Neural progenitors, neurons and oligodendrocytes from human umbilical cord blood cells in a serum-free, feeder-free cell culture Shawn J. Chua a,b , Ryszard Bielecki a , Christine J. Wong a , Nobuko Yamanaka a , Ian M. Rogers a, * , Robert F. Casper a,b, * a Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Ont., Canada M5T 3H7 b Institute of Medical Sciences, University of Toronto, Toronto, Ont., Canada M5S 1A8 article info Article history: Received 25 November 2008 Available online 25 December 2008 Keywords: Umbilical cord blood Neural progenitors Oligodendrocytes Differentiation abstract We have previously demonstrated that lineage negative cells (Lin neg ) from umbilical cord blood (UCB) develop into multipotent cells capable of differentiation into bone, muscle, endothelial and neural cells. The objective of this study was to determine the optimal conditions required for Lin neg UCB cells to dif- ferentiate into neuronal cells and oligodendrocytes. We demonstrate that early neural stage markers (nestin, neurofilament, A2B5 and Sox2) are expressed in Lin neg cells cultured in FGF4, SCF, Flt3-ligand reprogramming culture media followed by the early macroglial cell marker O4. Early stage oligodendro- cyte markers CNPase, GalC, Olig2 and the late-stage marker MOSP are observed, as is the Schwann cell marker PMP22. In summary, Lin neg UCB cells, when appropriately cultured, are able to exhibit character- istics of neuronal and macroglial cells that can specifically differentiate into oligodendrocytes and Schw- ann cells and express proteins associated with myelin production after in vitro differentiation. Ó 2008 Elsevier Inc. All rights reserved. Human UCB cells are an attractive source for cell transplantation because they are easy to harvest and survive long-term cryopreser- vation [1]. However, whole UCB samples contain stem and progen- itor cells of mixed lineage (blood and mesenchymal) as well as mature blood cells, resulting in cell cultures with multiple cell types. Several groups have reported the presence of mesenchymal progenitor cells from UCB. These reports state that the generation of mesenchymal progenitor cells could not be obtained from all UCB units [2–4]. A method to generate multi-potential stem cells from 100% of UCB units has been previously reported by us using lineage negative (Lin neg ) cells grown in fibroblast growth factor 4 (FGF4), stem cell factor (SCF) and fms-like tyrosine kinase recep- tor-3 ligand (Flt-3l) supplemented serum-free medium (FSFl med- ium) [5]. These UCB-derived multi-potential stem cells (MPSCs) have properties similar to multi-potential mesenchymal cells found in the bone marrow. There have been a number of in vitro and in vivo studies demonstrating the capacity of UCB cells to differen- tiate into neural and glial cells [6–10]. However, these groups re- port different sub-populations of blood with the capacity to differentiate into neurons and macroglial cells. Before UCB can be used in a stem cell-based therapy to treat neural diseases or spinal cord injuries, the ability to differentiate UCB stem cells into neural cells and oligodendrocytes must be accomplished reproducibly. In this study we focus on deriving neuron and glial cells from the line- age negative population of UCB (Lin neg ). This population is devoid of mature cells and contains an enriched population of hematopoietic stem cells. As reported by others, we have also found that we could only obtain positive results with cells that have spent time in cul- ture [11,12]. In this study, we have grown the MPSC cells in a fee- der-free and serum-free environment, which is important for establishing cells for clinical use. We found that when exposed to medium reported to support neural cell differentiation and growth, the MPSCs develop a neuronal or macroglial cell morphology and express neuron specific markers (neurofilament, b-III-tubulin) and macroglial specific markers, including the oligodendrocyte markers 2 0 ,3 0 -cyclic nucleotide 3 0 -phosphohydrolase (CNPase), oligodendro- cyte marker O4, galactocerebroside (GalC), myelin oligodendrocyte specific protein (MOSP), and the Schwann cell marker PMP22. Materials and methods Human umbilical cord blood collection. UCB was collected from informed consenting donors at Mt. Sinai Hospital (Toronto, Ontar- io) by qualified hospital personnel following protocols approved by the human ethics committee of that hospital and processed as de- scribed previously [5]. Enrichment and proliferation of UCB cells. A negative selection column was used to remove mature cells according to the manu- facturer’s instructions (Stem Cell Technologies) and as previously 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.12.045 * Corresponding authors. Address: Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Ont., Canada M5T 3H7. Fax: +1 416 586 5130 (I. Rogers). E-mail addresses: rogers@lunenfeld.ca (I.M. Rogers), rfcasper@aol.com (R.F. Casper). Biochemical and Biophysical Research Communications 379 (2009) 217–221 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc