Directed differentiation of embryonic stem cells into dorsal interneurons

Murashov, Alexander K.; Pak, Elena S.; Hendricks, Wesley A.; Owensby, John P.; Sierpinski, Paulina; Tatko, Lisa; Fletcher, Paul L.

doi:10.1096/fj.04-2251fje

Cited by 48 publications

(34 citation statements)

References 33 publications

(45 reference statements)

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“…Murine ES cells (D3, ATCC) were transfected in the presence of FuGENE 6 (Roche) to express green fluorescent protein (GFP) [10,13,22,23]. Transfected colonies were selected as described previously [13] (see Fig.…”

Section: Cell Culture and Differentiationmentioning

confidence: 99%

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Transplantation of GABAergic neurons but not astrocytes induces recovery of sensorimotor function in the traumatically injured brain

Becerra

Tatko

Pak

et al. 2007

Behavioural Brain Research

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Embryonic stem (ES) cells have been investigated in many animal models of injury and disease. However, few studies have examined the ability of pre-differentiated ES cells to improve functional outcome following traumatic brain injury (TBI). The purpose of the present study was to compare the effect of murine ES cells that were pre-differentiated into GABAergic neurons or astrocytes on functional recovery following TBI. Neural and astrocyte induction was achieved by co-culturing ES cells on a bone marrow stromal fibroblast (M2-10B4) feeder layer and incubating them with various mitogenic factors. Rats were initially prepared with a unilateral controlled cortical contusion injury of the sensorimotor cortex or sham procedure. Rats were transplanted 7 days following injury with ∼100K GABAergic neurons, astrocytes, fibroblasts, or media. Animals were assessed on a battery of sensorimotor tasks following transplantation. The stromal fibroblast cells (M2-10B4), as a control cell line, did not differ significantly from media infusions. Transplantation of GABAergic neurons facilitated complete and total recovery on the vibrissae-forelimb placing test as opposed to all other groups, which failed to show any recovery. It was also found that GABAergic neurons reduced the magnitude of the initial impairment on the limb use test. Histological analysis revealed infiltration of host brain with transplanted neurons and astrocytes. The results of the present study suggest that transplantation of pre-differentiated GABAergic neurons significantly induces recovery of sensorimotor function; whereas, astrocytes do not.

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Section: Cell Culture and Differentiationmentioning

confidence: 99%

“…Brains were cut with a cryostat into 30 micron coronal slices and were then processed as freefloating sections [13,22]. Immunostaining was performed using the Elite ABC Kit (Vector Laboratories, Burlingame, CA).…”

Section: Immunocytochemistry Immunohistochemistry and Immunofluoresmentioning

confidence: 99%

Transplantation of GABAergic neurons but not astrocytes induces recovery of sensorimotor function in the traumatically injured brain

Becerra

Tatko

Pak

et al. 2007

Behavioural Brain Research

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“…Cells were passaged at approximately 80% to 90% confluence, usually every other day. Our published 4-/4+ protocol was used for differentiation of ES cells (28,29). Briefly, during differentiation induction maintenance medium was exchanged for differentiation medium (Iscove's modified Dulbecco's medium, 15% FBS, L-glutamine [2 mM], nonessential amino acids [0.1 mM], 100 units/mL penicillin, and 100 µg/mL streptomycin, but no LIF or β-mercaptoethanol).…”

Section: Embryonic Stem Cell Culturementioning

confidence: 99%

“…At this point, the cells could be transplanted or plated for immunocytochemical analysis. We routinely producẽ 60% neurons and less than 40% of glial cells using this protocol (28)(29)(30).…”

Section: Embryonic Stem Cell Culturementioning

confidence: 99%

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Predifferentiated Embryonic Stem Cells Prevent Chronic Pain Behaviors and Restore Sensory Function Following Spinal Cord Injury in Mice

Hendricks

Pak

Owensby

et al. 2006

Mol Med

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Embryonic stem (ES) cells have been investigated in repair of the CNS following neuronal injury and disease; however, the efficacy of these cells in treatment of postinjury pain is far from clear. In this study, we evaluated the therapeutic potential of predifferentiated mouse ES cells to restore sensory deficits following spinal cord injury (SCI) in mice. The pain model used unilateral intraspinal injection of quisqualic acid (QUIS) into the dorsal horn between vertebral levels T13 and L1. Seven days later, 60,000 predifferentiated ES cells or media were transplanted into the site of the lesion. Histological analysis at 7, 14, and 60 days posttransplantation revealed that animals receiving ES cell transplants suffered significantly less tissue damage than animals receiving media alone. Transplanted cells provided immediate effects on both spontaneous and evoked pain behaviors. Treatment with ES cells resulted in 0% (n = 28) excessive grooming behavior versus 60% (18 of 30) in media-treated animals. In the acetone test (to assess thermal allodynia), mice recovered to preinjury levels by 12 days after ES cell transplant, whereas control animals injected with media after SCI did not show any improvement up to 60 days. Similarly, the von Frey test (to assess mechanical allodynia) and the formalin test (to assess nociceptive hyperalgesia) showed that transplantation of predifferentiated ES cells significantly reduced these pain behaviors following injury. Here we show that predifferentiated ES cells act in a neuroprotective manner and provide antinociceptive and therapeutic effects following excitotoxic SCI.

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A new method for generating high purity motoneurons from mouse embryonic stem cells

McCreedy

Brown

Butts

et al. 2014

Biotech & Bioengineering

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A common problem with using embryonic stem (ES) cells as a source for analysis of gene expression, drug toxicity, or functional characterization studies is the heterogeneity that results from many differentiation protocols. The ability to generate large numbers of high purity differentiated cells from pluripotent stem cells could greatly enhance their utility for in vitro characterization studies and transplantation preclinical injury models. Population heterogeneity is particularly troublesome for post-mitotic neurons, including motoneurons, because they do not proliferate and are quickly diluted in culture by proliferative phenotypes, such as glia. Studies of motoneuron biology and disease, in particular amyotrophic lateral sclerosis (ALS), can benefit from high purity motoneuron cultures. In this study, we engineered a transgenic-ES cell line where highly conserved enhancer elements for the motoneuron transcription factor Hb9 were used to drive puromycin N-acetyltransferase (PAC) expression in ES cell-derived motoneurons. Antibiotic selection with puromycin was then used to obtain high purity motoneuron cultures following differentiation of mouse ES cells. Purity was maintained during maturation allowing the production of consistent, uniform populations of cholinergic ES cell-derived motoneurons. Appropriate functional properties of purified motoneurons were verified by acetyl cholinesterase (AChE) activity and electrophysiology. Antibiotic selection, therefore, can provide an inexpensive alternative to current methods for isolating ES cell-derived motoneurons at high purity that does not require specialized laboratory equipment and provides a unique platform for studies in motoneuron development and degeneration.

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Directed differentiation of embryonic stem cells into dorsal interneurons

Cited by 48 publications

References 33 publications

Transplantation of GABAergic neurons but not astrocytes induces recovery of sensorimotor function in the traumatically injured brain

Transplantation of GABAergic neurons but not astrocytes induces recovery of sensorimotor function in the traumatically injured brain

Predifferentiated Embryonic Stem Cells Prevent Chronic Pain Behaviors and Restore Sensory Function Following Spinal Cord Injury in Mice

A new method for generating high purity motoneurons from mouse embryonic stem cells

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