Welcome

 to  MICB  302

Dr.  T.  Kion
Wesb.  125
tkion@mail.ubc.ca

TAs: Madison  Bolger-­‐Munro

Amy  Shim

Dr.  P.  Johnson  takes  over  in  late  October.

Tutorials  will  start  next  week.

Tips  to  doing  well  in  MICB  302
1. Check  Connect  for  the  iOnerary  for  each  topic:
-­‐ review  the  relevant  secOons  from  MICB  202,
-­‐ read  the  relevant  secOons  of  the  textbook,
-­‐ print  out  the  relevant  lecture  slides  -­‐  be  prepared  to
annotate  the  slides  in  class,
-­‐ look  at  the  relevant  learning  objecOves,
-­‐ come  to  class.

2. ARend  tutorials:
-­‐ most  of  the  tutorial  sessions  will  have  pracOce  quesOons
for  you  to  work  on,
-­‐ do  the  pracOce  quesOons,  discuss  with  your  classmates
what  you  could  add  to  make  your  answer  beRer/more
complete.
 Tips  to  doing  well  in  MICB  302
3. Office  hours:
-­‐ don’t  leave  your  visits  to  hour  office  hours  to  the  day
before  the  exam,
-­‐ write  out  your  quesOons  before  hand,
-­‐ printouts  should  have  font  no  smaller  than  12  point.

4. Start  studying  now:

-­‐ 3rd  year  courses  typically  have  more  reading  than  your
2nd  year  courses,
-­‐ instructors  expect  that  students  have  the  appropriate
background  in  cell  biology,  immunology,  microbiology,
biochemistry,  etc.,
-­‐ instructors  expect  students  to  take  responsibility  for  their
own  learning.
Exam  schedule  and  grade  distribuOon

Midterm  #1  (20%)  –  Sept.  30th,  early  evening.

Midterm  #2  (30%)  –  Oct.  28th,  early  evening.

Missed  exam  -­‐  a  doctor’s  notes  explaining  why  you  were  not  physically
able  to  write  the  exam  is  required.

Final  exam  (40%):  December  exam  period.

Do  not  finalize  travel  plans  unOl  the  final  exam  schedule  has
been  published  in  October.

iClicker  quesOons  (10%)  -­‐  grade  based  parOally  on  parOcipaOon

and  correctness.
 What  to  have  done  before  next  class
1. Check  that  you  are  registered  in  the  course  –  and  registered
for  a  tutorial  secOon.    As  space  is  limited,  you  should  go  to
the  session  you  are  registered  in.

2. Purchase  the  course  textbook  from  the  Bookstore.    Purchase

an  iClicker  if  needed.

3. Read  the  course  syllabus.    Register  your  iClicker  on  Connect.

4. Mark  the  midterm  dates  on  your  calendar.    Report  exam

conflicts  by  the  end  of  next  week.    A  conflict  is  when  you
have  another  exam  or  lecture  course  scheduled  at  the  same
Ome.    A  conflict  is  not  when  you  are  scheduled  to  work  a
shid.
Module  1:    Stem  cells  and  the  development  of  the  immune
system
What  is  the  hematopoieOc Development  of  immune Signaling  pathways  and
stem  cell? organs  during  embryogenesis hematopoiesis
–  descripOon,
–  idenOficaOon,
–  isolaOon.

Sidebar  I:
IdenOficaOon  of  HSCs

Sidebar  II:
CD  and  Lin  markers

Sidebar  III:
Enrichment  of  HSCs
from  mixed  populaOons

Sidebar  IV:
FACS
Module  1:    Stem  cells  and  the  development  of  the  immune
system

Learning  ObjecOves

Ader  compleOng  this  part  of  Module  1,  students  should  be  able  to:

• describe  the  cells  of  the  immune  system,

• define  the  nature  of  the  hematopoieOc  stem  cell,
• explain  how  hematopoieOc  stem  cells  can  be  enriched  in  a  mixed
populaOon  of  bone  marrow  cells.
Stem  cells
Stem  cells  are  defined  by  two  capaciOes:
–  the  ability  to  regenerate  (self-­‐renewal),
–  the  ability  to  differenOate  into  diverse  cell  types.

Embryonic  stem  cells:

–  pluripotent,
–  potenOal  to  develop  into  any  cell  type  in  the  organism.

