RESUMEN
BACKGROUND: Umbilical cord (UC) mesenchymal cells have the ability to differentiate into various cell types, which make them an easily obtainable source for therapeutic uses. Different approaches have been used to isolate mesenchymal stem cells (MSC). AIM: Here, we report a detailed enzymatic method where large number of cells can be efficiently isolated from the cord matrix and cryopreserved on the same day of arrival at the laboratory. METHODS/MATERIALS: Cells were successfully isolated from 12 samples, with a new procedure that uses the total length of the UC. MSC have been isolated using a detailed enzymatic method with collagenase and hyaluronidase followed by trypsin, without removing the vessels and without mincing the cord. Stem cells were measured with flow cytometry before cryopreservation and post-thaw. Cultured cells were assessed for MSC marker expression and adherence plasticity for three passages. Multilineage differentiation was performed. RESULTS: Nucleated cell yield was calculated at 0·95 × 10(6) /cm. MSC yield was calculated at 0·65 × 10(6) /cm of cord with flow cytometry while the mean length was 31 cm. Cultured cells expressed the mesenchymal markers CD29, CD90, CD105 and CD44. Mesenchymal marker expression remained intact over the three passages and post-thaw. Osteogenic and adipogenic differentiation was evaluated. CONCLUSIONS: Our findings provide a fast and efficient method for mesenchymal cell isolation from Wharton's jelly using the total length of the UC. This method resulted in a large number of cells while the cells retained their mesenchymal character after thawing. This method can be easily applied, along with UC blood, for UC banking.
Asunto(s)
Separación Celular/métodos , Células Madre Mesenquimatosas/citología , Cordón Umbilical/citología , Antígenos de Diferenciación/biosíntesis , Criopreservación/métodos , Femenino , Humanos , Masculino , Células Madre Mesenquimatosas/metabolismo , Factores de Tiempo , Cordón Umbilical/metabolismoRESUMEN
INTRODUCTION: Adipose tissue is recognized as an important source of postnatal mesenchymal stem cells for generative medicine applications. Moreover, cord blood stem cells have been shown to contain pluripotent stem cells called unrestricted somatic stem cells (USSCs). However, this population is rare and cannot be generated from every cord blood sample. In this study, we have presented a new method of co-culture of adipose-derived stem cells (ADPCs) and cord blood stem cells that results in pluripotent differentiation. MATERIALS AND METHODS: ADPCs were obtained from a piece of adipose tissue after treatment with 0.075% collagenase, which was subsequently inactivated with DMEM/10% FBS. The cellular pellet of centrifugation was plated at 5-7 x 10(6) cells/mL in T25 culture flasks in a low-glycose DMEM with 30% FCS. Cord blood stem cells were obtained by centrifugation following double-processing in the presence of 2% HES 200/0.5 and plated at 5-7 x 10(6) cells/mL in the same medium. To investigate the crucial role of ADPCs in pluripotent cord blood differentiation, we added a ADPCS as (1 x 10(4) cells/mL) to the cord blood cultures and analyzed the contribution of ADPCs using a microscope as well as with flow cytometry. RESULTS: After only 3 days, adherent cells (USSC colonies) of fibroblastic morphology were detected in all co-cultured samples, whereas this was observed later or not at all in the non-co-cultured samples. The greater density of colonies in the co-coltured samples was another point. Hematopoietic CD45 cells were no longer detected after the first passage. Pluripotent stem cells were obtained from all co-cultured samples that contained stem cells positive for CD29, CD44, CD49e, CD90, CD105, CD51 Stro, and C-kit antibodies but negative for CD34, CD45, CD133, and glycophorin A. CONCLUSION: Addition of ADPCs was crucial to generate pluripotent-derived stem cells from cord blood samples. This double culture may be a useful tool for a universal allogeneic stem cell source for tissue repair or regeneration.