Afroza Khatun

1

• Afroza Khatun
•                  09 November 2006

2
Introduction

• Neural stem cells
• -self-renewing precursors of neurons and glia
• -could also differentiate into a variety of
  hematopoietic cells
• Rodent bone marrow cells
• -migrate into the brain and differentiate
  into microglia and astrocytes
• HBM and RBM stromal cells can differentiate into
  cells bearing neuronal markers
• Mesodermal-derived cells can also differentiate
  within the mammalian system
• Bone marrow stem cells give rise to a variety of
  hematopoietic lineages and repopulate the blood
  throughout adult life

3
Objectives

• bone marrow-derived cells enter the brain and
  differentiate into cells that express neuronal
  markers, supporting the idea that
  mesodermal-derived cells can adopt neural cell
  fates. 

4
Materials and Method

• Mice homozygous for a mutation in the PU1 gene
  were used as bone marrow transplant recipients.
• These animals are born alive but require a bone
  marrow transplant within 48 hours after birth to
  survive and develop normally.
• There are no gross morphological differences in
  the brain cytoarchitecture of these mice versus
  wild-type mice.
• NeuN was used as a neuronal marker.
• Within 24 hours after bieth, PU1 homozygous
  recipients were given intraperitoneal injections
  of bone marrow cells from wild-type mice.
• Seven transplant recipient mice and
  nontransplanted control littermates were examined
  between 1 and 4 months of age.
• Analysed different organ tissues for the presence
  of donar-derived cells
• Y chromosome-positive male cells were identified
  by fluorescent in situ hybridization
  histochemistry.
• Immunohistochemistry to visualize the neuronal
  nuclear marker.

5
Results and Discussions

• No LacZ-expressing or ß-galactosidase-immunoposive
  cells were present, and there was no specific
  immunostaining for NG2 chondroitin sulfate
  proteoglycan or 04, antigens that are present in
  Schwann cells and oligodendrocytes.
• -These results strongly suggest that the bone
  marrow cell preparations were devoid of neurons
  and glia at the time of transplantation.

6
Supplemental Figure 1. Cultured bone marrow cells
express nestin in vitro. Bone marrow cells were
isolated from adult mice as described see (20)
in report and treated with ACK lysing buffer
(BioWhittaker) for 30 s to eliminate red blood
cells.
7
(No Transcript)
8
Supplemental Figure 2. Y chromosome staining in
the CNS. Coronal sections from a 4-month-old
nontransplanted (A to E) female and (F to J) male
brains that were mounted on the same slide and
processed together. The Y chromosome was detected
with the tyramide-FITC conjugate.
9
(No Transcript)
10
(No Transcript)
11
Supplemental Figure 3. Y chromosome-positive
neuronal nuclei in homozygous PU.1 female brains
following transplantation of male bone marrow.
High-magnification images of NeuN and Y
chromosome double staining in (A) the olfactory
cortex of 2-month-old, (D) the frontopolar cortex
of 1-month-old, and (G) the cortical amygdala of
2-month-old females transplanted at birth with
male bone marrow.
12
Supplemental Figure 4. A Y chromosome- and
NeuN-positive neuron in the parietal cortex (1.2
mm behind the bregma) of a 1-month-old homozygous
female PU.1 knockout transplanted at birth with
male bone marrow. The images were obtained using
a Zeiss confocal microscope (63 oil objective
and 2 digital magnification).
13
Supplemental Figure 5. Z series of a Y
chromosome- and NeuN-positive neuron in the
somatosensory cortex (1.2 mm behind the bregma)
of a 2-month-old homozygous female PU.1 knockout
transplanted at birth with male bone marrow. The
images were obtained with a Zeiss confocal
microscope (63 oil objective and 2 digital
magnification). (A to I) Nine different levels
through the section (1 mm thick each), overlaying
the NeuN (red), Y chromosome detected with the
tyramide-FITC conjugate (green), and DAPI (blue)
(to visualize cell nuclei) fluorescence.
14
Supplemental Figure 6. Examples of the presence
of Y chromosome-positive cells in the ventricular
system of transplanted mice. (A, D, and E) The
sections are stained with ethidium bromide to
show cell nuclei, and the Y chromosome is
detected using nonradioactive in situ
hybridization and TSA amplification with
FITC-tyramide. The Y chromosome is seen as a
green dot in the periphery of the nuclei. (A) is
a sagittal view of the lateral ventricle of a
female recipient 3 months after transplant. (D)
is a coronal section of the ventral portion of
the lateral ventricle from another 3-month-old
recipient.
15
Summary

• bone marrow cells -express neuron-specific
  antigens.
• the bone marrow can supply the brain with an
  alternative source of neural cells.
• neurons and macroglia (oligodendrocytes and
  astrocytes) are thought to arise from pluripotent
  neural stem cells.
• neural stem cells-continuous influx of bone
  marrow stem cells into the ependymal and
  subependymal zones that give to a variety of CNS
  neural cell types.
• serve as portals into the CNS for diseases that
  primarily originate in and affect the
  hematopoitic system (i.e.,leukemia and AIDS). 

16
Conclusions

• Bone marrow is more accessible than neural stem
  cells.
• Having inherent host compatibility-thereby
  obviating the need to screen for viral and
  foreign antigens.
• Only a small number of transplanted cells
  expressed neuronal antigens in the adult brain.
• Bone marrow cells might be expanded in vitro.
• Provide an unlimited source of cells for the
  treatment of CNS disease and injury.
• Hematopoitic and stromal stem cells have been
  isolated from bone marrow.