text:
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Tracking Stem Cell Differentiation in the Setting
of Automated Optogenetic Stimulation
ALBRECHT STROH,a,bHSING-CHEN TSAI,aLI-PING WANG,aFENG ZHANG,aJENNY KRESSEL,cALEXANDER ARAVANIS,a
NANDHINI SANTHANAM,aKARL DEISSEROTH,a,dARTHUR KONNERTH,bM. BRET SCHNEIDERa
aDepartment of Bioengineering anddDepartment of Psychiatry and Behavioral Sciences, Stanford University,
Stanford, USA;bInstitute of Neuroscience andcDepartment of Neuroradiology, Technical University Munich,
Munich, Germany
Key Words. Embryonic stem cells•Optogenetics•Channelrhodopsin-2•Neuronal differentiation
Membrane depolarization has been shown to play an
important role in the neural differentiation of stem cells
and in the survival and function of mature neurons. Here,
we introduce a microbial opsin into ESCs and develop
optogenetic technology for stem cell engineering applica-
tions, with an automated system for noninvasive modula-
tion of ESC differentiation employing fast optogenetic
control of ion flux. Mouse ESCs were stably transduced
with channelrhodopsin-2 (ChR2)-yellow fluorescent protein
and purified by fluorescence activated cell sorting (FACS).
Illumination of resulting ChR2-ESCs with pulses of blue
light triggered inward currents. These labeled ESCs
retained the capability to differentiate into functional
mature neurons, assessed by the presence of voltage-gated
sodium currents, action potentials, fast excitatory synaptic
transmission, and expression of mature neuronal proteins
and neuronal morphology. We designed and tested an ap-
paratusforoptically stimulating
chronic neuronal differentiation, with high-speed optical
switching on a custom robotic stage with environmental
chamber for automated stimulation and imaging over
days, with tracking for increased expression of neural and
neuronal markers. These data point to potential uses of
ChR2 technology for chronic and temporally precise non-
invasive optical control of ESCs both in vitro and in vivo,
ranging from noninvasive control of stem cell differentia-
tion to causal assessment of the specific contribution of
transplanted cells to tissue and network function.
ChR2-ESCs during
Disclosure of potential conflicts of interest is found at the end of this article.
ESCs or induced pluripotent stem cells (iPSCs) potentially
could be employed in the study and treatment of central nerv-
ous system (CNS) diseases such as Parkinson's disease [1–6],
stroke [7–9], and spinal cord injury [10–12]. Indeed, ESCs
can give rise to functional neurons capable of integrating into
the host after intracerebral transplantation [13–16], suggesting
that stem cell therapy has the potential to become a therapeu-
tic approach in brain disease if suitably differentiated cells
can be (a) generated [17–23], (b) integrated into native cir-
cuitry [5, 24], and (c) controlled [25–28]. All three of these
key processes are intrinsically activity-dependent [29–32], and
specific control of electrical activity in differentiating neural
cells and their progeny is therefore a central goal in CNS
regenerativemedicine. Channelrhodopsin-2 (ChR2) is
rapidly gated blue light-sensitive cation channel suitable for
noninvasive control of ion flux [33–41]. Here, we explore the
potential of this optogenetic [42] strategy for chronic and
temporally precise noninvasive optical control of electrical
activity in ESCs and their progeny. For this approach, it was
necessary to develop a set of automated high-speed optical
stimulation tools (hardware, software, and cell lines) to nonin-
vasively and specifically modulate ESCs and their progeny
with light.
Mouse ESC Culturing
Mouse ESCs (CRL-1934, ATCC, Manassas, VA, www.atcc.org)
were grown in Dulbecco's Modified Eagle's medium (DMEM)
medium (ATCC) containing medium conditioned by feeder cells
(CRL-1503, ATCC), 15% fetal calf serum (Gibco, Invitrogen,
Carlsbad, CA, www.invitrogen.com), 15 ng/ml leukemia inhibi-
toryfactor(LIF; Sigma-Aldrich,St. Louis,MO,
Author Contributions: A.S.: collection of data, conception and design, data analysis and interpretation, manuscript writing, final approval
of manuscript; H-C.T.: collection of data, data analysis and interpretation, manuscript writing; L.P.W.: collection of data, conception and
design, data analysis and interpretation; F.Z.: collection of data; J.K.: collection of data; A.A.: collection of data, data analysis and
interpretation; N.S.: collection of data; K.D.: conception and design, data analysis and interpretation, manuscript writing; A.K.:
conception and design; M.B.S.: conception and design, data analysis and interpretation, manuscript writing.
Correspondence: Albrecht Stroh, Ph.D., Institute of Neuroscience, Technical University Munich, Biedersteiner Str. 29, Munich 80802,
Germany. Telephone: 49-89-41403519; Fax: 49-89-41403352; e-mail: email@example.com
accepted for publication October 20, 2010; first published online in STEM CELLS EXPRESS November 18, 2010; available online without
subscription through the open access option. V
STEM CELLS 2011;29:78–88 www.StemCells.com
Received May 17, 2010;
C AlphaMed Press 1066-5099/2010/$30.00/0 doi: 10.1002/stem.558
Page 2
www.sigmaaldrich.com), 0.1 mM 2-mercaptoethanol (Sigma-
Aldrich), and 1% penicillin–streptomycin (Sigma-Aldrich). The
cells were cultured in 75-cm2cell culture flasks (Falcon, BD Bio-
sciences, Franklin Lakes, NJ, www.bdbiosciences.com) with 20
ml medium at 37?C and 5% CO2 and passaged every 3 days.
