Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Summary

Fibrin matrices containing growth factors were used to retain grafted neural stem cells into sites of complete spinal cord transection. Grafted cells completely filled the lesion cavity and differentiated into multiple neural cell types, including neurons that extended axons into host spinal cord over long distances.

Abstract

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord injury (SCI), and can form functional relays to re-connect spinal cord segments above and below a lesion. Previous studies grafting neural stem cells have been limited by incomplete graft survival within the spinal cord lesion cavity. Further, tracking of graft cell survival, differentiation, and process extension had not been optimized. Finally, in previous studies, cultured rat NSCs were typically reported to differentiate into glia when grafted to the injured spinal cord, rather than neurons, unless fate was driven to a specific cell type. To address these issues, we developed new methods to improve the survival, integration and differentiation of NSCs to sites of even severe SCI. NSCs were freshly isolated from embryonic day 14 spinal cord (E14) from a stable transgenic Fischer 344 rat line expressing green fluorescent protein (GFP) and were embedded into a fibrin matrix containing growth factors; this formulation aimed to retain grafted cells in the lesion cavity and support cell survival. NSCs in the fibrin/growth factor cocktail were implanted two weeks after thoracic level-3 (T3) complete spinal cord transections, thereby avoiding peak periods of inflammation. Resulting grafts completely filled the lesion cavity and differentiated into both neurons, which extended axons into the host spinal cord over remarkably long distances, and glia. Grafts of cultured human NSCs expressing GFP resulted in similar findings. Thus, methods are defined for improving neural stem cell grafting, survival and analysis of in vivo findings.

Introduction

Spinal cord injury (SCI) often damages not only white matter tracts that carry signals to and from the brain, but also the central gray matter, causing segmental loss of interneurons and motor neurons. The consequence of SCI is loss of both motor and sensory function below the lesion. Unfortunately, the adult central nervous system (CNS) does not spontaneously regenerate, resulting in permanent functional deficits¹. Therefore, reconstruction of the injured adult spinal cord and improvement in motor, sensory and autonomic function is an important goal of SCI research. Neural stem cells (NSCs), whether directly isolated from embryonic or adult CNS, are compelling candidate cells to replace lost neurons and glia. Moreover, these cells have the potential to form new functional relays to restore axonal conduction across a lesion site^2,3.

To date, there has not been a detailed elucidation of the anatomical, electrophysiological and behavioral effects of neuronal relay formation by transplanted NSCs after severe SCI. There are several reasons for this: first, transplanted NSCs or fetal CNS tissue survive poorly when grafted into large lesion cavities. Previous studies show substantial early cell loss, leaving large empty cystic lesion cavities^4,5. In some studies the grafted cells would subsequently divide and fill the lesion cavity^4,5, but this might occur after a delay of days to weeks, and subsequent lesion site filling might not be complete or consistent. Second, an efficient tracking system that provides sound data on cellular survival, differentiation/maturation, and outgrowth of transplanted NSCs was lacking. Most early studies utilized antegrade and retrograde labeling to trace axonal projections from transplants^2,3. However, these techniques only partially and often unclearly labeled axonal projections arising from grafted cells, and tracer methods are subject to artifacts caused by dye leakage beyond the implanted cells. Other groups used human specific neuronal markers to label axonal projections after transplantation of human fetal NSCs into injured rodent spinal cord^5,6. However, in those studies, the xenografts did not consistently survive well. Recently, viral  delivery of the GFP reporter gene was used to label cultured NSCs^7,8. However, GFP expression was often inconsistent and can be down-regulated⁷. Recently, the use of transgenic donor mice or rats stably expressing the reporter gene, GFP, or the human placental alkaline phosphatase, has dramatically improved the tracking of transplanted neural stem cells/progenitors in vivo^9,11. Third, several studies indicate that in vitro cultured rat NSCs derived from either embryonic or adult CNS exclusively differentiate into glial lineages when transplanted into the milieu of the intact or injured adult spinal cord^7,12,13, despite the fact that these neural stem cells are capable of differentiating into both neurons and glia in vitro, indicating that local environments may dictate the fate of stem cells. Alternatively, cultured NSCs, especially those derived from adult CNS, may have intrinsic defaulty property to differentiate into glial lineages in vivo¹³.

