An International Society for Cell and Gene Therapy Mesenchymal Stromal Cells (MSC) Committee perspectives on International Standards Organization/Technical Committee 276 Biobanking Standards for bone marrow-MSCs and umbilical cord tissue-derived MSCs for research purposes.

The rapidly growing field of mesenchymal stromal cell (MSC) basic and translational research requires standardization of terminology and functional characterization. The International Standards Organization's (ISO) Technical Committee (TC) on Biotechnology, working with extensive input from the International Society for Cells and Gene Therapy (ISCT), has recently published ISO standardization documents that are focused on biobanking of MSCs from two tissue sources, Wharton's Jelly, MSC(WJ) and Bone Marrow, MSC(M)), for research and development purposes and development. This manuscript explains the path towards the consensus on the following two documents: the Technical Standard ISO/TS 22859 for MSC(WJ) and the full ISO Standard 24651 for MSC(M) biobanking. The ISO standardization documents are aligned with ISCT's MSC committee position and recommendations on nomenclature because there was active input and incorporation of ISCT MSC committee recommendations in the development of these standards. The ISO standardization documents contain both requirements and recommendations for functional characterization of MSC(WJ) and MSC(M) using a matrix of assays. Importantly, the ISO standardization documents have a carefully defined scope and are meant for research use of culture expanded MSC(WJ) and MSC(M). The ISO standardization documents can be updated in a revision process and will be systematically reviewed after 3-5 years as scientific insights grow. They represent international consensus on MSC identity, definition, and characterization; are rigorous in detailing multivariate characterization of MSCs and represent an evolving-but-important first step in standardization of MSC biobanking and characterization for research use and development.

3D Multicellular Spheroid for the Study of Human Hematopoietic Stem Cells: Synergistic Effect Between Oxygen Levels, Mesenchymal Stromal Cells and Endothelial Cells.

INTRODUCTION: The human bone marrow microenvironment is composed of biological, chemical and physical factors that act in a synergistic way to modulate hematopoietic stem cell biology, such as mesenchymal stromal cells (MSCs), endothelial cells (ECs) and low oxygen levels; however, it is difficult to mimic this human microenvironment in vitro. METHODS: In this work, we developed 3D multicellular spheroid (3D-MS) for the study of human hematopoietic stem cells (HSCs) with some components of perivascular niche. HSCs were isolated from umbilical cord blood, MSCs were isolated from human bone marrow and a microvasculature EC line (CC-2811, Lonza®) was used. For the formation of a 3D structure, a magnetic levitation culture system was used. Cultures were maintained in 21%, 3% and 1% O2 for 15 days. Culture volume, sphericity index and cell viability were determined. Also, human HSC proliferation, phenotype and production of reactive oxygen species were evaluated. RESULTS: After 15 days, 3D-MS exhibited viability greater than 80%. Histology results showed structures without necrotic centers, and higher cellular proliferation with 3% O2. An increase in the expression of the CD34 antigen and other hematopoietic antigens were observed to 1% O2 with MSCs plus ECs and low ROS levels. CONCLUSION: These findings suggest that 3D-MS formed by MSCs, ECs and HSCs exposed to low concentrations of oxygen (1-3% O2) modulate human HSC behavior and mimics some features of the perivascular niche, which could reduce the use of animal models and deepen the relationship between the microenvironment of HSC and human hematological diseases development.

Impact of donor characteristics on the quality of bone marrow as a source of mesenchymal stromal cells.

In recent years, the therapeutic use of mesenchymal stromal cells (MSC) has generated a valuable number of scientific studies that delve into their biological characteristics and their potential in regenerative medicine; however, the impact of the clinical characteristics of tissue donors, from which these cells are isolated, on their potential in applied clinical research is not yet clear. The objective of this study was to evaluate the impact of the clinical characteristics of bone marrow donors on the quality of this tissue as a source of MSC for therapeutic use. Human MSC were isolated, characterized and cultured (according to ISCT criteria) from bone marrow samples from volunteer donors (n = 70) attending the Department of Orthopedics and Traumatology of the Hospital Universitario San Ignacio (Bogota, Colombia) for surgery of prosthetic hip replacement that agreed to participate voluntarily in the study. Donor data such as age, gender, weight, smoker and type of anesthesia used during the surgical procedure were recorded, and the impact of these characteristics on the volume of tissue collection, mononuclear cell count and confluence time of cells with fibroblastoid morphology was evaluated. Correlation coefficients between quantitative variables were calculated with Spearman's correlation test, and the association between qualitative and quantitative variables was evaluated with biserial correlation coefficient. A significant correlation was observed between the age of the donors and the time necessary to obtain confluent cells in vitro (r = 0.2489, P = 0.0377); similarly, the correlation between the volume of bone marrow collected and the number of mononuclear cells obtained was significant (r = 0.7101, P = 0.0001). Although a negative correlation tendency was observed between the mononuclear cell count and the confluence time, this was not significant (r = -0.2041, P = 0.0950). No significant associations were observed between gender, smoking status or type of anesthesia and the expansion characteristics of human mesenchymal stromal cells. Bone marrow donor age and the tissue collection volume impact the time of obtaining MSC in vitro and the mononuclear cell count with which the culture starts. These conditions must be considered when the bone marrow is selected as the tissue for obtaining MSC.