Only undifferentiated cells that could be easily suspended on
brief trypsination were used for the experiments. After washing
in phosphate-buffered saline (PBS; Gibco, Invitrogen), cells were
counted in a Neubauer counting chamber. The viability was
determined by staining with trypan blue solution (0.4%; Sigma-
Transduction of ESCs with ChR2
Lentiviruses carrying the ChR2-enhanced yellow fluorescent pro-
tein (EYFP) fusion gene under the control of the elongation fac-
tor 1-a (EF1a) promoter were generated as previously described
[35]. Viruses were concentrated via ultracentrifugation and redis-
solved in PBS at 1/1,000 of the original volume. The concen-
trated viruses were then incubated with ESCs for 24 hours and
transduction efficiency evaluated using fluorescent microscopy 1
week after transduction. To obtain a highly and homogenously
expressing ChR2-ESC colony, cells were sorted using fluores-
cence activated cell sorting (FACS); a subpopulation consisting
of the top 5% of yellow fluorescent protein (YFP)-expressing
cells was collected. A subpopulation of ChR2-ESCs was rein-
fected with lentiviruses on neuronal differentiation, to test
whether subsequent transduction affects viability and gene
expression. Transduction of ESC-derived neurons might be of im-
portance in future studies requiring further increase of ChR2-
expression or in cell populations, which cannot be sorted.
Neuronal Differentiation of ESCs
Neuronal differentiation was performed as previously described
[17, 22]. For optical stimulation, ESCs were plated on Matrigel-
coated dishes in embryoid body stage in complete ESC medium
(see above). After 24 hours, medium was changed to ESC me-
dium lacking LIF and including 0–5 lM retinoic acid (RA) and
changed every second day for 5 days.
Immunohistochemical Staining of Cultured Cells
Cells were fixed with 4% paraformaldehyde in PBS for 30
minutes at room temperature. Fixation was stopped by washing
cells three times with 0.1 M glycine/PBS. Cells were permeabil-
ized and blocked (4% bovine serum albumin (BSA)/0.4% sapo-
nin/PBS) for 30 minutes and incubated in primary antibody solu-
tion at 4?C overnight. Cells were washed four times and
incubated with secondary antibody at room temperature for 2
hours. Cells were washed three times with PBS, and at the final
washing step 40,6-diamidino-2-phenylindole (DAPI) was added
(1:50,000). Coverslips were mounted using anti-quenching Fluo-
romount. Primary antibodies were mouse anti-stage-specific em-
bryonic antigen-1 (SSEA1) (Chemicon, Millipore, Billerica, MA,
www.millipore.com; 1:300), mouse anti-nestin (Chemicon 1:200),
chicken anti-bIII tubulin (Chemicon 1:200), mouse anti-microtu-
bule-associated protein 2 (MAP2) antibodies (Sigma 1:500), rab-
bit anti-vGlut 2 (Chemicon 1:200), and rabbit anti-a 1C, rabbit
anti-a 1D, rabbit anti-a 1G, and rabbit anti-a 1H (all Alomone
Labs, Jerusalem, Israel, www.alomone.com; 1:200). Cy3- or Cy5-
conjugated donkey anti-mouse, -chicken and -rabbit secondary
antibodies (Jackson, West Grove, PA, www.jacksonimmuno.com)
were all used at 1:200.
Reverse Transcription Polymerase Chain Reaction
Cells were homogenized by homogenizer (Invitrogen). RNA iso-
lation was performed using Micro-to-Midi Total RNA Purification
System (Invitrogen). Prior to reverse transcription polymerase
chain reaction (RT-PCR), RNA samples were pretreated with
DNaseI (Invitrogen) and reverse transcription conducted per man-
ufacturer's protocol. Negative controls without reverse transcrip-
tase did not result in amplified sequences. Mouse hippocampal
total RNA was purchased from Clontech Laboratories (Mountain
View, CA, www.clontech.com) and the resulting cDNA served as
a positive control. For PCR analysis, primers targeted to coding
regions of two subunits each from both the L- and T-type volt-
age-gated Ca2þchannel (VGCC) families were used, they are as
follows (see Table 1 for primer sequences): L-type a1C; L-type
a1D; T-type a1G; T-type a1H; housekeeping gene (Actin). PCR
products of actin and L-type and T-type subunits were cloned
and sequenced to confirm identity. For analysis of stemness and
neural markers, total RNA was extracted from cells using Mini
RNeasy kit (Qiagen, Valencia, CA, www.qiagen.com). Primers
targeted to the coding regions of following markers were used
(see Table 1 for primer sequences): Pluripotency marker Oct4,
Sox2, Nestin, neuronal cell adhesion molecule 1, MAP, and
housekeeping gene GAPDH.
Long-Term Optical Stimulation of ESCs
Key components of the hardware interface include (a) Oasis4i
Controller (Objective Imaging, Cambridge, United Kingdom;
hardware for x-y-z 3-axis and focus control; http://www.objecti-
veimaging.com/Download/OI_Download.htm; software develop-
ment kit [SDK] for the Oasis4i Controller), (b) DG-4 Ultra High-
Speed wavelength switcher (Sutter, Novato, CA, www.sutter.
com), (c) Retiga SRV Camera (QImaging, Surrey, BC, Canada,
www.qimaging.com), and (d) Leica DM6000 Microscope con-
trolled by Abstract Hardware Model controller. The parallel port
is controlled using DLPORTIO library file (www.driverlinx.com/
DownLoad/DlPortIO.htm; Dlls for parallel port control) and cam-
era parameters (gain, exposure) set using QCam SDK (Ver.