Adult  stem  cells:

–  mulOpotent,
–  found  in  several  adult  organs,
–  potenOal  to  give  rise  to  mature  Ossue-­‐specific  cells.
HematopoieOc  stem  cells
MulOpotent*  hematopoieOc  stem  cells  (HSCs)  are  the  adult  stem  cells
that  give  rise  to  all  funcOonally  specialized  and  mature  blood    cells.

Some  types  of  mature  blood  cells  are  short-­‐lived  (hours  -­‐  days),  but  some
types  live  for  months  -­‐  years.

The  average  human  requires  approximately  one  hundred  billion  new

hematopoieOc  cells  each  day.

The  process  by  which  HSCs  differenOate  into  mature  blood  cells  is  called
hematopoiesis.

*Parham  textbook  -­‐  refers  to  HSCs  as  pluripotent  (this  is  not  the  best  term).
 Immune  system  cells  –  neutrophils
• found  mostly  in  the  blood  (but
can  migrate  into  the  Ossues  in
infecOons),

• phagocyOc  cells  that  kill  bacteria

(phagocytose  once),

• relaOvely  short-­‐lived  cells.

Parham  Figure  1.12

 Immune  system  cells  –  monocytes/macrophages
• found  in  the  blood  (monocytes),

• migrate  into  Ossue  during  an

infecOon,  differenOate  into
macrophages,

• long-­‐lived  cells,

• highly  phagocyOc  cells  that  kill

bacteria  (i.e.,  several  Omes),

• can  present  anOgens  to  T  cells  to

parOcipate  in  adapOve  immune
responses,

• other  roles  in  the  body.

Parham  Figure  1.12
 Immune  system  cells  –  dendriOc  cells
• immature  DC’s  are  found  in  the
blood  and  the  Ossues,

• highly  phagocyOc,

• ader  ingesOng  Ags,  DC’s  migrate

to  the  lymph  nodes  and
The  most  important  anOgen-­‐ differenOate  into  a  highly-­‐
presenOng  cell  for  T  cells, efficient  Ag-­‐presenOng  cell,
especially  in  primary  immune
responses. • expresses  lots  of  MHC  proteins
on  its  surface  to  interact  and
They  form  a  key  link  between acOvate  T  cells,
the  innate  immune  system  and
the  adapOve  immune  system • makes  chemoaRractant
hormones  (chemokines)  that
Parham  Figure  1.12 aRract  naïve  T  cells.
 Immune  system  cells  –  mast  cells
• found  in  the  Ossues  throughout
the  body,

• involved  the  innate  immune

responses  during  microbial
infecOon,

• release  chemicals  that  aRract

neutrophils  and  monocytes  to
the  site  of  infecOons,

• also  destroy  Ab-­‐coated

parasites,

• responsible  for  Ag-­‐specific

allergies.
Parham  Figure  1.12
 Immune  system  cells  –  T  cells  and  B  cells
CTLs  –  kill  virally  infected  cells  and
some  types  of  cancer  cells.

TH  cells  –  provide  cytokine  and  co-­‐

sOmulatory  signals  to  CTLs,
macrophages  (to  kill  intracellular
infecOons),  and  sOmulate  B  cell  to
produce  anObody.

Treg  cells  –  inhibit  T  cell  responses

and  are  involved  in  controlling
immune  reacOons  and  prevent
autoimmunity.

B  cells  –  synthesize  and  secrete

anObodies.
Parham  Figure  1.12
 Immune  system  cells  –  plasma  B  cells
• anOgen  acOvated  cell  -­‐
differenOated  from  B  cell

• synthesize  and  secrete

anObodies,

• large  numbers  of  ribosomes,  lots

of  ER,

• relaOvely  short  lived.

Parham  Figure  1.12

 Immune  system  cells  –  natural  killer  cells
• kill  virus  infected  cells,  but  using
a  different  recogniOons  system
than  CTLs  do.

Parham  Figure  1.12

HematopoieOc  stem  cells
HSCs  are  rare  cells:
–  fewer  than  one  per  5  x  104  cells  in  the  bone  marrow*,
–  numbers  are  controlled  by  a  balance  of  cell  division,  
     differenOaOon  and  apoptosis,
–  number  of  HSCs  seem  to  decrease  with  age.

Steady  state  condiOons  (homeostaOc,  no  immune  system  challenge):

–  most  HSCs  are  quiescent  but  a  small  number  of  HSCs  divide,  
–  some  daughter  cells  remain  as  HSCs,  others  differenOate  into  
     progenitor  cells.

