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or lentivirus-derived vectors[51]. It has been suggested that pseudotransduction
may result from VSVg-pseudotyping due to membrane fusion eiciency being
higher than the rate of integration of the transgene[61]. Nevertheless, most lenti-
viral vectors have been generated with VSV-G, as this glycoprotein makes it easy
to recover and concentrate the pseudotyped vectors [62].
We also showed that eGFP was produced in all colony subtypes. Clusters of
eGFP production were observed on luorescence microscopy, indicating that not
all the cells of a given positive colony - theoretically derived from a single cell -
produced eGFP. his result is consistent with those of Mikkola et al. concerning
murine HSC transduction by a VSVg-pseudotyped lentiviral vector, in which
a mismatch was reported between the transduction rate of cells (almost 25%)
and the transduction rate of myeloid colonies (almost 60%). hese authors high-
lighted the occurrence of mosaicism in GFP gene expression in colonies obtained
following the myeloid diferentiation of CD34+ cells[63], possibly due to a delay
in the integration of the transgene during diferentiation, resulting in the forma-
tion of clusters of GFP-positive cells within a single myeloid colony.
In our in vivo study, autologous HSC were injected into the bone marrow,
whereas intravenous injection is currently the most frequently used transplanta-
tion method. We aimed to increase seeding eiciency and homing, as only a lim-
ited number of stem cells were theoretically available. However, 2.5 × 106 to 5.0 ×
106 CD34+ cells is generally suicient to ensure engraftment, and we found that
less than 2.0 × 106 cells were suicient for long-term reconstitution in macaques.
As predicted[64,65], total-body gamma irradiation leads to a drastic decrease in
the number of hematopoietic progenitors, preventing the development of mature
cells [66]. Despite the occurrence of severe pancytopenia, a positive correlation
has been found between the number of CD34+ cells infused and time required
for immune reconstitution [42,67,68]. However, hematopoietic recovery may
take longer if fewer than 2.0 × 106 CD34+ cells/kg are infused. his notion is
consistent with our observation that CD34+ cell transplantation decreases both
the severity and duration of irradiation-induced cytopenia. Clonogenic activity
also reappeared more strongly in transplanted animals. We also showed that the
animals recovered B cells, T cells, monocytes and granulocytes. Nevertheless, the
functional activity of these cells requires conirmation, particularly for lympho-
cytes. However, although we observed long-term reconstitution with lentiviral
vector-transduced cells of diferent lineages, its proportion remained below 1%.
Hanawa et al., provided the irst evidence that SIV-based vectors can success-
fully transduce rhesus macaque repopulating hematopoietic stem cells, with an
average of 16% of peripheral blood leukocytes containing the SIV vector ge-
nome. However, this study was carried out with HSC from mobilized peripheral
blood cells, making it possible to obtain larger numbers of HSC than can be har-
vested from bone marrow. Nevertheless theoretically, these cells contained more
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