STEM CELL TRANSPLANTATION:

STEM CELLS HARVEST, CD34 COUNTS AND

CRYOPRESERVATION

Paras, Ruzzelle Mae F.

FATHERS OF STEM CELL SCIENCE
James
Till & Ernest
McCulloch
1961
Ontario Cancer
Institute

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HISTORY OF STEM CELL

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HEMATOPOIETIC STEM CELL
TRANSPLANTATION
Stem cells are a population of
undifferentiated cells capable of self-renewal
and generation of a functional progeny of
different specialized cells
CD34 positive
Live in the bone marrow
CXCR4
CXCR4 antagonist, plerixafor
HEMATOPOIETIC STEM CELL
TRANSPLANTATION
Hematopoietic stem cell transplantation
(HSCT) is the intravenous infusion of
hematopoietic stem cells designed to engraft
the bone marrow and establish hematopoietic
and immune function in patients with a
variety of diseases.
HEMATOPOIETIC STEM CELL
TRANSPLANTATION
HSCT mortality and morbidity rates have
decreased significantly due to
improved conditioning regimens
better HLA typing
improved supportive care and treatment of
infections
PURPOSE OF HSCT
Hematopoietic progenitor cells are collected
and transplanted for three main reasons:
(1) to serve as bone marrow replacement
following total body irradiation or chemotherapy
given to treat primary marrow and nonmarrow
disorders
(2) to provide a graft-versus-leukemia (or tumor)
reaction
(3) to replenish diseased or destroyed bone
marrow
TYPES OF STEM CELL
TRANSPLANTATION
Autologous
Allogeneic
Reduced-intensity
Syngeneic
AUTOLOGOUS HSCT
Most effective when there is direct correlation between
chemotherapy dose and tumor response
The rescue with the px own harvested HSCs
Disadvantages:
Not being able to mobilize adequate number of stem cells
The possibility of infusing malignant cells, which could have
been collected during the stem cell harvesting and pheresis
collection, back into the patient during the rescue phase->this is
minimized by purging of tumor cells from the graft by either
chemotherapy or monoclonal antibodies
Lack of graft vs tumor effect
Long term survivors also have slightly higher chance of therapy
related leukemia
 http://www.bonemarrowmx.com/autologous-bone-marrow-transplant/
ALLOGENEIC HSCT
Chemotherapy, either myeloblative or
nonmyeloblative, conditioning regimens serve
to eradicite malignant cells, as well as, the
host immune system which may initially reject
the donor stem cells
Used for the treatment of autoimmunity,
immunodeficiency, and congenital or acquired
bone marrow failures
ALLOGENEIC HSCT
Disadvantages
Higher treatment related mortality than
autologous HSCT
Graft vs host disease
Time needed to find a donor if no sibling related
donor is available
 http://www.bonemarrowmx.com/allogeneic-bone-marrow-transplant/
COMPLICATIONS OF ALLOGENEIC
STEM CELL TRANSPLANT
Graft vS Host Disease
As acute GvHD and chronic GvHD share common
features, the new National Institute of Health
(NIH) Consensus Conference now has proposed
definitions of acute GvHD and chronic GvHD to be
based on signs and symptoms rather than time of
onset
ACUTE GvHD
Risk factors for acute GvHD include
HLA disparity between donor and recipient
Increasing age of host
Graft type (umbilical cord blood low rate and PBSC
higher rate than bone marrow derived grafts)
Gender disparity between donor and recipient
Alloimmunization of donor (multiparous women)
ACUTE GvHD
Acute GvHD may involve the skin, gastointestinal
tract, and liver
Caused by cytokine storm which causes
infiltration of T-effector and NK cells into target
tissue
GvHD prophylactic regimens are usually a
calcineurin inhibitor (tacrolimus or cyclosporine)
plus short course of methotraxate to be tapered
by 2nd and 3rd month post transplant and off by
6th month post transplant
ACUTE GvHD
May consider depleting T-cells but that would
decrease GvT as well
Treatment
High dose steroids (2mg/kg/day)
Mycophelanilate, anti-TNF alpha, anti-CD52 antibody,
extracorporeal photopheresis
CHRONIC GVHD
It resembles autoimmune disease (such as SLE,
sicca syndrome and scleroderma)
It includes skin hardening, sclerotic features,
esophageal strictures, and webs, bronchiolitis
obliterans, alopecia, nail loss, joint stiffness, dry
eyes, dry mouth, muscle weakness, hematuria,
and hematopoietic system (thrombocytopenia)
Major cause of death: opportunistic infections
from immunosuppression from both the
medicines as well as graft vs immune system
STEM CELL TRANSPLANTATION HLA
TYPING
Human Leukocyte Antigen types
Major Histocompatibility Complex
A stem cell transplantation from an HLA
mismatched donor can result in the recipients
immune system recognizing the transplanted
cells as non-self and attacking the stem cells
as foreign entities
STEM CELL TRANSPLANTATION HLA
TYPING
An HLA type consists of two main groups:
1. Class I antigens (HLA-A, B, C) and
2. Class II antigens (HLA-DR, DQ, DP)
Six HLA antigens: two A antigens, two B
antigens and two DR antigens
HLA typing
blood samples
buccal swabs
STEM CELL TRANSPLANTATION HLA
TYPING
Methods:
HLA serologic typing
Molecular HLA typing
A match is noted when the major class I
antigens (HLA A, B, C loci) as well as, class II
antigens (HLA DR, DQ) are the same as those
of the donor.
SOURCES OF HEMATOPOIETIC GRAFTS
BONE MARROW TRANSPLANT
Bone marrow are harvested from either
posterior or anterior iliac crest and must be
infused within 24 hours
If there is ABO blood type mismatch between the
donor and recipient, plasmpaheresis may remove
anti-a and anti-b antibodies
BONE MARROW TRANSPLANT
PERIPHERAL BLOOD STEM CELLS
Since PBSCs are found fewer in numbers, it
could require multiple leukopheresis for
adequate stem cell collection
Pheresed products may be cryopreserved in
5% dimethlysulfoxide for frozen storage.
PERIPHERAL BLOOD STEM CELLS
UMBILICAL CORD STEM CELLS
Umbilical cord stem cells given the
immunologic immaturity of umbilical cord
blood cells of newborn babies
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UMBILICAL CORD STEM CELLS
AUTOLOGOUS ADIPOSE STEM CELL
TREATMENT

