Bone Marrow-Derived Stem Cells Can Differentiate into Retinal Cells

in Injured Rat Retina

By Anna Begelfer-OstrovskiMinoru Tomita1,2,Yasushi Adachi1,2,3,Haruhiko Yamada2,

Kanji Takahashi2,Katsuji Kiuchi2,5,Haruki Oyaizu1,

Kazuya Ikebukuro1,Hiroyuki Kaneda1,

Miyo Matsumura2,3,4,Susumu Ikehara M.D.1,3,

Bone marrow-

• spongy, fatty tissue houses stem cells.

• located inside a few large bones.

• It’s stem cells transform themselves into white and red blood cells and platelets.

• essential for immunity and circulation

Bone marrow cells (BMCs) differentiate into various cells:

• hepatocytes

• endothelial cells of the blood vessel

• epithelial cells of the stomach, esophagus, small intestine, large intestine and bronchus

• cardiac muscle

• skeletal muscle

• neural cells

This experiment:

examined whether bone marrow stem

cells can differentiate into

retinal neural cells in adult

rats.

Experiment overview:

• Collection of BMC’s stem cells.

• Injury of Rat’s eye.

• Injection of BMC’s stem cells

• Sectioning of the eye

• Data Analysis

• Conclusion

MATERIALS AND METHODS

Preparation of Stem Cell-Enriched BMCs for grafting:

• 5-fluorouracil injected into the peritoneal cavity 4-week old male rat (inside abdominal cavity)

• BMCs were obtained from femurs and tibias (leg)

• Fractionated by density centrifugation using Lymphoprep™ density solution

• collected and labeled.

MATERIALS AND METHODS

• Rats and Grafting Procedure

• rats were anesthetized with pentobarbital sodium.

• pupils were dilated with 0.5% tropic amide

• the corneas were further anesthetized with drops of 0.4% oxybuprocaine hydrochlorid

MATERIALS AND METHODS

Injury:

• eyeballs were perforated at the equator with a 30-gauge needle

• retina was injured by scratching (under a surgical microscope)

• special care was taken to injure the whole layer of the retina

MATERIALS AND METHODS

Post injury:

• 20 μl of the cell suspension, containing 200,000 cells, were slowly injected into the intravitreous space.

• 6 rats, 6 injected eyes with BMC’s stem cells.

• Control-20 μl of normal saline solution injected into the same place of injured eyes.

MATERIALS AND METHODS

• Tissue Sectioning

• The animals were sacrificed 2 weeks after injection

• eyes were enucleate and prepared for cutting.

• Samples were stained and observed under confocal microscope.

RESULTS:

• BMCs had been built into the host retina in 83% of the experimental eyes (5/6).

• Saline treated retina showed progressing damage.

• BMC’s treated cells showed cell proliferation.

Image I:Saline injectionProliferated due to BMC’s injectionDistribution of BMC’s using green fluorescenceC without Flourescence

Image I:

• The grafted cells( green fluorescence) were distributed around the injured sites.

• Grafted cells were observed at the site of injury and at normal retinal structure.

• Most of the grafted cells were in the injured site

• BMCs had migrated into the retina, followed by proliferation and differentiation into retinal neural cells.

Image II

Immunohistochemistry on Sections after Transplantation

Image II

• Injected cells labeled with expressed green fluorescence.

• Astrocytes, carbindin, vimentin, and rhodopsin, red fluorescence.

• Incorporated bone marrow-derived cells, yellow fluorescence.

• Incorporated bone marrow cells had differentiated into retinal cells.

DISCUSSION:

• stem cell-enriched BMCs injected into injured eyes differentiated into retinal neural cells in vivo.

• Injection of retinal tissue of newborn and brain-derived cell lines are other methods that did not bring same results.

Conclusion:

• stem cell-enriched BMCs have the ability to differentiate into retinal neural cells.

• Injection of stem cell-enriched BMCs into the retina would help repair damaged retinal cells.

Take home message:

• injection of BMC’s into the eye can potentially rescue injured retinal tissue, even in humans.