Immune  system  challenge:

–  HSCs  display  enormous  proliferaOve  capacity.

*  EsOmate  of  HSC  in  mouse  bone  marrow.

 HematopoieOc  stem  cells
HSC  are  rare  cells  –  they
HSC can  undergo  self-­‐renewal
or  they  can  differenOate.
CLP CMP
CLPs  and  CMPs  are
progenitor  cells  –  they  can
expand  temporarily  but
always  conOnue  to
differenOate.

Parham  Figure  1.14

How  did  we  learn  about  HSCs?
Atomic  bombings  of  Hiroshima  and  Nagasaki,  1945.

Many  people  were  killed  instantly.

Many  people  suffered  fatal  injuries  from

the  radiaOon  and  died  in  the  following  weeks.

Some  vicOms  that  were  exposed  to

“lower  doses”  of  radiaOon  (but  sOll  lethal
 doses)  had  compromised  hematopoieOc  systems.

These  people  could  not  regenerate  sufficient  white  blood  cells  to  protect
against  otherwise  nonpathogenic  infecOons  or  enough  platelets  to  clot
their  blood.

Image:  Hiroshima  Peace  Memorial,  hRp://en.wikipedia.org/wiki/Hiroshima_Peace_Memorial

In  vivo  HSC  assays  –  Experiment  1
The  hematopoieOc  system  is  very  sensiOve  and  can  be  destroyed  by  the
relaOvely  low  doses  of  lethal  X-­‐irradiaOon.

For  mice,  a  lethal  dose  of  X-­‐rays  is  950  Rads  (9,500  mSv).

No  treatment  -­‐  death  within  10  days.

(RadiaOon  syndromes)

If  a  bone  or  the  spleen  was  shielded  from  radiaOon,

the  mice  did  not  develop  the  radiaOon  syndromes.

Infuse  with  104  -­‐  105  bone  marrow  cells  completely

restores  hematopoieOc  system.

*  X-­‐ray  of  bone  (extremity)  -­‐  0.001  mSv,  X-­‐ray  of  lower  g.i.  tract  -­‐  8  mSv
**  Mice  have  approximately  3  x  108  bone  marrow  cells.
In  vivo  HSC  assays  –  Experiment  2
Knowing  that  the  mouse  can  be  rescued  by  an  infusion  of  bone  marrow
cells  allows  the  opportunity  to  try  to  idenOfy  the  HSC.

In  a  another  set  of  experiments,  mice  that

had  been  exposed  to  lethal  irradiaOon  were
given  low  numbers  of  bone  marrow  cells
(not  enough  to  rescue  the  animal  from
hematopoieOc  failure).

Ader  death  (10  -­‐  15  days  post  exposure),

colonies  of  myeloid  and  erythroid  cells  were
observed  in  their  spleens  (about  1  colony
per  7,000  bone  marrow  cells  injected).

Image  adapted  from  :  hRp://stemcells.nih.gov/info/RegeneraOve_Medicine/Pages/2006Chapter2.aspx

In  vivo  HSC  assays
How  would  you  know  that  the  colonies  arose  from  the  donor  bone
marrow  cells  or  cells  that  had  somehow  survived  in  the  recipient?

How  would  you  know  that  the  colonies  arose  from  a  single  precursor
cell?
In  vivo  HSC  assays  –  Experiment  3
Bone  marrow  donor  mice  were  irradiated  with  low  doses  of  irradiaOon
–  induces  unique  chromosome  breaks  in  most  hematopoieOc  cells  (but
allows  some  cells  to  survive).

The  irradiated  bone  marrow  injected

mice  that  had  been  exposed  to  lethal
irradiaOon.

Each  colony  displayed  its  own  unique

chromosomal  marker  (radiaOon  induced
and  repaired  chromosomal  breaks)  seen
in  its  dividing  cells.

Image  adapted  from  :  hRp://stemcells.nih.gov/info/RegeneraOve_Medicine/Pages/2006Chapter2.aspx

In  vivo  HSC  assays
CollecOvely,  these  experiments  established  the  fact  that  cell  types  that
can  both  proliferate  and  generate  most  (if  not  all)  of  the  cell  populaOons
in  the  blood  must  exist  in  bone  marrow.