Aislamiento y caracterización de células "stem" mesenquimales de médula ósea humana según criterios de la Sociedad Internacional de Terapia Celular / Isolation and characterization of mesenchymal stem cells from human bone marrow according to the criteria of the International Society for Cellular Therapy / Isolamento e caracterização de células-tronco mesenquimais de medula óssea humana de acordo com critérios da Sociedade Internacional de Terapia Celular

La médula ósea (MO) es una fuente importante para el aislamiento de células "stem" mesenquimales (CSM) útiles en terapias de regeneración tisular e inmunomodulación. Objetivo. Aislar y caracterizar células "stem" mesenquimales obtenidas de MO según los criterios exigidos por la Sociedad Internacional de Terapia Celular. Materiales y métodos. Se recolectaron muestras de MO de donantes voluntarios que asistían al Servicio de Ortopedia del Hospital Universitario San Ignacio, Bogotá (Colombia). Se evaluaron las características morfológicas por microscopia invertida y se determinó el inmunofenotipo por citometría de flujo. Se establecieron protocolos de inducción adipogénica, osteogénica y condrogénica mediante el uso de medios de cultivo de específicos y se evaluó la diferenciación utilizando tinciones de aceite rojo 'O', fosfatasa alcalina y safranina, respectivamente. Resultados. Se recolectaron 24 muestras de MO de pacientes sometidos a reemplazo total de cadera (volumen de muestra de MO: 5 - 45 ml). Se aislaron células con morfología fibroblastoide a partir de 21 muestras de MO (eficiencia de aislamiento: 87,5 %). En la determinación inmunofenotípica (CSM de MO) no existe diferencia estadísticamente significativa entre los antígenos hematopoyéticos (CD34 y CD45, p>0,05). Entre el antígeno hematopoyético CD45 y los antígenos mesenquimales (CD13, CD44, CD73, CD90, CD105, HLA-I y HLA-DR) existe diferencia estadísticamente significativa (p=0.006). La tinción de aceite rojo 'O' reveló la presencia de adipocitos multiloculares, en la inducción osteogénica se observaron centros de mineralización y la condrogénesis fue positiva para la coloración con safranina. Conclusión. A partir de MO se aislaron satisfactoriamente y caracterizaron CSM según los criterios internacionales exigidos.

Bone marrow (BM) is an important source for isolating mesenchymal stem cells (MSC) useful in immunomodulation and tissue regeneration therapies. Objective. To isolate and characterize mesenchymal stem cells obtained from BM meeting the requirements of the International Society for Cellular Therapy. Materials and methods. BM samples were collected from volunteer donors attending the Orthopedics Service of the San Ignacio University Hospital (Bogotá, Colombia). Morphological characteristics were evaluated by inverted microscopy and the immunophenotype was determined by flow cytometry. Protocols were developed for adipogenic, osteogenic and chondrogenic differentiation using the Oil Red O, alkaline phosphatase and safranin stains, respectively. Results. We collected 24 samples of BM from patients with total hip replacement (volume of BM sample: 5-45 ml). Cells with a fibroblastoid morphology were isolated from 21 BM samples (isolation efficiency: 87.5%). No statistical significant differences were found between the hematopoyetic antigens (CD34 and CD45, p>0.05) in the immunophenotypic evaluation (of MSC from BM); on the contrary, there were differences (p=0.006) between the hematopoyetic antigen CD45 and the mesenchymal antigens (CD13, CD44, CD73, CD90, CD105, HLA-I, and HLA-DR). Oil Red O stain revealed the presence of multilocular adipocytes, in the osteogenic induction we observed localized mineralization nodules, and chondrogenesis was positive as revealed by the safranin stain. Conclusion. MSC were satisfactorily isolated from BM and characterized according to the international standards.