Five.1.1.14; http://www.qimaging.com/support/downloads/; SDK
to control the Retiga SRV/ Exi Cameras). The custom software
user interface to the optogenetic stimulation setup was developed
using the Microsoft Foundation Library (MFC; Ver. Eight.0) and
is available on request. Briefly, regions of interest (ROI; for
example, an embryoid body or a small well in a multiwell plate)
to be stimulated and/or imaged are selected using the Oasis4i
Controller, and their locations saved using the MFC interface.
Table 1. Primer sequences
GenePrimer forwardPrimer reverse
L-type a1C
L-type a1D
T-type a1G
T-type a1H
Abbreviations: MAP, microtubule-associated protein; NCAM1, neuronal cell adhesion molecule 1; GAPDH, glyceraldehyde-3-phosphate
Stroh, Tsai, Wang et al.
Page 3
Stimulation parameters (excitation filter wavelength, the duration
of the excitatory pulse, and the frequency and duty cycle of exci-
tation) are then set in the custom graphical user interface (GUI).
To allow stimulation space to be mapped, each region of interest
can be readily programmed to receive a different stimulus pattern
to operate over the many days of stimulation and imaging. Simi-
larly, imaging parameters can also be varied for selected regions,
including number of images per region and exposure, gain, exci-
tation, and emission filters.
Undifferentiated cells were seeded on Matrigel (BD)-coated
coverslips in 24-well plates in complete ESC medium at a density
of 100,000 cells/well. Both native ESCs and ChR2-expressing
ESCs were used in different wells on the same plate. Twenty-
four hours after seeding, medium was changed to the various ex-
perimental conditions including complete ESC medium, ESC me-
dium lacking both LIF and conditioned media from feeder cells
(differentiation medium), differentiation medium with 1 lM RA
(Sigma), and differentiation medium with 2.5 lM RA. Optical
stimulation was conducted using the described custom hardware
and software (Fig. 4). Up to 30 ROIs were defined per well,
ensuring that all cell-containing regions on the coverslip were
stimulated. ROIs were illuminated every hour around the clock
over 5 days with blue light (470 nm) pulsing at 15 Hz for 10 sec-
onds, using a ?10 objective (NA 0.3). Every 8 hours, a photomi-
crograph was programmed to be taken of the selected ROIs. At
the end of the experiment, coverslips were removed from the
plates and immediately fixed with paraformaldehyde in PBS and
stained as described earlier. Mounted slides were labeled with
coded numbers by a colleague so that the investigators conduct-
ing confocal analysis were blind to treatment condition.
Confocal Microscopy and Image Analysis
Confocal imaging was conducted using the Leica SP2 (Leica,
Wetzlar, Germany,www.leica-microsystems.com)
microscope with a ?40 oil objective (NA 0.75) and an Olympus
FV1000 (Olympus, Center Valley, PA, www.olympusfluoview.
com)for analysis of neuronal differentiation, with a ?60 oil
objective (NA 1.42). For DAPI excitation, a 402-nm diode laser
was used; Cy5-nestin was excited using a 633 nm HeNe laser.
Six ROIs were randomly and blindly selected for analysis per
coverslip, and 1,024 ? 1,024 8-bit confocal images were
obtained. For each ROI, a z-stack with 8–12 x-y-sections and a z-
step size of 0.98 lm were collected, thereby including all cells
present in the ROI. Data analysis was conducted using ImageJ
(NIH) software, and after unblinding, confocal images of all
ROIs of all coverslips of each condition (e.g., ChR2-ESCs, opti-
cally stimulated, 2.5 lM RA) were converted into a single z-
stack. Fluorescence intensity histograms were calculated [43] for
DAPI and nestin channels. DAPI histograms reflecting the cell
numbers allowed for a normalization of nestin histograms. All
nestin voxel numbers have been divided by this DAPI factor. For
analysis of neuronal differentiation, Cy3-b3-tubulin was excited
using a 559-nm laser. Again, six ROIs were randomly and blindly
selected for analysis per coverslip, and 1,024 ? 1,024 8-bit con-
focal images were obtained. b3-Tubulin-expressing cells consti-
tuted a monolayer, individual cells could be easily discriminated.
Therefore, a z-stack with three x-y-sections and a z-step size of
1.4 lm was collected in each ROI, covering all b3-tubulin-
expressing cells. The Cy3-channel was merged with the DAPI
channel using ImageJ software. b3-Positive cells were counted
manually for all ROIs. After unblinding, cell counts from each
condition were normalized by the cell count from the ''native
ESC with 2.5 lM RA w/o stimulation'' condition. Statistical anal-
www.spss.com) software. To statistically compare histograms, the
employed, and to compare means, statistical significance was cal-
culated using the t test.
(K-S)test [44]was
Stereotactic Cell Transplantation
Rats (male Wistars, 250–350 g) were the subjects of these experi-
ments. Animal husbandry and all aspects of experimental manipu-
lation of our animals were in strict accord with guidelines from
the National Institute of Health and approved by members of the
Stanford Institutional Animal Care and Use Committee. Rats
were anesthetized by i.p. injection (90 mg ketamine and 5 mg
xylazine per kg of rat body weight). For cell transplantation, a 1-
mm craniotomy was drilled over motor cortex. One microliter of
ESCs-expressing ChR2-EYFP fusion protein at a density of 50 ?
103cells per microliter suspended in PBS was injected (26 g
Hamilton syringe) into rat motor cortex (anterior-posterior, þ 1.5
mm; medial-lateral, þ 1.5 mm; dorsal-ventral, ? 1.5 mm). The
injection duration was 10 minutes; an additional 10-minute delay
followed before syringe withdrawal, and electrophysiology was
conducted after 1 week.