However,  in  the  last  two  experiments,  each  colony  contained  different
types  (but  not  necessarily  all)  of  the  blood  cells.

Did  they  really  idenOfy  the  mulOpotent  HSC?

Sidebar  I  :  IdenOficaOon  of  HSCs.
How  do  you  idenOfy  a  HSC  from  a  mixed  populaOon  of  bone  marrow
cells?

HSCs  have  an  idenOty  problem.

HSCs  look  like  lymphocytes.    They  are  non-­‐adherent,  with  a  rounded

nucleus  and  low  cytoplasm–to–nucleus  raOo.

They  are  extremely  difficult  to  grow  in  culture.

How  do  you  know  that  ader  some  Ome  in  culture,  that  it  sOll  is  a  HSC?
Sidebar  II:  CD  and  Lin  markers
The  cluster  of  differenOaOon  (CD)  is  a  cell  membrane  molecule  used  to
classify  leukocytes  into  subsets.

In  terms  of  physiology,  CD  molecules  can  act  in  numerous  ways.    Some
CD  molecule  funcOon  in  cell  adhesion  whereas  others  act  as  receptors  or
ligands  involved  in  cell  signaling.

Lineage  (Lin)  markers  are  cell  membrane  molecules  that  can  be  used
idenOfy  cells  of  a  parOcular  lineage  (i.e.,  myeloid  cells  vs.  lymphoid  cells).

Cells  that  do  not  express  these  anOgens  (or  express  that  at  extremely  low
levels)  are  said  to  be  Lin–.
Sidebar  II:  CD  and  Lin  markers
Commercially  available  ready  to  use  Lin-­‐anObody  mixtures  are  available.

Some  use  5  -­‐  7  different  Ab,  others  as  many  as  15  different  Ab.

Lin–  populaOons  look  like  a  “shoulder”  or  “tail”  shape  on  a  FACS  plot.

Examples  of  lineage  markers:

CD3  -­‐  T  cells

CD14  -­‐  monocytes/macrophages
CD16  and  CD56  -­‐  natural  killer  cells
CD19  and  CD20  -­‐  B  cells

Image  from  hRp://stemcellassays.com/2009/11/what-­‐is-­‐lineage-­‐negaOve-­‐cells/

Sidebar  I  :  IdenOficaOon  of  HSCs.
The  common  approach  to  idenOfy  HSC  is  through  markers  –  cell  surface
proteins  that  selecOvely  bind  or  adhering  to  other  cell  surface  proteins  or
signaling  molecules.

HSCs  are  commonly  characterized  by  the  absence  of  lineage-­‐specific  (Lin)
marker  expression.

Human  HSCs  are  described  as  Lin–,CD34+,CD59+,CD90/Thy1+,CD38low/–,

c-­‐Kit–/low  cells.

Lin–,  CD34–,  CD38–  HSCs  have  also  been  described.

Sidebar  III:  Enrichment  of  HSCs  from  mixed  populaOons
HSCs  are  rare  cells  –  fewer  than  one  per  5  x  104  cells  in  the  bone  marrow.

AnObodies  against  Lin  markers  can  be  used  to  negaOvely  select  against
mature  blood  cells  and  enrich  for  HSCs.

Buy  a  cocktail  of  anObodies  to  Lin  markers.

–  may  have  5  -­‐  8  different  anObodies,
–  anObodies  are  tagged  with  a  magneOc  bead.

Add  Ab  to  a  suspension  of  bone  marrow  cells  –  incubate  –  place  magnet
on  side  of  container  -­‐  pour  off  suspension.

Suspension  is  “enriched”  for  HSCs

Sidebar  III:  Enrichment  of  HSCs  from  mixed  populaOons

Different  beads  have  bound

Ab  that  recognize  one  of  the
Lin  markers

Ab-­‐bead  binds  to  mature  blood

cell  expressing  the  Lin  marker

Enriched
for  HSCs

Adapted  from:  hRp://www.stemcell.com/en/Products/All-­‐Products/Easy-­‐50-­‐EasySep-­‐Magnet.aspx

Sidebar  IV:  FACS
FACS  has  been  a  parOcularly  useful  technique  in  the  enrichment  of  HSCs.

FACS  can  sort  cells  into  different  populaOons  based  on  what  proteins
they  express  and  determine  the  number  of  cells  in  each  populaOon.