Because of the limitations discussed above, our group recently developed a new protocol to improve embryonic NSC tracking, survival, and differentiation/maturation in the severely injured adult spinal cord. Briefly, we began with a stable transgenic Fischer 344 rat inbreed line expressing a GFP reporter gene that sustains GFP expression after in vivo transplantation¹⁴. Next, we used freshly isolated NSCs from embryonic day 14 Fischer 344 spinal cord, a stage of development that retains the potential to generate both neurons and glia. Finally, we embedded freshly dissociated NSCs into a fibrin matrix containing growth factors^15-17 to retain the cells and evenly distribute them within a large lesion cavity, aiming to support graft cell survival, differentiation and integration. Grafts were placed into sites of T3 complete transection, two weeks after spinal cord injury. These grafted cells consistently filled complete transection sites and differentiated into abundant neurons that extended large numbers of axons into host spinal cord over long distances¹⁸. Similar results were obtained using cultured human neural stem cell grafts to immuno-deficient rats¹⁸.

Protocol

All animal protocols are approved by VA-San Diego Institutional Animal Care and Use Committee (IACUC). NIH guidelines for laboratory animal care and safety are strictly followed. Animals have free access to food and water throughout the study and are adequately treated for minimizing pain and discomfort.

1. Preparation of Fibrin Components Containing Growth Factor Cocktails

1. Dissolve 25 mg rat fibrinogen in 0.5 ml PBS to obtain 50 mg/ml 2x stock solution (see Materials table). Fibrinogen is difficult to dissolve and should be placed into a 37 °C incubator or waterbath and shaken every 5-10 min for 1-2 hr.
2. Dissolve 100 UN rat thrombin in 2 ml of 10 mM sterile CaCl₂ to obtain 50 U/ml 2x stock solution.
3. Dissolve growth factors in PBS at the following concentrations to obtain 100 μl stock solution: 1 mg/ml: BDNF; NT-3 (high stock concentration as intrathecal infusion in vivo¹⁹); 200 μg/ml: GDNF; IGF; bFGF; EGF; PDGF; aFGF; HGF (about 1,000x higher than used for cell culture).
4. Dissolve 25 mg MDL28170 (calpain inhibitor) in 1.3 ml DMSO to obtain 50 mM DMSO stock solution, and then dilute 50x in PBS to obtain 1 mM stock solution.
5. Mix 100 μl of each growth factor component and 100 μl of MDL28170 (1 mM stock solution) to produce 1,000 μl growth factor cocktail solution (Figure 1).
6. Mix 500 μl fibrinogen 2x stock solution with 500 μl growth factor cocktail to obtain 25 mg/ml fibrinogen working solution containing growth factors and aliquot into 10 or 20 μl for storage at -70 °C. The solution is stable for up to 12 months at -70 °C.
7. Similarly, mix 500 μl thrombin 2x stock solution with 500 μl growth factor cocktail to obtain 25 U/ml thrombin working solution containing growth factors and aliquot into similar 10 or 20 μl volumes for storage at -70 °C for up to 12 months.
8. On the day of grafting, place fibrinogen- and thrombin-containing growth factor cocktails on ice until mixed with cells.

2. T3 Spinal Cord Transection

1. Anesthetize adult female Fischer 344 rats or athymic nude rats using acceptable methods. Subjects are deeply anesthetized, when responsiveness to tail and paw pinch are completely absent. Note: We typically use 150-200 g Fischer rats or 180-220 g athymic rats, and an anesthesia cocktail (2 ml/kg) of ketamine (25 mg/ml), xylazine (1.3 mg/ml) and acepromazine (0.25 mg/ml).
2. Apply eye ointment to prevent dryness or injury of eyes while the animals are under anesthesia.
3. Shave the upper thoracic area and clean the skin with Betadine.
4. Cut the skin and muscle using #15 blade, expose T3 vertebra using a surgical retractor, and perform a laminectomy at T3 vertebra to expose T3/4 spinal cord using a rongeur.
5. Make a longitudinal midline incision in the dura of approximately 2 mm length using a #11 blade.
6. Place iridectomy scissors underneath the dura to cut the right lateral entire spinal cord. Make another cut on the same side 1.5 mm more cadually.
7. Aspirate spinal cord tissue between the two cut segments with a blunt 23 G needle connected to moderate intensity vacuum. Use visual verification to ensure complete transection ventrally and laterally. This will complete a lateral hemisection.
8. To extend the lesion to a full-width spinal cord transection, place mirror incisions into the left hemicord. Attempt to leave the dura intact, other than the first cut, to retain the firm graft/fibrin matrix in the lesion site when grafted two weeks later.
9. Suture the muscle, apply antibiotic powder and staple the skin.
10. Inject lactated Ringer's (3 ml), Bannamine (2.5-5 mg/kg) and Ampicillin (80-100 mg/kg) (see Materials table) immediately after surgery and maintain rats in a warm incubator until thermoregulation is re-established.
11. Empty the bladder and inject the Ringer's and Ampicillin solution twice a day for about two weeks until the establishment of reflex bladder emptying (Figure 1B).