A medula óssea (MO) é uma importante fonte para o isolamento de células-tronco mesenquimais (CTM) úteis na terapia de regeneração dos tecidos e imunomodulação. Objetivo. Isolar e caracterizar células-tronco mesenquimais obtidas de MO de acordo com os critérios exigidos pela Sociedade Internacional de Terapia Celular. Materiais e métodos. Foram coletadas amostras de MO de doadores voluntários que assistiram ao Serviço de Ortopedia do Hospital Universitário de São Ignacio, Bogotá (Colômbia). As características morfológicas foram avaliadas por microscopia invertida, e o imunofenótipo foi determinado por citometria de fluxo. Foram estabelecidos protocolos de indução adipogênica, osteogênica e condrogênica usando meios de cultura específicos e a diferenciação foi avaliada por meio da coloração com o "Oil Red O", fosfatase alcalina e safranina, respectivamente. Resultados. Foram coletadas 24 amostras de MO em pacientes submetidos â artroplastia total do quadril (volume de amostra de OM: 5 - 45 ml). Foram isoladas células com morfologia fibroblastóide a partir de 21 amostras de MO (eficiência de isolamento: 87,5%). Na determinação immunofenotípica (CTM de MO), não existe diferença estatisticamente significativa entre os antigénios hematopoiéticos (CD34 e CD45, p>0,05). Entre o antigénio hematopoiético CD45 e os antigénios mesenquimais (CD13, CD44, CD73, CD90, CD105, HLA-I e HLA-DR), existe diferença estatisticamente significativa (p =0,006). A coloração com o "Oil Red O" revelou a presença de adipócitos multiloculares, na indução osteogénica observaram-se centros de mineralização e a condrogênese foi positiva para a coloração com safranina. Conclusão. A partir de MO foram isoladas satisfatoriamente e caracterizaram CTM segundo os critérios internacionais exigidos.

Análisis inmunofenotípico de muestras normales de médula ósea: aplicaciones en el control de calidad en los laboratorios de citometría / Immunophenotypic analysis of normal cell samples from bone marrow: applications in quality control of cytometry laboratories / Análise imunofenotípica de amostras de medula óssea normal: aplicações no controle de qualidade em laboratórios de citometria

Objetivo. Describir un protocolo estandarizado mediante citometría de flujo para cuantificar en términos absolutos y relativos distintas subpoblaciones celulares de médula ósea normal y analizar la expresión de diferentes marcadores celulares específicos de linaje cuya reactividad está asociada a la diferenciación celular para ser usado como parte del control de calidad de rutina en los laboratorios de citometría. Materiales y métodos. El análisis inmunofenotípico de distintas subpoblaciones celulares se realizó en muestras de MO normal empleando un panel de anticuerpos monoclonales y policlonales útiles para la caracterización fenotípica de leucemias agudas en 4 fluorescencias distintas, con un protocolo que combina marcaje celular de antígenos de membrana y de citoplasma. El análisis de expresión se realizó en términos de intensidad media de fluorescencia. Para el cálculo de recuentos absolutos se adicionaron esferas fluorescentes de concentración conocida. Resultados. El panel de anticuerpos utilizado permitió la identificación y cuantificación de las distintas subpoblaciones leucocitarias normales de origen linfoide y mieloide incluyendo células precursoras CD34+, y poblaciones celulares más diferenciadas incluidas en las líneas granulocítica, monocítica y eritroide. Se establecieron los valores de referencia de las poblaciones celulares y los rangos de expresión de los diferentes marcadores celulares importantes como parte del control de calidad de rutina en los laboratorios de citometría. Conclusión. Los patrones inmunofenotípicos identificados y la determinación de los valores absolutos y relativos de referencia de las distintas poblaciones leucocitarias normales en MO podrán ser utilizados por los laboratorios de citometría como modelo para establecer parámetros de referencia en el análisis fenotípico de neoplasias hematológicas.

Objective. To describe a standardized flow cytometry protocol for the relative and absolute quantification of hematopoietic cell subpopulations from normal bone marrow, and to evaluate the expression of different lineage-specific cell markers with a reactivity associated to cell differentiation to be used as part of the routine quality control in cytometry laboratories. Materials and methods. The immunophenotypical analysis of different cell subpopulations was done with samples from normal bone marrow using a panel of monoclonal and polyclonal antibodies useful in the characterization of acute leukemias with four different fluorescences, by means of a protocol that combines cell labeling of membrane and cytoplasm antigens. Expression analysis was done in terms of mean fluorescence intensity (MFI). Fluorescent beads at a known concentration were added for calculating the absolute count of cells. Results. The antibody panel used allowed the identification and quantification of different normal leukocyte subpopulations of lymphatic and myeloid origin, including CD34+ stem cells and more differentiated cell populations in the granulocytic, monocytic, and erythroid cell lines. We established reference values for cell populations and cell marker expression ranges as part of routine quality control of cytometry laboratories. Conclusion. Immunophenotypic patterns identified as well as absolute and relative reference values for the different normal leukocyte populations from bone marrow can be used by cytometry laboratories as a basis for establishing reference parameters in phenotypic analyses of hematologic neoplasia.