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 AUTOLOGOUS ADIPOSE STEM CELL
TREATMENT
Adipose stem cell therapy is approximately a four hour outpatient procedure involving
the following:

Harvest: Using a proprietary but simple procedure similar to tumescent

liposuction, 100-300cc's of adipose tissue is harvested from the patient
Breakdown: The adipose tissue is then broken down using a phospholipid solution,
and a proprietary Cell Preparation Medium.
Separate: Using standard techniques, the Stromal Vascular Fraction (SVF) which
contains your Mesenchymal Stem Cells (MSC) is separated from the fat cells.
Isolate: Mesenchymal Stem Cells and other progenitor cells are then isolated from
the SVF using standard techniques.
Wash: The stem cells are then triple-washed with saline to remove any traces of
the phospholipid solution and cell preparation medium.
 AUTOLOGOUS ADIPOSE STEM CELL
TREATMENT
Activate: The isolated MSCs are then suspended in the
persons own platelet-rich-plasma (PRP) growth factors and
activated with a red/yellow/green laser. This awakens or
activates the dormant mesenchymal stem cells.
Infuse: The cells are then administered back to the patient
through one or more of the following modes of
administration:
Intravenous: The mesenchymal stem cells are administered
through a standard intravenous drip
Fat Transfer: The mesenchymal stem cells are mixed with the
fat filler-biomaterial mix before reintroduction
Local/Topical: The mesenchymal stem cells are administered
directly onto a localized area different from their tissue of origin
AUTOLOGOUS ADIPOSE STEM CELL
TREATMENT
CONDITIONING REGIMEN
The chemotherapy and/or radiation therapy
prior to transplant which serves two purposes:
1. Eradicate recipient immune system so there is
no donor graft rejection
2. Eradicate cancer calls (less important in
allogeneic transplants due to graft vs tumor
effect of donor immune system)
TRANSFUSION SUPPORT AFTER STEM
CELL TRANSPLANT
Transfuse if Hgb <8g/dL or platelets <10000/mm3
Platelet and blood components to be transfused should
be irradiated (to reduce donor lymphocyte induced
graft vs host disease) and filtered (to reduced HLA
alloimmunization to donor antigens and minimize CMV
transmission)
ABO mismatched HSCT recipients have slightly slower
neutrophil recovery. Mature erythroid progenitors do
express ABO antigens, and immune-mediated delayed
erythropoiesis with reticulocytopenia can occur,
leading to prolonged transfusion dependence up to 1
year after transplantation
TRANSFUSION SUPPORT AFTER STEM
CELL TRANSPLANT
A more important concern is the phenomenon
of immune hemolysis from mature,
competent passenger lymphocytes transfused
with the HSC component; hemolysis can occur
abruptly, from days 7 to 14 after HSCT, and
can be very severe or even fatal
INFECTIONS RELATED TO STEM CELL
TRANSPLANTATION
Engraftment after autologous HSCT occurs within
7 to 14 days
Engraftment after allogeneic HSCT occurs within
14 to 28 days
Risk of infection is related to the timing of
transplantation:
Pre-engraftment (less than 3 weeks)
Immediate post engraftment (3 weeks to 3 months)
Late post engraftment (>3 months)
INFECTIONS RELATED TO STEM CELL
TRANSPLANTATION
Autologous transplant recipients are at
highest risk during, pre and immediate post
engraftment; whereas, allogeneic transplant
recipients were at risk during all three phases
In initial post conditioning and post transplant
period (pre-engraftment and neutropenic
period), bacterial infections are the most
common, prophylax and treat early
INFECTIONS RELATED TO STEM CELL
TRANSPLANTATION
Bacteria commonly arise from:
Skin (coagulase negative staphylococcus, MRSA)
Oropharynx (Streptococcus viridans)
GI tract (enterococcus and gram negative bacilli)
Watch out for Pseudomonas aeroginosa,