3. Preparation of Freshly Dissociated Embryonic Day 14 Spinal Cord Neural Stem Cells

1. Obtain inbreed transgenic GFP F344 rats (F344-Tg(UBC-EGFP)F455Rrrc) from Rat Resource and Research Center, University of Missouri.
2. Inject 40 μg des-Gly¹⁰, [D-Ala⁶]-Leuteinizing Hormone Releasing Hormone Ethylamide (LHRH) diluted in PBS at a concentration of 200 μg/ml intraperitoneally (IP) per mature female rat (8 weeks or older) after 2-3 hr light induction for synchronization of embryo collection.
3. Mate with a mature GFP homozygous or heterozygous male rat overnight 4 days after injection.
4. Collect embryos at days 13.5-14.5 postcoitus (p.c.) in cold HBSS buffer, examine GFP expression under a "NightSea" flashlight and filter glasses (BLS1 - BlueStar Flashlight and Model VG1 barrier filter) or a fluorescent microscope.
5. Dissect spinal cord out aseptically from each GFP-positive fetus and remove overlying meninges and attached spinal ganglia with iridectomy scissors and fine jewelers forceps.
6. Collect the dissected spinal cords into 2 ml cold HBSS buffer in a 15 ml cornical tube and keep on ice.
7. Follow reference #20 to dissociate dissected spinal cords:
   1. Briefly, add 2 ml of 0.25% Trypsin-EDTA to the tube containing dissected spinal cords (several cords can be digested together, 1 cord can generate enough cells for graft of one subject) and digest at 37 °C for 10-12 min.
   2. Stop the trypsin reaction by adding 10 ml of DMEM containing 10% FBS to the tube and centrifuge the tissue at 2,500 rpm for 2 min.
   3. Resuspend tissue in 2 ml NeuroBasal medium containing 2% B27, and triturate the spinal cord tissue using progressively smaller fire-polished Pasteur pipets.
   4. Centrifuge the cells at 2,500 rpm for 2 min, resuspend in 2 ml of NeuroBasal medium containing B27, and filter cells with a 40 μm cell filter strainer.
8. Count the cells with a hemocytometer and divide the cells equally into two Eppendorf tubes.
9. Centrifuge the cells and completely remove supernatant (1-2 min are required to withdraw all supernatant with a low vacuum tip) so the growth factor cocktails will not be diluted by remaining supernatant after cell re-suspension.
10. Resuspend each half of the cells in the fibrinogen or thrombin working solution containing growth factors, respectively, at a concentration of 250,000 cells/μl (cell pellet counts for about ⅓ final volume) and place them on ice prior transplantation (Figure 1A). Note that fibrinogen may spontaneously gel when mixed with cells before combining with thrombin at the lesion/graft site; to avoid premature gellation, mix cells in fibrinogen immediately before cell grafting in vivo.

4. Transplantation

1. Reexpose the lesioned T3/4 spinal cord two weeks after spinal cord transection.
2. 5 μl fibrinogen-cells and 5 μl thrombin-cells could be directly injected into nine points of the lesion site through sealed scar tissue and intact dura without re-opening the lesion site.
3. Use a 27 G needle to create 9 holes on the surface of the scar tissue and intact dura 1-2 weeks post lesion: 3 in center (one in middle point and two in laterals, and spaced 0.5 mm apart) and 3 in both rostral and 3 in caudal interface (about 0.5 mm rostral or caudal to the center of the lesion).
4. Then inject half volume of fibrinogen cell solution into three central points of the lesion site and a quarter volume into 3 points of the rostral interface and a quarter volume into 3 points of the caudal interface.
5. Then, immediately inject throbmbin cells into the same spots. The mixture of fibrinogen cells and thrombin cells inside the lesion site will form fibrin gel to hold cells into lesion site and the growth factor cocktails will support the survival of cells. Cells are injected using pulled glass pipettes connected to a PicoSpritzer (General Valve, Inc.).
6. Transplant cultured human NSCs²¹ in the same procedure described above, and use fibrin and growth factor cocktails made from human reagents.