FACS  can  select  cells  based  on  several  parameters  simultaneously  (e.g.,
Linneg,  CD34pos,  CD90pos)  –  10,000  –  50,000  cells  can  be  sorted  per
second.

FACS  allows  rare  cells  (i.e.,  less  than  1  in  10,000  in  bone  marrow,  less
than  1  in  100,000  in  peripheral  blood)  to  be  purified  into  populaOons  of
near  100%  purity.    This  might  not  be  possible  in  the  case  of  HSCs  though.

What  is  needed  is  an  anObody  to  a  parOcular  protein  that  is  tagged  with
a  fluorochrome.
 Fluorescent  cell  sorOng  machines  “inspect”  each  cell  individually  to  see
what  fluorochomes  are  aRached  to  it.

Cell  suspension  –  cells  labeled  with

either  “anO-­‐A”  anObody  or  “anO-­‐B”
anObody  –  the  anObodies  are  tagged
with  different  fluorochromes.
A  vibraOng  nozzle  ensures
that  droplets  leaving  the
nozzle  contain  a  single  cell.

detectors

laser
+ + + + +

–
charger
Then  sort  the  cells  into  separate  containers  depending  on  what  is
detected  -­‐  the  machine  also  counts  the  number  of  cells  going  into  each
container.

laser
+ + + ++

–
charger
– +
deflecOon  plates – +
+
– – +
+
– +
– + – +
– +

cells  without
bound
anObody
 Data  for  a  FACS  experiment  using  one
Top:    the  machine  was  asked  to fluorescent-­‐labeled  anObody
sort  and  count  the  number  of  cells

RelaOve  number
depending  on  whether  a  single A– A+

of  cells
anObody  had  bound  to  them  (yes
or  no).

AnO-­‐A  anObody
fluorescence

detectors Data  for  a  FACS  experiment  using  two

fluorescent-­‐labeled  anObodies
A–  B+ A+  B+

AnO-­‐B  anObody
fluorescence
A–  B– A+  B–

AnO-­‐A  anObody
fluorescence
Sources  of  HSCs
There  are  three  main  sources  of  HSCs:

–  bone  marrow,
–  mobilized  peripheral  blood,
–  umbilical  cord  blood.

Each  source  has  some  advantages  associated  with  it,  as  well  as  some
disadvantages.

Cytokine  mobilized  peripheral  blood  seems  to  becoming  a  more  popular

method  of  isolaOng  HSCs  from  donors.
Sources  of  HSCs  -­‐  bone  marrow  and  mobilized  peripheral  blood
In  adults  under  steady-­‐state  condiOons  (i.e.,  no  infecOons),  most  of  the
HSCs  do  reside  in  the  bone  marrow.

HarvesOng  HSCs  from  the  bone  marrow  is  a  fairly  invasive  procedure  (for
the  donor).

Cytokine  mobilizaOon  can  result  in  the  release  of  large  numbers  of  HSCs
into  the  blood.
Sources  of  HSCs  -­‐  bone  marrow  and  mobilized  peripheral  blood
Bone  marrow  and  mobilized  peripheral  blood  contains  a  mixture  of
hematopoieOc  stem  and  progenitor  cells  as  well  as  other  cell  types  (e.g.,
T  cells).

The  collected  cells  are  oden  passed  through  a  device  that  selects  for  the
CD34  marker.

The  resulOng  cell  suspension  is  enriched  for  both  HSCs  and  progenitor
cells  (the  major  component).
Sources  of  HSCs  -­‐  umbilical  cord  blood
Blood  from  the  placenta  and  umbilical  cord  is  a  rich  source  of  HSCs.

Umbilical  cord  blood  can  be  harvested,  frozen  and  stored  in  cord  blood
banks.

Advantages  to  using  umbilical  cord  blood  as  a  source  of  HSCs:
–  Availability  (how  many  babies  are  born  each  day  in  Vancouver?),
–  Ease  of  harvest  (don’t  have  to  pre-­‐treat  the  donor  with  cytokines),
–  May  have  greater  proliferaOve  capacity  compared  to  adult  HSCs,
–  Reduced  risk  of  grad-­‐vs-­‐host  disease  in  the  recipient.

Disadvantages  to  using  umbilical  cord  blood  as  a  source  of  HSCs:
–  Limited  number  of  cells  that  can  be  harvested  and  provide  to  the
recipient,
–  Delayed  immune  reconsOtuOon  as  a  result.