stenotrophomonas, Clostridium difficile, and
Listeria monocytogenes
INFECTIONS RELATED TO STEM CELL
TRANSPLANTATION
Reduced intensity (nonmyeloblative) conditioning
regimens which cause engraftment to occur more
quickly, decrease risk of bacterial infections
Umbilical cord transplant, which takes longer to
engraft, has potentially higher risk of bacterial
infections
High risk for Pneuomocycstis carinii pneumonia
two months after transplantation, prophylax with
trimethoprim/sulfamthoxazole (bactrim) or
pentamidine
INFECTIONS RELATED TO STEM CELL
TRANSPLANTATION
Fungal infections are a problem in allogeneic
stem cell transplanted patients (especially
candida and invasive aspergillosis)
Remember the GvHD and the immune
suppression required to treat it will prolong
immune dysfunction and increase risk of
infections
INFECTIONS RELATED TO STEM CELL
TRANSPLANTATION
If patients develops chronic GvHD, then will need
prophylactic antibitocs longer
Viral infections such as herpes simplex virus 1, 2,
6, Ebstein-Barr virus and cytomegalovirus are not
uncommon
Herpes viral infections are typically due to
reactivation rather than primary new infection
Median time onset
HSV 2 to 3 weeks post transplant
CMV 100 days post transplant
STEM CELL HARVESTING (CD34+)
CD34 is a surface antigen present on 1% to 3%
of human bone marrow cells and on 0.05% of
nucleated circulating cells
serves as a marker for identifying and
separating hematopoietic stem and progenitor
cells because it is not found on fully
differentiated, or mature, hematopoietic cells
most accurate and recommended method to
predict the HSC yield
STEM CELL HARVESTING (CD34+)
Harvest: chemotherapy + cytokines (G-CSF)
Stem cell collection is initiated when peripheral
CD34+ stem cells exceed 5 to 10 cells/mL
A minimum of 1-2 million CD34+/kg peripheral
blood stem cells are need for successful
engraftment in the recipients bone marrow
Ideally 3-5 million CD34+ cells/kg body weight
CD34+ peripheral stem cells should be collected
CRYOPRESERVATION
Early theoreticians of cryopreservation was Jame
Lovelock known for Gaia theory
He suggested that damage to RBCs during
freezing was due to osmotic stress
1949-Ernest John Christopher Polge, an English
biologist, who solve the mystery of how to
preserve living cells and tissues at very low temp
He accidentaly discovered the cryprotective
properties of glycerol on fowl sperm
CRYOPRESERVATION
Two classes of cryopreservatives are
recognized
intracellular
glycerol, dimethyl sulfoxide [DMSOl, ethanol,
methanol, trimethylamine acetate [TMAA] and
ammonium acetate
to preserve cells at slow freezing rates (1 to 5 CV min)
extracellular
sucrose, glucose, polyvinylpyrrolidone [PVP] and
hydroxyethyl starch (HES1)
in vitro rapid freezing techniques
CRYOPRESERVATION
Glycerol
a trihydric alcohol and product of lipid
metabolism, is a clear, colorless, syrup-like fluid
with a sweet taste.
miscible with both water and alcohol and absorbs
water
large concentrated doses, in which case hemolysis
can occur
frozen preservation of sperm.
CRYOPRESERVATION
DMSO
a by-product of petroleum distillation, is a
colorless liquid with a sulfur smell
highly polar and dissolves many water and
lipid soluble substances
a low acute systemic toxicity and when given
intravenously in low doses it may cause
nausea, vomiting, local vasospasm, and an
objectionable garlic-like odor and taste
cryobiologic preservation of bovine RBCs
CRYOPRESERVATION
Storage of HSCs
510 years
80C for about 6 months
below 130C (liquid nitrogen)
rapidly thawed at 37C and immediately infused
CRYOPRESERVATION
 Reference
Videos
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wk
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https://www.youtube.com/watch?v=jqw3r-musrE
https://www.cordlife.ph/cord-blood-processing-
and-storage