For acute slice electrophysiological experiments, 1 week postcell
transplantation, 250-lm cortical slices were prepared in ice-cold
cutting buffer (64 mM NaCl, 25 mM NaHCO3, 10 mM glucose,
120 mM sucrose, 2.5 mM KCl, 1.25 mM NaH2PO4, 0.5 mM
CaCl2, and 7 mM MgCl2, equilibrated with 95% O2/5% CO2)
using a vibratome (VT 1,000 S; Leica). After a recovery period
of 30 minutes in cutting buffer at 32–35?C, slices were gently
removed to a recording chamber mounted on an upright micro-
scope (DM LFSA, Leica) and continuously perfused at a rate of
3–5 ml/minute with carbonated artificial cerebrospinal fluid
(ACSF) (124 mM NaCl, 3 mM KCl, 26 mM NaHCO3, 1.25 mM
NaH2PO4, 2.4 mM CaCl2, 1.3 mM MgCl2, 10 mM glucose) ven-
tilated with 95% O2/5% CO2. ChR2-YFP-ESCs were identified
on an upright fluorescence microscope (DM LFSA, Leica) with a
?20, 0.5 NA water immersion objective and a YFP filter set.
Images were recorded with a CCD camera (Retiga Exi, Qimag-
ing) by Qimaging software. Electrophysiological recordings in
cultured ChR2-YFP ESCs were
described [35, 39], in Tyrode solution containing (in mM): NaCl
125, KCl 2, CaCl23, MgCl21, glucose 30, and HEPES 25 (pH
7.3 with NaOH). Membrane currents were measured with the
patch-clamp technique in whole-cell mode [45] using Axon Mul-
ticlamp 700B (Axon Instruments, Molecular Devices, Sunnyvale,
CA, www.moleculardevices.com) amplifiers. Pipette solution con-
sisted of: 97 mM potassium gluconate, 38 mM KCl, 6 mM NaCl,
0.35 mM sodium ATP, 4 mM magnesium ATP, 0.35 mM EGTA,
7 mM phosphocreatine, and 20 mM HEPES (pH 7.25 with
KOH). Pipette resistance was 4–8 MX. Membrane potential was
noted at the time of establishing the whole-cell configuration. We
employed pClamp nine acquisition software (Axon Instruments),
a DG-4 high-speed optical switch with 300 W xenon lamp (Sutter
Instruments) and a GFP filter set (excitation filter HQ470/40x,
dichroic Q495LP; Chroma, Bellows Falls, VT, www.chroma.com)
to deliver blue light for ChR2 activation. Through a ?20 objec-
tive lens, power density of the blue light was 8–12 mW/mm2,
measured by power meter (Newport, Irvine, CA, www.newport.
com). All experiments were performed at room temperature (22–
Functional Expression of ChR2 in ESCs
To assess the potential of optogenetics in stem cells, mouse
ESCs were transduced with a lentiviral ChR2-YFP-construct
under the control of the EF1a promoter [46]; after sorting for
the top 5% based on YFP fluorescence intensity (Fig. 1A), we
found that the population doubling time and vitality of the
resulting ChR2-YFP-ESCs did not differ significantly com-
pared with nontransduced ESCs (supporting information Fig.
Tracking Stem Cell Differentiation
Page 4
1), and confocal microscopy demonstrated membrane local-
ization of ChR2-YFP with high, uniform expression levels in
the ESC population (Fig. 1B). ChR2-ESCs continued to
express the ESC markers Oct4 and Sox2 as measured by RT-
PCR (Fig. 1C) and SSEA1 (supporting information Fig. 1),
maintaining the undifferentiated state as in nontransduced
control cells. Electrophysiologically, the ChR2-ESCs dis-
played typical outwardly rectifying and passive currents,
whereas illumination with blue light (470 nm, 500-ms pulse
duration) evoked inward photocurrents (Fig. 1D, 1E); steady-
state photocurrents showed little inactivation, whereas peak
photocurrents showed inactivation and recovery with kinetics
similar to that previously shown in neurons [35] (Fig. 1F).
To test whether transplanted ChR2-ESCs could still
respond to optical stimulation, 5 ? 105ChR2-YFP-expressing
ESCs were stereotaxically injected into the cortex of healthy
rats. One week after transplantation, animals were sacrificed
and in acute slices, transplanted cells could be identified by
YFP fluorescence (Fig. 1G, 1H). Patch clamp recordings were
conducted, revealing inward currents on illumination with
blue light (Fig. 1I) that displayed typical inactivation of the
peak current and stability of the steady-state current.
ChR2-ESCs Express VGCCs
Intracellular Ca2þis a major mediator of differentiation and
survival in stem cells and their progeny, especially in neural
lineages [29]. ChR2 itself is a nonselective cation channel
that directly allows Ca2þentry into cells [47, 48]. Additional
routes of photo-evoked Ca2þentry could include activation of
VGCCs by virtue of ChR2-induced membrane voltage
Figure 1.
