From Stem Cells to Beta Cells: From Stem Cells to Beta Cells: Possible Cure for Diabetes MellitusPossible Cure for Diabetes Mellitus

By Ryan ScavinskiBy Ryan Scavinski

Diabetes Mellitus Diabetes Mellitus Type 1 Diabetes is Type 1 Diabetes is

caused by the caused by the autoimmune autoimmune destruction of destruction of ββ--cells within the cells within the pancreas. pancreas.

No No ββ-cells, no -cells, no insulininsulin

Diabetics need to Diabetics need to monitor blood monitor blood glucose and control glucose and control it with insulin.it with insulin.

Past TreatmentsPast Treatments

Transplantation of Transplantation of pancreatic islet cellspancreatic islet cells

ProblemsProblems– Patient requires Patient requires

more than 600 more than 600 islets/kg body weight islets/kg body weight = two deceased = two deceased organ donorsorgan donors

– Immunological Immunological rejectionrejection

Using Stem Cells Using Stem Cells

Two approaches used to differentiate Two approaches used to differentiate Embryonic Stem Cells into Embryonic Stem Cells into ββ-cells or -cells or Insulin Producing Cells (IPCs)Insulin Producing Cells (IPCs)– Embryoid Body FormationEmbryoid Body Formation– Definitive Endoderm FormationDefinitive Endoderm Formation

Embryoid Body FormationEmbryoid Body Formation

An embryoid is the arrangement of An embryoid is the arrangement of stem cells destined to differentiate stem cells destined to differentiate into the ectoderm, mesoderm and into the ectoderm, mesoderm and endoderm. endoderm.

With multiple treatment of growth With multiple treatment of growth factors, Embryonic stem cells give factors, Embryonic stem cells give rise to Nestin cells, which in turn rise to Nestin cells, which in turn differentiate into IPCs.differentiate into IPCs.

ProblemsProblems

Low efficiency for producing IPCsLow efficiency for producing IPCs Also showed development of tumors Also showed development of tumors

in the kidney and spleen in some in the kidney and spleen in some transplanted mice.transplanted mice.

Definitive Endoderm Definitive Endoderm ApproachApproach

This approach bypasses the This approach bypasses the Embryoid formation and generates Embryoid formation and generates the endoderm, in which produce the the endoderm, in which produce the cells needed for insulin production. cells needed for insulin production.

Differentiation StepsDifferentiation Steps

1. ESCs were placed onto a culture 1. ESCs were placed onto a culture dish with a chemically defined dish with a chemically defined medium (CDM) containing 50 ng/mL medium (CDM) containing 50 ng/mL Activin A for 4 daysActivin A for 4 days

With and without With and without Activin AActivin A

PCR of expression PCR of expression of endoderm genes of endoderm genes – Gapd, sox17, pdx1, Gapd, sox17, pdx1,

hlxb9, hnf4a and hlxb9, hnf4a and insulininsulin

gapd sox17 pdx1 hlxb9 hnf4α insulin

continuedcontinued

2. Then the cells were transferred 2. Then the cells were transferred onto a CDM with 10onto a CDM with 10-6-6 M Retinoic acid M Retinoic acid (RA) for another 4 days(RA) for another 4 days

Without Activin A Without Activin A and RAand RA

With Activin A and With Activin A and RARA

small clusters of small clusters of differentiated ESdifferentiated ES

PCR PCR

A+/RA+A+/RA+

A-/RA-A-/RA-

A+/RA-A+/RA-

A-/RA+A-/RA+

gapd sox17 pdx1 hlxb9 hnf4α insulin

continuedcontinued

3. Then the CDM was changed to 3. Then the CDM was changed to modified islet maturation medium modified islet maturation medium containing bFGF- a pancreatic cell containing bFGF- a pancreatic cell maturation factor for 3 daysmaturation factor for 3 days

4. Finally the differentiated cells were 4. Finally the differentiated cells were switched to a islet maturation switched to a islet maturation medium containing nicotinamide and medium containing nicotinamide and the bFGF for another 3 days. the bFGF for another 3 days.

In this final stage, many In this final stage, many differentiated cells formed spherical differentiated cells formed spherical clustersclusters

Also expressed the pancreatic Also expressed the pancreatic ββ cell cell markers such as pdx1, INSULIN, markers such as pdx1, INSULIN, glucokinase and glut2 shown in PCRglucokinase and glut2 shown in PCR

Differentiated Embryonic Stem Differentiated Embryonic Stem CellsCells

gapd sox17 pdx1 hlxb9 hnf4α insulin glut2 Amy SST Sur1 GCG GCK

MaturationControl

To test cells for insulin release, cells To test cells for insulin release, cells were incubated in buffer containing were incubated in buffer containing 2.5 mM glucose for 15 min 2.5 mM glucose for 15 min

Then incubated with 27.5 mM Then incubated with 27.5 mM glucose for another 15 minglucose for another 15 min

Tested for insulin release with a Tested for insulin release with a Rat/Mouse insulin ELISARat/Mouse insulin ELISA

Insu

lin s

ecr

eti

on (

ng/m

g)

Suspension Adhesion

2.5 mM 27.5 mM

Transplantation Transplantation

Differentiated cells were Differentiated cells were transplanted under the renal capsule transplanted under the renal capsule (kidney) of diabetic mice. (kidney) of diabetic mice.

30% showed normal blood glucose 30% showed normal blood glucose levels for 6 weekslevels for 6 weeks

They removed the cell transplanted They removed the cell transplanted kidney-mice regained hyperglycemiakidney-mice regained hyperglycemia

0 7 14 21 28 35 42 49 56 Days after transplantation

30

25

20

15

10

5

0

Blo

od g

luco

se (

mM

)

Results and DiscussionResults and Discussion

The combination of Activin A and The combination of Activin A and Retinoic Acid is an effective method Retinoic Acid is an effective method to induce Embryonic Stem Cells to to induce Embryonic Stem Cells to differentiate into insulin producing differentiate into insulin producing cellscells

Further research in needed to see if Further research in needed to see if the difference between human and the difference between human and mouse will impair the function of mouse will impair the function of transplanted ESC derived cellstransplanted ESC derived cells

My Opinion My Opinion

Reference:Reference: Soria, B., Skoudy, A., and Martin, F. 2001. From stem Soria, B., Skoudy, A., and Martin, F. 2001. From stem

cells to beta cells: new strategies in cell therapy of cells to beta cells: new strategies in cell therapy of diabetes mellitus. diabetes mellitus. Diabetologia 44 407-415Diabetologia 44 407-415

Raikwar, S. and Zavazava, N. 2009. Insulin Raikwar, S. and Zavazava, N. 2009. Insulin producing cells derived from embryonic stem cells: producing cells derived from embryonic stem cells: are we there yet?. are we there yet?. Journal of Cellular PhysiologyJournal of Cellular Physiology, 218 , 218 256-263256-263

Jiang, W., Shi, Y., Zhao, D., Chen, S., Youg, J., Zhang, Jiang, W., Shi, Y., Zhao, D., Chen, S., Youg, J., Zhang, J., Qing, T., Sun, X., Zhang, P., Ding, M., Li, D., and J., Qing, T., Sun, X., Zhang, P., Ding, M., Li, D., and Deng, H. 2007. Deng, H. 2007. In vitroIn vitro derivation of functional derivation of functional insulin producing cells from human embryonic stem insulin producing cells from human embryonic stem cells. cells. Cell ResearchCell Research. 17 333-344 . 17 333-344