMSCs. Overall, these results suggest that UCMSCs and CBMSCs preserve different functional potentials, which have to be carefully considered before clinical treatment.


Assuntos
Citocinas/metabolismo , Sangue Fetal/citologia , Células-Tronco Mesenquimais/metabolismo , Cordão Umbilical/citologia , Células Cultivadas , Sangue Fetal/metabolismo , Perfilação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Cultura Primária de Células , Cordão Umbilical/metabolismo
17.
Methods Mol Biol ; 1940: 97-107, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30788820

RESUMO

Human umbilical cord blood contains highly proliferative circulating endothelial colony-forming cells (ECFC). These cells have promising therapeutic potential for various cardiovascular diseases by possessing robust in vitro clonal expansion potential and the ability to form functional blood vessels in vivo upon transplantation into recipient immunodeficient mice. However whether similar cells also exist in murine blood remains unresolved, which impedes the study of circulating ECFC biology using murine models. Here we describe a method to identify and culture murine embryonic peripheral blood-derived circulating ECFC through co-culture with OP9 stromal cells. Using this method, embryonic circulating ECFC can be identified by the formation of sheet-like or network-like endothelial colonies upon OP9 stromal cell monolayers.


Assuntos
Células-Tronco Embrionárias/citologia , Células Progenitoras Endoteliais/citologia , Sangue Fetal/citologia , Animais , Técnicas de Cultura de Células , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL
18.
Cell Tissue Res ; 376(2): 233-245, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30610451

RESUMO

Large expansion of human mesenchymal stem cells (MSCs) is of great interest for clinical applications. In this study, we examine the feasibility of human fibroblast-derived extracellular matrix (hFDM) as an alternative cell expansion setting. hFDM is obtained from decellularized extracellular matrix (ECM) derived from in vitro cultured human lung fibroblasts. Our study directly compares conventional platforms (tissue culture plastic (TCP), fibronectin (FN)-coated TCP) with hFDM using umbilical cord blood-derived MSCs (UCB-MSCs). Early cell morphology shows a rather rounded shape on TCP but highly elongated morphology on hFDM. Cell proliferation demonstrates that MSCs on hFDM were significantly better compared to the others in both 10 and 2% serum condition. Cell migration assay suggests that cell motility was improved and a cell migration marker CXCR4 was notably up-regulated on hFDM. MSCs differentiation into osteogenic lineage on hFDM was also very effective as examined via gene expression, von Kossa staining and alkaline phosphatase activity. In addition, as the MSCs were expanded on each substrate, transferred to 3D polymer mesh scaffolds and then cultivated for a while, the data found better cell proliferation and more CXCR4 expression with MSCs pre-conditioned on hFDM. Moreover, higher gene expression of stemness and engraftment-related markers was noticed with the hFDM group. Furthermore when UCB-MSCs expanded on TCP or hFDM were injected into emphysema (a lung disease) animal model, the results indicate that MSCs pre-conditioned on hFDM (with 2% serum) retain more advanced therapeutic efficacy on the improvement of emphysema than those on TCP. Current works demonstrate that compared to the conventional platforms, hFDM can be a promising source of cell expansion with a naturally derived biomimetic ECM microenvironment and may find some practical applications in regenerative medicine.


Assuntos
Enfisema/terapia , Matriz Extracelular , Sangue Fetal/citologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/fisiologia , Regeneração , Animais , Materiais Biomiméticos , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Humanos , Pulmão/citologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores CXCR4/metabolismo , Medicina Regenerativa , Engenharia Tecidual , Tecidos Suporte
19.
Biomed Mater ; 14(2): 025009, 2019 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-30609413

RESUMO

AIMS: The need for small caliber vessels to treat cardiovascular diseases has grown. However, synthetic polymers perform poorly in small-diameter applications. Chitosan hydrogels can provide a novel biological scaffold for vascular engineering. The goal of this study was to explore host cell and tissue behavior at the interface with chitosan-based scaffolds in vitro and in vivo. METHODS AND RESULTS: in vitro, we assessed the ability of endothelial cells lining chitosan hydrogels to produce tissue factor (TF), thrombomodulin (TM) and nitric oxide. We showed that endothelial cells behave as a native endothelium since under stimulation, TF and TM expression increased and decreased, respectively. Endothelial cells seeded on chitosan produced nitric oxide, but no change was observed under stimulation. After in vivo subcutaneous implantation of chitosan hydrogels in rats, macrophage activation phenotypes, playing a crucial role in biomaterial/tissue, were explored by immunohistochemistry. Our results suggested a balance between pro- and anti-inflammatory signals since we observed an inflammatory response in favor of macrophage M2 phenotype. CONCLUSION: in vitro exploration of endothelial cell response at the interface with chitosan hydrogel showed a functional endothelium and in vivo exploration of tissue response revealed a biointegration of chitosan hydrogels.


Assuntos
Materiais Biocompatíveis/química , Prótese Vascular , Quitosana/química , Hidrogéis/química , Engenharia Tecidual/métodos , Animais , Células Cultivadas , Células Endoteliais/citologia , Endotélio Vascular/citologia , Sangue Fetal/citologia , Humanos , Imuno-Histoquímica , Macrófagos/citologia , Óxido Nítrico/química , Fenótipo , Ratos , Trombomodulina/química , Tromboplastina/química , Tecidos Suporte
20.
Blood ; 133(9): 927-939, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30622121

RESUMO

Recent advances in single-cell molecular analytical methods and clonal growth assays are enabling more refined models of human hematopoietic lineage restriction processes to be conceptualized. Here, we report the results of integrating single-cell proteome measurements with clonally determined lymphoid, neutrophilic/monocytic, and/or erythroid progeny outputs from >1000 index-sorted CD34+ human cord blood cells in short-term cultures with and without stromal cells. Surface phenotypes of functionally examined cells were individually mapped onto a molecular landscape of the entire CD34+ compartment constructed from single-cell mass cytometric measurements of 14 cell surface markers, 20 signaling/cell cycle proteins, and 6 transcription factors in ∼300 000 cells. This analysis showed that conventionally defined subsets of CD34+ cord blood cells are heterogeneous in their functional properties, transcription factor content, and signaling activities. Importantly, this molecular heterogeneity was reduced but not eliminated in phenotypes that were found to display highly restricted lineage outputs. Integration of the complete proteomic and functional data sets obtained revealed a continuous probabilistic topology of change that includes a multiplicity of lineage restriction trajectories. Each of these reflects progressive but variable changes in the levels of specific signaling intermediates and transcription factors but shared features of decreasing quiescence. Taken together, our results suggest a model in which increasingly narrowed hematopoietic output capabilities in neonatal CD34+ cord blood cells are determined by a history of external stimulation in combination with innately programmed cell state changes.


Assuntos
Antígenos CD34/metabolismo , Linhagem da Célula , Sangue Fetal/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Proteoma/análise , Análise de Célula Única/métodos , Diferenciação Celular , Células Cultivadas , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Humanos , Proteoma/metabolismo
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