ChR2-ESCs before sorting (two-dimensional dot plot, SSC versus green fluorescence [FITC]). A significant variation of YFP-expression in cell popu-
lation becomes apparent. A gate enclosing 5% of cells with the strongest fluorescence was applied for sorting. (B): Confocal micrograph of ChR2-
ESCs revealing membrane-localized expression of ChR2-YFP. Vitality (assessed by trypan blue exclusion), morphology, and population doubling
times of ChR2-YFP ESC were indistinguishable from native cells. Scale bar ¼ 50 lm. (C): Reverse transcription polymerase chain reaction analysis
of pluripotency marker expression of both ChR2-ESCs and native ESCs revealed strong expression of Oct4 and Sox2, compared with mouse hippo-
campal cells HT22 (negative control). The housekeeping gene GAPDH was used as loading control. (D): Pulsed light (473 nm) evoked inward cur-
rents in a ChR2–YFP ESC in voltage clamp (light pulse indicated by blue bar). (E): Summary data on evoked photocurrents (mean 6 SEM, n ¼ 15
cells). (F): Kinetics of deinactivation of the peak ChR2 current in ESCs. Ten overlaid photocurrent traces obtained in voltage clamp are shown; pairs
of 0.5-second light pulses (indicated by blue bars) were separated by increasing intervals from 1 to 10 seconds; traces are aligned to the initiation of
the first pulse in each sweep. Note that steady-state current is constant, whereas the peak current shows inactivating behavior. (G–I): Light evoked
currents in transplanted ChR2-YFP ESCs in rat cortical brain tissue. (G): YFP fluorescence of ChR2-YFP-ESCs 7 days after transplantation observed
in an acute cortical slice. Scale bar ¼ 500 lm. (H): Higher magnification reveals cellular morphology and membrane localization of ChR2-YFP.
Scale bar ¼ 80 lm. (I): Electrophysiological recordings in transplanted ChR2-ESC in acute brain slice revealed blue light-evoked inward photocur-
rents. As in culture, the steady-state current is stable and the peak current displays inactivation. Abbreviations: ChR2, channelrhodopsin-2; YFP, yel-
low fluorescent protein; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; SSC, sideward scattering.
Functional expression of ChR2 in ESCs in vitro and in brain slices after transplantation in vivo. (A): FACS analysis of a sample of 105
Stroh, Tsai, Wang et al.
Page 5
changes. Notably, we found that mouse ESCs express four
major VGCCs assessed by RT-PCR and immunoreactivity
(Fig. 2A), and this supplementary mechanism for photoacti-
vated Ca2þentry could become increasingly potent as cells
proceed down the neuronal lineage and develop hyperpolar-
ized membrane potentials. Moreover, the known Ca2þflux of
ChR2 itself suggested the potential for optical control of stem
cell processes.
ChR2-ESCs Differentiate into Functional
Excitatory Neurons
We first verified that ChR2-ESCs were capable of neural line-
age differentiation, using a RA-based neural differentiation
protocol. RT-PCR revealed gene expression of neural and
neuronal markers, complemented with immunofluorescent
stainings for neuronal cytoskeletal proteins b3-tubulin and
MAP2 (Fig. 2B). Reinfection with EF1a-ChR2 lentivirus did
not alter gene expression profile. By day 28, the resulting
ChR2-ESC-derived neurons displayed strong ChR2-expression
(Fig. 2B) and mature neuronal morphology. Generated neu-
rons were expressing the vesicular glutamate transporter II
(Fig. 3A). Electrophysiologically, cells displayed sodium
currents, action potentials, and excitatory postsynaptic cur-
rents, which could be blocked by excitatory synaptic trans-
missionglutamate receptor
quinoxaline-2,3-dione (CNQX) and D-2-amino-5-phosphono-
pentanoate (D-AP5) (Fig. 3B–3D).
Automated Optical Stimulation
One challenge in deriving replacement tissues from ESCs is
that differentiation takes place over many days and therefore,
also the patterning and differentiation stimuli. Hence, to be
applicable, optogenetic stimulation must be deliverable in
chronic fashion. In designing the system to meet this chal-
lenge, it is also important to consider that as knowledge of
the precise combinations and timing of signaling events
required for stem cell differentiation is limited, a multiwell
configuration would in principle be desirable, to allow for fast
optical mapping of cell lines, conditions, and ''differentiation
space'' in the laboratory. We therefore devised an automated
multiwell optogenetic stimulation approach designed to pre-
cisely revisit and optically stimulate multiple ROIs in defined
patterns over extended periods of time (Fig. 4A).
ROIs in multiwell plates were user-defined in a custom
GUI and their locations saved for rapid and reproducible
access by a robotic stage. Stimulation parameters (excitation
Figure 2.
known Ca2þpermeability of ChR2 itself, reverse transcription polymerase chain reaction analysis revealed multiple voltage-gated Ca2þ-channels in
ESCs and ChR2-ESCs. The transcript for the L-type voltage-gated Ca2þchannel a 1C subunit is present in HPC RNA, ESCs, and ChR2-ESCs but not in
HEK 293 cells (left). This transcriptional pattern was also observed for the L-type voltage-gated Ca2þ-channel a 1D subunit and the T-type voltage-gated
Ca2þchannel subunits a 1G and a 1H (right). All cells show strong expression of housekeeping gene b-actin. Immunocytochemical detection of mem-
brane-localized Ca2þ-channel subunit expression in ChR2-ESCs (L-type Ca2þ-channel subunit a 1C and T-type Ca2þ-channel subunit a 1G; [right]).
Cellular nuclei were visualized by DAPI staining. Scale bar ¼ 25 lm. (B): Transcriptional analysis of neural lineage markers of differentiated ChR2-
ESCs at day 22. ChR2-ESCs were compared with cells reinfected with EF1a-ChR2 lentivirus at neural stage (day 15) and mouse hippocampal HT22
cells. Transcripts of neural markers nestin were present in ChR2-ESCs and in reinfected ChR2-ESCs, in contrast to HT 22 cells. NCAM1 transcript could
be detected in all three cell populations, MAP2 is only strongly transcribed in both ESC populations. GAPDH was used as loading control. Immunocyto-
chemistry for stage-specific markers (right). After 20 days, cells adapt bipolar morphology, a dense network of b3-tubulin-expressing cells can be
observed indicating progression down the neuronal lineage. Scale bar ¼ 50 lm. After 30 days, cells express the mature neuronal marker MAP2. Scale
bar ¼ 100 lm. Reinfected ChR2-ESC-derived neurons display strong expression of ChR2. Scale bar ¼ 10 lm. Abbreviations: ChR2, channelrhodopsin-
2; DAPI, 40,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HEK, human embryonic kidney; HPC, hippocampus;
MAP2, microtubule-associated protein 2; NCAM1, neuronal cell adhesion molecule 1; YFP, yellow fluorescent protein.
