Expansion of hematopoietic stem cells
A technology of hematopoietic stem cells and hematopoietic cells, applied in the direction of blood/immune system cells, animal cells, extracellular fluid diseases, etc., can solve the problems of enhancing HSC populations and unclearness
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Embodiment 1
[0185] Example 1: Immunoselection of Mesenchymal Lineage Precursor or Stem Cells (MLPSCs)
[0186]Bone marrow (BM) was harvested from healthy normal adult volunteers (20-35 years old). Briefly, pipette 40 ml of BM from the posterior iliac crest into a tube containing lithium heparin anticoagulant.
[0187] Bone marrow mononuclear cells (BMMNC) were obtained using Lymphoprep TM (Nycomed Pharma, Oslo, Norway) prepared by density gradient separation as previously described by Zannettino et al., 1998. After centrifugation at 400 x g for 30 minutes at 4°C, the buffy layer was pipetted and washed three times in "HHF" containing Hank's Balanced Salt Solution (HBSS, Life Technologies, Gaithers Fort, MD) with 5% fetal calf serum (FCS, CSL Limited, Victoria, Australia).
[0188] STRO-3+ (or TNAP+) cells were then isolated by magnetic-activated cell sorting as previously described by Gronthos & Simmons, 1995; and Gronthos, 2003. In short, about 1-3x10 8 Each BMMNC was incubated on i...
Embodiment 2
[0190] Example 2: Co-cultivation of HSC and mesenchymal lineage precursor or stem cells (MLPSC)
[0191] Cells: CB CD34+ (Stem Cell Technologies)
[0192] Medium: StemSpan SFEM (Stem Cell Technologies), supplemented with:
[0193] Human LDL (Stem Cell Technologies) 10 μg / ml
[0194] Growth Factors (‘SFT’):
[0195] οrHu SCF 100ng / ml
[0196] οrHu-FLT3 ligand 100ng / ml
[0197] οrHu TPO 50ng / ml
[0198] ο (all recombinant cytokines were from R&D Systems)
[0199] Small molecule:
[0200] SR1 (500 nM); UM171 (35 nM); Trichostatin A (TSA, 50 nM); Valproic acid (VPA, 500 μM) (all from Stem Cell Technologies)
[0201] Determination conditions:
[0202] Day -1 MPCs (MCBCC006) were plated at 50,000 / well in two 24-well plates in α-MEM / 10% FBS
[0203] On day 0, 10,000 CD34+ cells per well were plated in each well of two 24-well plates. Wells containing MPCs were first washed to remove FBS medium.
[0204] The remaining CD34+ cells were cultured at the same concentration in S...
Embodiment 3
[0243] Example 3: Expansion of CD34+ cells from peripheral blood or cord blood
[0244] About 25 million MPCs are cultured and expanded in an animal component-free medium for about 5 days to obtain a cell population comprising about 400-500 million MPCs. The MPCs were then washed and co-cultured with approximately 50 million CD34+ cells obtained from the peripheral blood of subjects in need of HSC transplantation or from umbilical cord blood. At this stage, there were approximately 2.5 million primary HSCs of the phenotype CD34+CD45RA-CD90+CD49f+ in the total CD34+ population. These cells were then co-cultured in serum-free medium in the presence of HDAC inhibitors for a period of about 10 days, after which HSCs of the phenotype CD34+CD45RA-CD90+CD49f+ had expanded to about 100,000,000 cells.
[0245] At this stage, the total cell population is available for administration to a subject in need of HSC transplantation.
[0246] Alternatively, CD34+CD45RA-CD90+CD49f+ cells can ...
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