Objetivo. Descrever um protocolo padronizado por citometria de fluxo para quantificar em termos absolutos e relativos diferentes subpopulações de células de medula óssea normal e analisar a expressão de diferentes marcadores celulares de linhagem específica, cuja reatividade é associada com a diferenciação celular para ser usado como parte do controle de qualidade de rotina nos laboratórios de citometria de fluxo. Materiais e métodos. A análise imunofenotípica das subpopulações de células foi realizada em amostras de MO normais utilizando um painel de anticorpos monoclonais e policlonais úteis para a caracterização fenotípica de leucemia aguda em quatro fluorescências, com um protocolo que combina rotulagem celular de antígeno de membrana celular e de citoplasma. A análise de expressão foi realizada em termos de intensidade média de fluorescência. Para calcular a recontagem absoluta foram adicionadas esferas fluorescentes de concentração conhecida. Resultados. O painel de anticorpos utilizado permitiu a identificação e quantificação das subpopulações de leucócitos normais de origem linfóide e mielóide incluindo as células precursoras CD34+, e populações de células mais diferenciadas incluídas nas linhas granulocítica, monocítica e eritróide. Foram estabelecidos os valores de referência das populações celulares e os intervalos de expressão dos diferentes marcadores celulares importantes como parte da rotina de controle de qualidade em laboratórios de citometria. Conclusão. Os padrões imunofenotípicos identificados e a determinação dos valores absolutos e relativos de referência das diferentes populações de leucócitos normais em MOM podem ser utilizados pelos laboratórios de citometria como um modelo para estabelecer parâmetros de referencia na análise fenotípica de neoplasias hematológicas.

Evaluación de características morfológicas e inmunofenotipo de células madre mesenquimales en cultivo obtenidas a partir de sangre de cordón umbilical y médula ósea / Assessment of morphologic characteristics and immunophenotype of mesenchimal stem cell culture obtained from umbilical cord blood and bone marrow

La sangre del cordón umbilical y la médula ósea humana son una alternativa para el aislamiento y cultivo de células madre mesenquimales, útiles en terapias de regeneración tisular e inmunomodulación. El objetivo de este trabajo fue aislar y cultivar células madre mesenquimales a partir de la sangre del cordón umbilical y de la médula ósea. Se recolectaron muestras de sangre de cordón umbilical en el servicio de Gineco-Obstetricia del Hospital Occidente de Kennedy en Bogotá, Colombia. La recolección de médula ósea se realizó en los servicios de ortopedia y traumatología del hospital universitario San Ignacio de Bogotá, Colombia. Se evaluó la tasa de generación celular, características morfológicas por microscopia invertida y tinción de Wright, asi como el inmunofenotipo de las poblaciones celulares por citometría de flujo. La eficiencia de aislamiento de las células madre mesenquimales a partir de sangre de cordón umbilical fue del 30/100 con una tasa de generación entre 20 y 50 minutos. A partir de médula ósea el aislamiento fue del 100/100, con un tiempo de generación entre 16 y 39 minutos. Se observaron diferencias morfológicas por tinción de Wright y la presencia de progenitores hematopoyéticos durante el cultivo primario (3.54/100 de CD34+/CD45+), que disminuían cuando se realizaba el primer pase del cultivo (0.19/100 de CD34+/CD45+). El aislamiento de células madre mesenquimales es más eficiente a partir de medula ósea. Se observan diferencias morfológicas por microscopia invertida y tinción de Wright entre células aisladas de sangre de cordón umbilical y médula ósea. El antígeno CD105 constituye un marcador importante en el establecimiento del perfil inmunofenotípico de células madre mesenquimales.

Células madre mesenquimales: características biológicas y aplicaciones clínicas / Mesenchymal stem cells: Biological characteristics and clinical applications

Las células madre mesenquimales son células pluripotentes y adultas con morfología fibroblastoide y plasticidad hacia diversos linajes celulares como condrocitos, osteocitos y adipocitos entre otros. Estas células pueden ser aisladas principalmente de médula ósea, sangre de cordón umbilical y tejido adiposo de donde se han logrado establecer cultivos que han permitido estudiar sus propiedades funcionales y fenotípicas. Aunque la información obtenida hasta la fecha no brinda un conocimiento completo, se espera que con el desarrollo de próximas investigaciones se aclaren diversos aspectos biológicos para implementar su uso en medicina regenerativa. Esta revisión presenta una visión general sobre las células madre mesenquimales: morfología e inmunofenotipo, ontogenia, fuentes de obtención y aplicaciones clínicas.