ChR2-ESCs express multiple potential routes of evoked Ca2þentry and can differentiate into excitatory neurons. (A): In addition to the
Tracking Stem Cell Differentiation
Page 6
filter wavelength, optical switch pulse duration, and fre-
quency/duty cycle of excitation) were set in the custom soft-
ware, which also controls the microscopic stage in all three
spatial dimensions, and controls operation of the DG-4 optical
switch, which employs spinning galvanometers to deliver light
with submillisecond precision. The microscope itself is sur-
rounded by a climate-controlled Plexiglas chamber wherein
both temperature and CO2-level are tightly regulated and tem-
porally precise imaging can proceed in parallel with optical
stimulation (Fig. 4B). ESCs can be cultured and photostimu-
lated in this environment rather than in a standard incubator
for many weeks, allowing us to investigate the effect of
optogenetic stimulation on the differentiation of ESCs in a
controlled, reproducible manner.
Optogenetic Modulation of ESC Neural
and Neuronal Differentiation
Using a multiwell setup, differentiation space could be effi-
ciently mapped while controlling for nonspecific effects
related to the rig or to illumination. Cells were stimulated for
5 days with blue light (470 nm at 15 Hz for 10 seconds)
delivered every 60 minutes using a ?10 objective. The sur-
vival and morphology of the cells were monitored using time-
lapse imaging every 8 hours. Cells incubated without RA and
with 2.5 lM RA showed no decrease in cellular viability,
however, incubation with 5 lM RA resulted in significant
reduction of cell viability of both ChR2-ESCs as well as
native ESCs. Consequently, cells incubated with 5 lM RA
were not used for subsequent analysis.
To identify rapidly acting effects of optical stimulation on
ESC differentiation, cells were simultaneously assayed fol-
lowing the conclusion of stimulation. Immunostaining for the
neural marker nestin and for the neuronal marker b3-tubulin
followed by analysis of fluorescence histograms and direct
quantification of neuronal cells obtained by confocal micro-
scopy was used to quantify neural and neuronal lineage dif-
ferentiation, along with imaging of cellular nuclei using
DAPI. Figure 5A and 5B shows a three-dimensional projec-
tion of two typical confocal z-stacks of single ROIs, display-
ing both DAPI (blue) and nestin (red) fluorescence. Optically
stimulated cells consistently showed higher nestin immunore-
activity (Fig. 5B) compared with nonstimulated cells (Fig.
5A), whereas optical stimulation interestingly was ineffective
in the absence of RA (Fig. 5C). To quantify this effect, we
generated fluorescence intensity histograms of all ROIs across
all wells in each condition (resulting in more than 150 confo-
cal images per condition). These intensity histograms revealed
considerable differences between stimulated and nonstimu-
lated ChR2-ESCs (Fig. 5F; p < .01, Kolmogorov-Smirnov
test [44]). We next conducted an experiment to test the possi-
bility that the nestin distributions of unmodified (''native'')
optically stimulated ESCs (Fig. 5D) and ChR2-YFP optically
stimulated ESCs (Fig. 5F) could represent samples from the
repeated as in the earlier experiment and subsequent blinded
analysis, we found that this hypothesis could be rejected (p <
.001; two-tailed K-S Z ¼ 5.43); Figure 5F and 5G shows the
observed increase in high levels of optically induced nestin
expression in the ChR2-YFP cells. We calculated the mean
nestin fluorescence intensity in each condition, and comparing
optically stimulated with nonoptically stimulated cells across
all conditions revealed that only ChR2-YFP ESCs incubated
with 2.5 lM RA showed a significant optogenetically induced
increase in mean nestin expression (p < .01, two-tailed t test;
Fig. 5G). In the presence of 1 lM RA, a nonsignificant trend
toward higher nestin expression in the setting of optical stim-
ulation was observed, whereas in 0 lM RA, no effect of opti-
cal stimulation was observed (e.g., Fig. 5C). To exclude the
hypothesis that optogenetic stimulation only modulates neu-
ral, but not neuronal differentiation, we conducted an inde-
pendent quantification of the early neuronal marker b3-tubu-
lin. Blinded analysis of confocal images after staining with
b3-tubulin allowed for a direct quantification of b3-tubulin-
positive cells (Fig. 5H, 5I). Neuronal, b3-tubulin-expressing
cells could be easily distinguished from non-neuronal cells.
optical stimulation,
Figure 3.
mature dendritic morphology and vGlutII expression. Scale bar ¼ 30 lm. (B): Active membrane currents were observed in ChR2-ESC-derived neurons.
After establishing the whole-cell configuration, voltage steps were applied from a holding potential of ?80 mV (in 10 mV steps up to 20 mV); fast volt-
age-activated inward currents were evoked. (C): Corresponding to the fast active inward currents, action potentials could be recorded in current clamp in
response to current injection (150 pA). (D): Spontaneous excitatory synaptic activity was recorded (left panel), abolished with coapplication of the exci-
tatory glutamate receptor antagonists CNQX (10 lM) and D-AP5 (25 lM; middle panel), and restored after drug washout (right panel). Abbreviations:
CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; D-AP5, D-2-amino-5-phosphonopentanoate; DAPI, 40,6-diamidino-2-phenylindole.