Results

GFP immunohistological labeling demonstrates that grafted rat NSCs resuspended in phosphate buffered saline (PBS) (lacking a fibrin matrix and growth factors) survived poorly in the T3 transection site, only attaching to the lesion/host margin and leaving most of the lesion site empty without grafted cells (Figure 2A). The survival of NSCs improved when co-grafted with fibrin matrices alone, but the graft did not completely fill up a large lesion cavity (data not shown). Therefore, we embedded NSCs into ...

Discussion

One of the major hurdles for NSC transplantation in the injured spinal cord is poor survival in the lesion center. Any gaps or cavities in the lesion site could potentially reduce or weaken the formation of functional neuronal relays between supraspinal axons and separated spinal cord segments below the injury. In addition, poor survival of grafted NSCs may affect their integration with host tissue, and therefore reduce connectivity of grafted neurons with host neurons. Potential mechanisms to explain cell loss include t...

Materials

Name	Company	Catalog Number	Comments
Fibrinogen (rat)	Sigma	F6755-25MG	2 hr at 37 °C to dissovle Stock Concentration: 50 mg/ml Final Concentration: 25 mg/ml
Thrombin (rat)	Sigma	T5772-100UN	Dissovle in 10 mM CaCl2 Stock Concentration: 50 U/ml Final Concentration: 25 mg/ml
bFGF (human)	Sigma	F0291 (25 μg)	Stock Concentration: 200 ng/μl Final Concentration: 10 ng/μl
EGF (murine)	Sigma	E1257 (0.1 mg)	Stock Concentration: 200 ng/μl Final Concentration: 10 ng/μl
BDNF (human)	Peprotech	452-02 (1 mg)	Stock Concentration: 1,000 ng/μl Final Concentration: 50 ng/μl
NT-3 (human)	Peprotech	452-03 (1 mg)	Stock Concentration: 1,000 ng/μl Final Concentration: 50 ng/μl
GDNF (rat)	Sigma	G1401 (10 μg)	Stock Concentration: 200 ng/μl Final Concentration: 10 ng/μl
IGF-1 (mouse)	Sigma	I8779 (50 μg)	Stock Concentration: 200 ng/μl Final Concentration: 10 ng/μl
aFGF (human)	Sigma	F5542 (25 μg)	Stock Concentration: 200 ng/μl Final Concentration: 10 ng/μl
PDGF-AA (human)	Sigma	P3076 (10 μg)	Stock Concentration: 200 ng/μl Final Concentration: 10 ng/μl
HGF (human)	Sigma	H9661 (5 μg)	Stock Concentration: 200 ng/μl Final Concentration: 10 ng/μl
MDL28170	Sigma	M6690 (25 mg)	Stock in DMSO Stock Concentration: 1 mM Final Concentration: 50 μM
PBS	Millipore	BSS-1005-B	Stock Concentration: 1x Final Concentration: 1x
DMSO	Sigma	D2650
Ketamine	Putney	26637-411-01	40-80 mg/kg Stock Concentration: 100 mg/ml Final Concentration: 25 mg/ml
Xylazine	Lloyd	0410,	2.5-8 mg/ml Stock Concentration: 100 mg/ml Final Concentration: 5.8 mg/ml
Acepromazine	Butler	003845	0.5-4 mg/ml Stock Concentration: 10 mg/ml Final Concentration: 0.25 mg/ml
Betadine	Healthpets	BET16OZ
Ringers	Abbott	04860-04-10	2-3 ml/inj
Banamine	Schering-Plough	0061-0851-03	2.5-5 mg/kg Stock Concentration: 50 mg/ml Final Concentration: 0.5 mg/ml
Ampicillin	Sandoz	0781-3404-85	80-100 mg/kg Final Concentration: 50 mg/ml
LH-RH	Sigma	L4513	200 μg/kg Final Concentration: 200 μg/ml
HBSS	Gibco	14175-096
Trypsin	Gibco	25200056	Stock Concentration: 0.25 % Final Concentration: 0.125 %
DMEM	Gibco	11995073
FBS	Gibco	16000044	Stock Concentration: 100 % Final Concentration: 10 %
Neurobasal Medium	Gibco	21103049
B27	Gibco	17504044	Stock Concentration: 100x Final Concentration: 1x
Note, use human reagents for grafts of human NSCs