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Development
of a perfusion bioreactor for culturing hematopoietic stem cells |
Principal Investigator : Asok Mukhopadhyay Project Associates/Assistants
Ph D
Students Hematopoietic
stem cell (HSC) transplantation can provide cure to a variety of malignant and
non-malignant hematological diseases. Recently, it has also been observed that
bone marrow stem cells are multipotent in nature, that transdifferentiate into
various cell types. Inspite of promising new applications, the success of HSC
transplantation in different clinical setting largely depends on its
availability. Getting sufficient numbers and acceptable quality of progenitor
stem cells in right time is difficult and in some time impossible due to
various reasons. HSC transplantation would be greatly facilitated by ex
vivo expansion of a small number of cells from bone marrow, or peripheral
blood, or a cord blood sample. At present, no suitable system is available for
ex vivo expansion of stem cells. Thus, the main theme of the project is
to develop a prototype bioreactor for culturing adult stem cells. The project
aims at development of a bioreactor and its operation for cultivation of
hematopoietic stem cells, mimicking bone marrow microenvironment. The
objectives of the project are (i) development of a biocompatible
three-dimensional (3-D) matrix from natural and/or synthetic polymers and ex
vivo expansion of HSC, (ii) study on grafting and transdifferential
potential of HSC in bone marrow and in other organ, (iii) molecular control
for self-renewal of HSCs, and (iv) development of a prototype bioreactor and
its operation for expansion of progenitor stem cells. Hematopoietic
stem cell transplantation is generally carried out after irradiation or
treatment with cytotoxic drugs in the notion that host bone marrow
‘niches’ become available to the donor cells for engraftment. How bone
marrow stromal cells respond to the radiation, which ultimately modulate
grafting of donor cells is poorly understood. We examined homing and marrow
retention of PKH-26 labeled donor cells in age matched C57BL/6J mice
conditioned at different doses of radiation. When injected donor cells into
mice that received lethal radiation of 900 cGy, the percent homing was highest
(15.8 ±1.5%) as compared to the lower doses (450 and 200 cGy) of radiation.
Despite highest homing of donor cells in these mice, about 70% (p <
0.005) homed cells were detached from the marrow within 72 h of
transplantation. In contrast, 2- to 2.5-fold (p < 0.03)
multiplication of homed PKH-26+Sca-1+
cells was observed in sub-lethally irradiated mice. The poor retention of
homed cells in lethally irradiated mice was believed to be due to the damage
of marrow stromal cells that ultimately support the graft. In order to
investigate that, the population of non-hematopoietic (stoma) and VCAM-1
positive cells (CD45-CD106+)
in bone marrow at different doses of radiation was determined. It was revealed
that in lethally irradiated (900 cGy) mice, more than 80% stromal cells were
depleted within 72 h of radiation, implicating radiation-induced degeneration
of stroma as one of the factors for such poor retention of donor cells.
Another important observation made in this study was that the grafted cells
confer protection to the endogenous marrow cells from the radiation-induced
damage. In order to study whether such observation was transplant
dose-dependent, two different dose ranges (5-8 and 12-15 x 106)
of cells labeled with PKH-26 dye were injected in sub-lethally irradiated (450
cGy) mice. It was found that in control mice (without grafting) the bone
marrow cellularity was declined from 279.2 ± 8.9 x 106
to 106.5 ± 11.7 x 106
within 24 h of radiation. However, following transplantation at two doses of
cells, the bone marrow cellularity was increased by 33 and 41%, respectively
as compared to the control. The colony forming assay confirmed that in
irradiated host the myeloid colony was partially restored by the transplanted
cells in dose dependent manner. In
vivo grafting study for 28 days
experiment demonstrated that the expression of three integrin adhesion
molecules (VLA-4/5/6) in the bone marrow grafted cells were up-regulated in
time dependent manner. This probably justifies the physiological requirement
of VLA-4/5/6 molecules in the long-term retention of primitive cells in bone
marrow. Earlier, it was shown by different groups of investigators that
VLA-4/5 adhesion molecules are important for grafting of donor cells to the
marrow. Therefore, ex vivo expansion of murine stem cells was carried
out in the notion that the expression of these adhesion molecules will be
restored, if not up-regulated. It was found that the expression of VLA-5 and
–6 were increased up to 2 to 3-fold, where as that of o1-integrin (CD29) was
restored in 5 day culture in presence of cytokines. Contrary to that, the
expression of VLA-4 in the cultured cells was decreased. How the expression of
o4-integrin in the cultured cells modulated is the subject of present
investigated. Evidence
from recent reports suggest that hematopoietic stem cells may have unexpected
developmental plasticity, which means that these cells differentiate in to
cells of other tissues under suitable culture conditions. It is crucial to
comprehend the underlying mechanisms governing the cell fate decisions, so
that the clinical benefits of HSCs in tissue regeneration and gene therapy can
be maximized. To determine such mechanism PKH-26 labeled cells derived from
bone marrow were transplanted into age matched mice. It was revealed that
about 60 ±
5.4% donor cells homed to the liver and stayed there at least for one
month without multiplication. By cell cycle analysis, it was confirmed that
about 92% of the grafted cells were maintained in the G0-G1
phase. The homed cells were uniformly distributed in the liver, as confirmed
by histological analysis of the cryosection. The phenotypes of the homed cells
were gradually changed, as the characteristic hematopoietic cell markers like
CD45.1 and CD11b were dropped from 94 % to 20% and 40% to 20%, respectively.
Colony forming cell assay also indicated that the functional property of the
bone marrow specific cells was modulated in the liver. In comparison, the
cells grafted in the bone marrow multiplied and maintained typical properties
of the cells, indicating that the cellular microenvironment is influential in
determining the fate of the cell. Though it is clear from the present study
that the phenotypes of hematopoietic cells grafted in the liver is changed,
but it is yet to be known whether these cells acquire hepatocyte specific
phenotype and/or hepatocyte specific function.
Publications Original peer-reviewed articles 1.
*Mashusadan T, Richhariya A, Mazumder S and Mukhopadhyay A (2003) An in vitro
model for grafting of hematopoietic stem cells predicts bone marrow
reconstitution of a myeloablative mice. J Hematother Stem Cell Res
12:243-252 (*in press last year, since published). 2.
†Takada Y, Mukhopadhyay A, Kundu GC, Mahabeleshwar GH, Singh S and Aggarwal
BB (2003) Hydrogen peroxide activates NF-kB through tyrosine phosphorylation
of IkBa and serine phosphorylation of P65: evidence for the involvement of IkB
kinase and Syk protein tyrosine kinase. J Biol Chem 278:24233-24241
(†on deputation/work done elsewhere). 3.
Raju GMK, Guha S, Mukhopadhyay A, Kumar L, Kale VP, Mittal S, Deka D, Mohanty
S and Kochupillai V (2003) Colony-stimulating activity of fetal liver
cells:synergistic role of stem cell factor in bone marrow recovery from
aplastic anemia. J Hematother Stem Cell Res 12:491-498. 4.
Mashusadan T, Mazumder S and Mukhopadhyay A (2004) Degeneration of stroma
reduces retention of homed cells in bone marrow of lethally irradiated mice. Stem
Cells Dev (in press).
Reviews/Proceedings 1.
*Mukhopadhyay A, Madhusudhan T and Kumar R (2003) Hematopoietic stem cells:
Clinical requirements and developments in ex vivo culture. Adv Biochem Eng
Biotechnol 86:215-253 (*in press last year, since published). |