Electrophysiological maturity of ChR2-ESC-derived neurons. (A): ChR2-ESC-derived neurons after 36 days of differentiation, displaying
Stroh, Tsai, Wang et al.
Page 7
For normalization, the number of b3-tubulin-positive cells of
the experimental condition native ESCs, without stimulation,
with 2.5 lM RA was defined as 100%. Across all stimulation
conditions, no or only single b3-tubulin-expressing cells
could be detected without the presence of RA. Incubation of
native ESCs with 1 lM RA lead to about 30% b3-tubulin-
positive cells, with or without optical stimulation. A nonsigni-
ficant increase of neuronal differentiation became apparent
when comparing optically stimulated and nonstimulated
ChR2-ESCs. At 2.5 lM RA, again no increase in neuronal
cells in response to optical stimulation could be observed
(Fig. 5J) in native ESCs. In contrast, optically stimulating
ChR2 ESCs in presence of 2.5 lm RA led to a significant
increase of neuronal cells (Fig. 5K).
Depolarization has been reported in other studies to modulate
neural differentiation processes in dividing cells [49–52], and
depolarization and calcium waves have both been observed in
proliferating CNS progenitors in situ [30, 53]. The specific
signal transduction cascades mediating the influence of mem-
brane depolarization events in early development remains
unclear, but Ca2þand Ca2þchannels may play a key role and
ChR2 is well suited to recruit these mechanisms; indeed,
emerging evidence points to the expression of VGCCs during
early stages of embryonic development [54, 55], which may
allow ChR2 to recruit Ca2þ-dependent cellular processes not
Figure 4.
well plate-containing cells is placed on the stage of a fluorescence microscope rig with robotic stage, millisecond scale optical switching, autofo-
cus, and environmental control of temperature and CO2. Custom software controls all functions of the stage and microscope as well as the CCD
camera and the light source (methods). (B): Photomicrographs obtained by automated setup of identical regions of interest every 8 hours. Time
course of differentiation and survival of mESCs on incubation with 2.5 lM retinoic acid can be monitored. Scale bar ¼ 300 lm.
Spatiotemporally precise long-term optogenetic control of ESCs. (A): Schematic of automated optical stimulation setup. The multi-
Tracking Stem Cell Differentiation
Page 8
only via its own light-activated Ca2þflux but also by activat-
ing native VGCCs as differentiating cells mature. Krtolica
et al. [56] reported enhancement of hematoendothelial differ-
entiation on chronic depolarization of human ESCs. In all of
these cases, as we observed with the RA gating of optogenetic
modulation, depolarization or Ca2þinflux will likely depend
on other patterning and lineage-specific differentiation factors,
and more studies are clearly needed to investigate the precise
temporal patterns and combinatorial interactions of Ca2þwith
other signaling messengers.
Studies have shown the induction of iPSCs from somatic
cells [57–61], significantly expanding the possible sources of
stem cells in regenerative medicine but further highlighting
the ongoing need for selective and highly sensitive stem cell
differentiation and control tools. Globally applied stimuli such
as growth factors and organic compounds will affect all cells
Figure 5.
(B, C) or without (A) optical stimulation. (A, B): Cells were incubated with 2.5 lM retinoic acid; as a negative control, in (C) no neural lineage-
inducing agents were added. Cells were stained for cellular nuclei (DAPI; blue fluorescence) and the neural marker nestin (red fluorescence). The
3D reconstruction consists of 10 x-y sections, z-step size is 0.98 lm. Scale bar ¼ 50 lm. (D–G): Summary of blinded experiments comparing
response with optical stimulation of native ESCs (D, E) and ChR2-ESCs (F, G). (D, F): Histograms of nestin fluorescence intensities of optically
stimulated versus nonstimulated cells. All individual x-y sections of all embryoid body-containing regions of interests (ROIs) in each condition
were used to calculate the intensity histograms (8-bit images gave rise to 256 intensity bins). (D): Optical stimulation of native ESCs did not sig-
nificantly affect nestin expression. (F): Increased nestin expression in the optically driven ES cells; histograms were normalized revealing an opti-
cally driven increased fraction of nestin-positive voxels, as evaluated by nonparametric statistical testing. DAPI histograms reflecting the number
of cells did not reveal any significant changes on light stimulation, no proliferative effect could be observed. (E, G): Summary data of mean nes-
tin-positive voxels per embryoid body-containing ROI (6SEM, n ¼ 10 ROIs, two independent experiments). (E): Optical stimulation of native
ESCs did not significantly affect nestin expression. (G): Optically stimulated ChR2-ESCs gave rise to significantly increased nestin expression in
the presence of 2.5 lM RA (two tailed t test, p ¼ .03). (H, I): Confocal images of ChR2-ESCs with (I) or without (H) optical stimulation. Cells
were incubated with 2.5 lM retinoic acid. Cells were stained for cellular nuclei (DAPI; blue fluorescence) and the neuronal marker b3-tubulin
(red fluorescence). Scale bar ¼ 30 lm. (J, K): Normalized percentage of b3-tubulin-expressing cells incubated with 2.5 lM RA, two independent
experiments per condition, mean 6 SEM. (J): No significant change in cell numbers on optically stimulated native ESCs can be detected. (K): In
contrast, stimulation of ChR2-ESCs results in a significant increase (p ¼ .01, two tailed t test) of the number of b3-tubulin-expressing cells of
about 25%. Abbreviations: ChR2, channelrhodopsin-2; DAPI, 40,6-diamidino-2-phenylindole; RA, retinoic acid.
Optogenetic modulation of ESC neural differentiation. (A–C): Three-dimensional (3D) image stacks of ChR2-ESCs after 5 days with
Stroh, Tsai, Wang et al.
