Early appearance of stem/progenitor cells with neural-like characteristics in human cord blood mononuclear fraction cultured in vitro.
OBJECTIVE
The exposure of human umbilical cord blood mononuclear cells devoid of hematopoietic stem cells (HUCB-MNCsCD34-) to defined culture condition promotes their conversion into neural lineage. We have asked the question if observed fate change of HUCB-MNCsCD34- results from direct conversion of hematopoietic precursors into neural-like phenotypes due to expression of overlapping genetic program or, alternatively, these neural phenotypes arise from sequential differentiation of more primitive progenitors (embryonic-like cells) preexisting in HUCB-MNCsCD34- fraction.


METHODS
HUCB-MNCs negatively selected for CD34 antigens were cultured in vitro up to 14 days. Changes in stem/neural cell genes and proteins were successively evaluated during this period and after evoked neuronal differentiation of cells in the presence of RA or BDNF or cocultured with neonatal rat brain astrocytes.


RESULTS
Freshly isolated HUCB-MNCsCD34- expressed pluripotent cell markers: Oct3/4, Sox2, and Rex1 genes. During 24 hours of culture the frequency of Oct3/4 immunopositive cells increased markedly with parallel enlargement of "side population" and CD133+ cell appearance. Concomitantly, cultured cells start to form aggregates and express pro-neural genes, i.e., enhanced Sox2, OTX1, Nestin, GFAP, and NF-200. During the next days of culture immunoreactions for beta-tubulin III, MAP2, GFAP, S100beta, Doublecortin, and GalC were induced with reciprocal lowering of stem cell gene and protein markers. At this stage cells successively adhered to the bottom, dispersed, and decreased proliferation rate (Ki67 expression). Additional treatments with neuromorphogenes or coculturing with rat brain primary culture induced further differentiation of these neural precursors toward more advanced neuronal phenotypes.


CONCLUSIONS
HUCB-MNCs(CD34-) fraction contains embryonic-like stem/progenitor cells which increase rapidly but transiently in culture, then differentiate spontaneously after cell aggregate adhesion toward neural lineage. Neurally promoted cells from 10-14 DIV culture acquire three main neural-like phenotypes, i.e., neurons, astrocytes, and oligodendrocytes. In this respect they are promising candidates for experimental treatment of neuronal injury; however, the final proof for conversion of HUCB cells to neural cells can be obtained through transplantation experiments.
Habich
Aleksandra
A
NeuroRepair Department, Medical Research Institute, Polish Academy of Sciences, Warsaw, Poland.
Jurga
Marcin
M
Markiewicz
Inga
I
Lukomska
Barbara
B
Bany-Laszewicz
Urszula
U
Domanska-Janik
Krystyna
K
D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D019208:Brain-Derived Neurotrophic Factor / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D018920:Coculture Techniques; D017931:DNA Primers; D000089662:Doublecortin Protein; D005312:Fetal Blood / Q000166:cytology*; D006801:Humans; D007150:Immunohistochemistry; D009000:Monocytes / Q000166:cytology*; D009474:Neurons / Q000166:cytology*; D010641:Phenotype; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000737:chemistry*; D014212:Tretinoin / Q000494:pharmacology
D019208:Brain-Derived Neurotrophic Factor; D017931:DNA Primers; C000714848:Dcx protein, rat; D000089662:Doublecortin Protein; D012333:RNA, Messenger; D014212:Tretinoin