Method for purification of human hematopoietic stem cells by flow cytometry.

Human hematopoietic stem cells (HSCs) and progenitors can be isolated by enriching for a rare cell population with a combination of monoclonal antibodies (MAbs). Such an isolation scheme involves multi-step procedures including ficoll-density fractionation and presort enrichment followed by cell sorting. Over the past decade, various cell-surface and metabolic markers have been identified and used to isolate human HSCs and progenitors as summarized in Table 1. Among them, CD34 has become the most critical cell-surface marker for positively selecting a rare cell population (1,2). Within the CD34(+) cell population, the differential expression of Thy-1, CD38, and AC133 have been used to fractionate HSCs and progenitors. In order to subfractionate CD34(+) cells by these markers, the cells can be further purified by flow cytometry. HSCs can be further enriched into a Thy-1(+) (3-7), CD38(-lo) (8-10), Thy-1+ CD38(-lo) (11), or AC133+ (12,13) fraction of CD34(+) cells. Table 1 Commercially Available Cell-Surface and Metabolic Markers for Isolation of Human HSC and Progenitor Cells Marker Expression/Remark Fluorochrome conjugate recommended Reference Positive marker CD34 Positive FITC, PE, APC, BIO 1,2,33 Thy-1 Positive PE, BIO 3,4 AC133 Positive PE 12,34 Negative/low marker CD38 Negative /low FITC, PE, APC 8,9 HLA-DR( a ) Negative to low FITC, PE 35,36 Mature lineage marker, Lin- CD2 T-cell lineage FITC, PE, BIO 3 CD3 T-cell lineage FITC, PE, APC, BIO 3 CD19 B-cell lineage FITC, PE, APC, BIO 3 CD16 NK-cell lineage FITC, PE, APC, BIO 3 CD14 Myeloid lineage FITC, PE 3 CD15 Myeloid lineage FITC, PE 3 Glycophorin A Erythroid lineage FITC, PE 3 2nd Step reagent Avidin/Streptavidin For BIO MAb FITC, PE, APC, TXRD, PharRed, Cy-chrome( d ) Metabolic marker( b ) Rhodamine 123( c ) Low Mitochondria-binding dye 37,38 Hoechst 33342( c ) Low DNA-binding dye 39,40 Pyronin Y Low RNA-binding dye 39,40 Propidium iodide Negative to low Dead-cell exclusion Abbreviations: FBM, fetal bone marrow; MPB, mobilized peripheral blood; ABM, adult bone marrow; HSC, hematopoietic stem cells; FITC, fluorescein; PE, phycoerythrin; APC, allophycocyanin; TXRD, Texas red; BIO, biotinylated. ( a ) FBM, MPB HSCs express HLA-DR (41,42). ( b ) To isolate quiescent HSC. ( c ) Substrates for p-glycoprotein, encoded by MDR-1. HSC possess high levels of p-glycoprotein efflux activity. ( d ) Recommended for single laser flow cytometry only, lineage marker positive and PI positive cells can be excluded simultaneously.

Neuroprotection of host cells by human central nervous system stem cells in a mouse model of infantile neuronal ceroid lipofuscinosis.

Infantile neuronal ceroid lipofuscinosis (INCL) is a fatal neurodegenerative disease caused by a deficiency in the lysosomal enzyme palmitoyl protein thioesterase-1 (PPT1). Ppt1 knockout mice display hallmarks of INCL and mimic the human pathology: accumulation of lipofuscin, degeneration of CNS neurons, and a shortened life span. Purified non-genetically modified human CNS stem cells, grown as neurospheres (hCNS-SCns), were transplanted into the brains of immunodeficient Ppt1(-/)(-) mice where they engrafted robustly, migrated extensively, and produced sufficient levels of PPT1 to alter host neuropathology. Grafted mice displayed reduced autofluorescent lipofuscin, significant neuroprotection of host hippocampal and cortical neurons, and delayed loss of motor coordination. Early intervention with cellular transplants of hCNS-SCns into the brains of INCL patients may supply a continuous and long-lasting source of the missing PPT1 and provide some therapeutic benefit through protection of endogenous neurons. These data provide the experimental basis for human clinical trials with these banked hCNS-SCns.

Engraftment of sorted/expanded human central nervous system stem cells from fetal brain.

Direct isolation of human central nervous system stem cells (CNS-SC) based on cell surface markers yields a highly purified stem cell population that can extensively expand in vitro and exhibit multilineage differentiation potential both in vitro and in vivo. The CNS-SC were isolated from fetal brain tissue using the cell surface markers CD133(+), CD34(-), CD45(-), and CD24(-/lo) (CD133(+) cells). Fluorescence-activated cell sorted (FACS) CD133(+) cells continue to expand exponentially as neurospheres while retaining multipotential differentiation capacity for >10 passages. CD133(-), CD34(-), and CD45(-) sorted cells (approximately 95% of total fetal brain tissue) fail to initiate neurospheres. Neurosphere cells transplanted into neonatal immunodeficient NOD-SCID mice proliferated, migrated, and differentiated in a site-specific manner. However, it has been difficult to evaluate human cell engraftment, because many of the available monoclonal antibodies against neural cells (beta-tubulin III and glial fibrillary acidic protein) are not species specific. To trace the progeny of human cells after transplantation, CD133(+)-derived neurosphere cells were transduced with lentiviral vectors containing enhanced green fluorescent protein (eGFP) expressed downstream of the phosphoglycerate kinase promoter. After transduction, GFP(+) cells were enriched by FACS, expanded, and transplanted into the lateral ventricular space of neonatal immunodeficient NOD-SCID brain. The progeny of transplanted cells were detected by either GFP fluorescence or antibody against GFP. GFP(+) cells were present in the subventricular zone-rostral migrating stream, olfactory bulb, and hippocampus as well as nonneurogenic sites, such as cerebellum, cerebral cortex, and striatum. Antibody against GFP revealed that some of the cells displayed differentiating dendrites and processes with neurons or glia cells. Thus, marking human CNS-SC with reporter genes introduced by lentiviral vectors is a useful tool with which to characterize migration and differentiation of human cells in this mouse transplantation model.