Small molecule-driven direct conversion

of human pluripotent stem cells into

functional osteoblasts

Heemin Kang, Yu-Ru Shih, Manando Nakasaki, Harsha Kabra, and Shyni Varghese
Material Science Engineering Department and Bioengineering Department
University of California San Diego
Science Advances, August 2016
Stem cells

 Divide and renew

 Unspecialized
 Potential to develop into different types of cells

 Potency: Totipotent, pluripotent, multipotent, and oligopotent

 Source: embryonic stem cell, adult or induced pluripotent stem cell

 hPSCs: hESC and hiPSCs

 Optimal sources for regenerative medicine
 hiPSCs are clinically advantageous (can be derived from the patient’s own cells)
Differentation of hPSCs

 Generated by genetic manipulation, use of biomaterials, growth factors, small molecules,

and cocktails thereof
 Long list of ingredients and steps make the process costly and inefficient to be applied in a
clinical setting
Small molecules

 Simplicity
 Ease of synthesis
 Cost-effectiveness
 Scalability
 Stability
Biomaterials containing calcium
phosphate

 Triggered the phosphate-ATP-adenosine A2b receptor (A2bR) axis signaling

Source: doi 10.1073/pnas.1321717111

Objectives and method

 Prove that adenosine can drive osteogenic differentiation of hPSCs

 Confirm that the adenosine-driven osteogenesis of hPSCs involve A2bR
 Test if hiPSC-derived osteoblasts can produce calcified bone tissue in vitro
 These osteoblasts can directly participate in the healing of critical-sized cranial defect
Adenosine can drive osteogenic
differentiation of hPSCs
Adenosine can drive osteogenic
differentiation of hPSCs

 Time-resolved quantitative analyses of gene expression of osteogenic genes by qPCR

Adenosine can drive osteogenic
differentiation of hPSCs

 Immunofluorescence staining for osteocalcin and

staining with Alizarin Red S
Both adenosine-supplemented growth medium and conventional
osteogenic medium can drive osteogenic differentiation of hPSCs.
Adenosine-driven osteogenesis of hPSCs
involves A2bR signaling pathway

 Quantitative analyses of gene expression of adenosine receptor genes by qPCR

Adenosine-driven osteogenesis of hPSCs
involves A2bR signaling pathway

 Addition of A2bR antagonist PSB 603 and quantitative analyses of gene expression of
osteogenic genes
Adenosine-driven osteogenesis of hPSCs
involves A2bR signaling pathway

 Immunofluorescence staining for osteocalcin and

staining with Alizarin Red S
Adenosine-driven osteogenesis of hPSCs
involves A2bR
hiPSC-derived osteoblasts can produce
calcified tissue in vitro
hiPSC-derived osteoblasts can produce
calcified tissue in vitro

 Micro-CT imaging after 3 weeks of culturing

hiPSC-derived osteoblasts can produce calcified
tissue in vitro in the absence of any other
osteoinductive molecules
hiPSC-derived osteoblasts directly participate
in the healing of critical-sized cranial defects
hiPSC-derived ostepblasts directly participate
in the healing of critical-sized cranial defects

 Micro-CT imaging after 16 weeks of culturing

hiPSC-derived ostepblasts directly participate
in the healing of critical-sized cranial defects

 H&E staining, osteocalcin, and TRAP staining

hiPSC-derived ostepblasts directly participate
in the healing of critical-sized cranial defects

 Immunostaining of lamin A/C of the hiPSCs implanted

hiPSC-derived osteoblasts can participate in the healing of critical-
sized cranial defects in vivo, producing a bone tissue that is
homogenous and integrated with the native tissue, and
morphologically similar to the native bone
Summary

 Adenosine can direct osteogenic differentiation of hPSCs

 Devoid of any other osteoinductive factors
 Through the A2bR axis signaling pathway
 hiPSC-derived osteoblasts can form bone tissue in vitro and in vivo and contributed to the
repair of critical-sized bone defect
 Without teratoma formation or causing infections
Significance

 Adenosine
 Simple molecule
 Easy to synthesize
 Naturally occurring in the body
 Exogenous injection is a potential cost-effective and scalable method for regenerating bone

 Ease of development and cost-effectiveness allow accessible regenerative treatments for

patients with critical bone defects and soldiers who have suffered bone injuries
 Hasn’t been proven if the therapy would work
 Milieu of different molecular players inside the bone
 More research is needed before doctors could apply this in the clinical setting
Thank you!
Supplementary slides

Heemin Kang, Yu-Ru Shih, Manando Nakasaki, Harsha Kabra, and Shyni Varghese
Material Science Engineering Department and Bioengineering Department
University of California San Diego
Science Advances, August 2016
Small molecules that drive osteogenic
differentiation

 Puromorphamine and N6 benzoyladenosine monophosphate

 CHIR99021 and cyclopamine
 Calcium phosphate
Making embryonic stem cells

Source: https://s-media-cache-ak0.pinimg.com/originals/5b/82/58/5b82588b4dc8b7eab38e81b53bdbb006.jpg
Making induced pluripotent stem cells

Source: http://axiogenesis.com/images/science-technology/ipscartoon.png