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Biological properties and enucleation enucleation of of red red Biological properties and blood cells from from human embryonic stem cells human embryonic stem cells blood cells Shi-Jiang Lu, Qiang Feng, Jennifer S. Park, Loyda Vida, Bao-Shiang

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Biological properties and Biological properties and enucleation enucleation of

f red

red blood cells blood cells from from human embryonic stem cells human embryonic stem cells

Speaker: Jui‐Yun Weng Advisor: Ching‐Tsan Huang, PhD 2009/03/17

Shi-Jiang Lu, Qiang Feng, Jennifer S. Park, Loyda Vida, Bao-Shiang Lee, Michael Strausbauch, Peter J. Wettstein, George R. Honig, and Robert Lanza Blood 112(12):4475-4484, 2008.

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Blood transfusion

Blood donation
Surgery , serious accident, Sickle Cell Anemia
RhD(‐) blood type (western:~15%；Asia: <0.3%)
Pathogen (HIV, hepatitis B virus…)

RhD(+) patient RhD(‐) patient

?

B A B A

RBC

RhD

RBC

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HIV

15~20% 0.1~0.5%

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Using stem cells to generate red blood cells

Less rejection response
Enucleated cells‐‐problem of using stem cells

Carcinogenesis!! Carcinogenesis!!

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hematopoiesis

Red blood cell

Using stem cells to generate red blood cells

Scale up the production:

reproducible sources

Universal red blood cells‐‐

O/ O/RhD RhD( (‐ ‐) )

Pathogen‐free

Hemangioblast

Stem cell

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framework

Human embryonic stem cells (hESCs)

Erythroid cells

Enucleation Characterization

Hemoglobin FACS analysis

Day 19-21

Functional analysis

O2 equilibrium curves

Further Maturation ABO?RhD?

Day 36-42 Day 28

Mature/immature marker Morphology Erythroid cell + + stromal stromal cell cell + IL3 + IL3 hemagioblasts hemagioblasts

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Culturing hESCs

WA01 MA01 MA99 HuES-3

Embryoid body formation

&

Hemangioblast precursor induction

Day -3.5~0

Hemangioblast expansion

Day 0~10

Erythroid cell differentiation and expansion Day 11~20

Enrich of erythroid cells Day 21

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1010‐1011 cell/6‐well plate of hESCs
12‐16 μm in diameter (day 21)

Culturing hESCs

1000x From 2x106 hESCs

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Hemoglobin of erythroid cells

Electrospray ionization mass spectra Adult hemoglobin: 2α+2β Fetal hemoglobin: 2α+2γ Embryo: α, γ, ζ, ε No β-globin Early developmental stage

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FACS analysis

(fluorescence activated cell sorter)

(Fetal Hb)

Cells labeled with antibody

Immature cell marker

% %

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Functional analysis

O2 equilibrium curves

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Enucleation

Normal RBCs OP9 co-cultured Day 42

1000x 1000x

Condition Enucleation %

w/o co‐cultured 10‐30 Human mesenchymal stem cell 30 OP9 mouse stromal cell 30 30‐ ‐65 65 Hemangioblast (day 7) + IL3 + stromal cell co-cultured

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Further maturation

Mature Immature

Immunofluorescence staining Further maturation

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Further maturation

DAPI: dye for nucleotide

Further maturation Immunofluorescence staining

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Enucleation and maturation

Hemangioblast Enucleated N/C=nuclear to cytoplasm ratio

(6-8 μm)

normal RBC: ~7 μm

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Further maturation

Benzidine + Wright-Giemsa stain

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ABO and RhD Genotyping

RhD(-)

RhD--PCR for the intron 4 of RhD sequence

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ABO and RhD Genotyping

O

(O, O)= O type (B, O)= A type (B, B)= B type

B A B B A A

ABO--PCR+RE based on

polymorphism of glycosyltransferase B+ MA09 A+ MA01 A+ WA09 B+ WA07 O+ WA01 A‐ MA133 B‐ MA99

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19 hESCs Erythroid cells 19-21 days

Protein mass Protein mass Fetal/embryo hemoglobin Enucleation Enucleation O/ O/RhD RhD( (-

) ? O O2

2 equilibrium curve

equilibrium curve Normal ability FACS FACS Fetal Hb:65% CD71:76% 30~65% CD71↓ ↓, , CD253a↑ ↑ β-hemoglobin ↑ ↑ Further maturation Further maturation

OP9 stromal cell

To be continued To be continued… …

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