Page 9
present, including nondividing constituents of the stem cell
niche as well as the stem cells and their progeny, but it is
unlikely that these growth factors will have the same desired
effect in all of the very different cells present in the typical
differentiation milieu. By targeting optical control to either
the proliferating cells or to niche constituents like astrocytes,
optogenetic control of intracellular signaling will allow selec-
tive control of the desired cell type [41]. Future studies may
further capitalize on the genetic targeting of optical control to
selectively drive differentiating cells within complex multiple
cell-type environments.
Indeed, this optical specificity principle extends to the selec-
tive control of fully differentiated stem cell progeny in situ.
Minimally invasive fiberoptic strategies [33, 34, 37] have
brought optogenetics to the fully intact, behaving mammal.
Transplanted cells may require electrical activity to drive the
final stages of phenotype consolidation and to fully integrate
into host neural circuitry, representing the central goal of
stem cell-based regeneration medicine. Yet the lack of precise
tools to drive only the transplanted cells has hindered progress
on this front. Compared with conventional electric stimulation
or drugs, the genetic targeting of ChR2 makes it possible to
specifically and reversibly drive precise amounts of activity in
the transplanted ESCs and their progeny, which moreover do
not require addition of chemical cofactors in vivo for ChR2
function. Finally, optically driving only the transplanted cells,
with behavioral readouts or noninvasive imaging readout
modalities like functional magnetic resonance imaging (fMRI)
(and without the serious problem of signal interference from
metal electrodes), opens the door to imaging and tuning the
specific contribution of transplanted cells in the restoration of
network activity and circuit dynamics, for example, in Parkin-
son's disease [62]. With these approaches and others, optoge-
netic technologies have the potential to become valuable tools
in stem cell biology and regenerative medicine.
We thank B. Cord and K. Schrenk-Siemens for assistance with
ESC culture, K. Lee for assistance with confocal imaging, W.
Beisker for assistance with FACS analysis, and C. Zimmer for
support. This work was supported by CIRM, NSF, NIDA,
NIMH, and the NIH Director's Pioneer Award, as well as by the
Snyder, Kinetics, Culpeper, Coulter, Klingenstein, Whitehall,
McKnight, Albert Yu and Mary Bechmann Foundations (K.D.),
CIRM (L.P.W.), the Bavarian State Ministry of Sciences,
Research and the Arts (''ForNeuroCell''; A.S. and J.K.), and the
Stanford Graduate Fellowship (H.-C.T.). A.S., H.-C.T., and
L.P.W.contributedequally to this article.
The authors indicate no potential conflicts of interest.
Figure 5.
Tracking Stem Cell Differentiation
Page 10
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Tracking Stem Cell Differentiation


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Florida Couple Sentenced for Rolesin Procurement Contract Bribery Scheme
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A Florida man was sentenced to serve 15 months in prison, and his wife was sentenced to 24 months of probation, for their roles in a bribery and fraud scheme involving federal procurement contracts, announced Acting Assistant Attorney General Mythili Raman of the Justice Department's Criminal Division and U.S. Attorney David B. Barlow of the District of Utah.
On Feb. 26, 2013, Sylvester Zugrav, 70, of Sarasota, Fla., pleaded guilty to conspiracy to commit bribery and procurement fraud, and his wife, Maria Zugrav, 67, also of Sarasota, pleaded guilty to misprision of a felony related to her efforts to conceal the conspiracy.
The Zugravs were charged in an October 2011 indictment along with Jose Mendez, 51, of Farr West, Utah.
Mendez, a procurement program manager for the U.S. Air Force Foreign Materials Acquisition Support Office (FMASO) at Hill Air Force Base, in Ogden, Utah, was charged in the indictment with conspiracy, bribery and procurement fraud, and has since pleaded guilty to all charges and agreed to forfeit more than $180,000 he received as part of the bribery scheme.
Sentencing for Mendez is scheduled for Jan. 29, 2014.
According to court documents, the Zugravs owned Atlas International Trading Company, a business that contracted to provide foreign military materials to the U.S. government through FMASO.
In his plea agreement, Sylvester Zugrav admitted that, from 2008 through August 2011, he gave Mendez more than $180,000 in bribe payments and offered Mendez more than $1 million in additional bribe payments contingent upon Atlas's receipt of future contracts with FMASO.
In exchange for Sylvester Zugrav's bribe payments and offers, Mendez ensured that Atlas and Sylvester Zugrav received favorable treatment in connection with procurement contracts by, among other things, assisting Atlas in obtaining and maintaining procurement contracts; assisting Atlas in receiving payments on such contracts; and providing Atlas with contract bid or proposal information or source selection information before the award of procurement contracts.
In her plea agreement, Maria Zugrav admitted that she was aware of Sylvester Zugrav's bribe payments to Mendez and assisted with concealing the crime.
According to court records, Sylvester Zugrav provided bribe payments to Mendez in three ways: cash payments via Federal Express to Mendez's residential address; in-person payments of cash and other things of value; and electronic wire transfers to a bank account in Mexico opened by and in the name of Mendez's cousin.
Between November 2009 and August 2011, Sylvester Zugrav sent nine FedEx packages to Mendez's home address.
Each package contained $5,000 in cash, except the last package, which contained $3,000 and was seized by law enforcement.
Maria Zugrav assisted her husband and Mendez's bribe scheme by limiting cash withdrawals from Atlas's bank account to not more than $5,000 to avoid scrutiny by banking officials and law enforcement.
According to the plea documents, on multiple occasions when Sylvester Zugrav and Mendez traveled to the same location, Sylvester Zugrav would give Mendez cash payments and other things of value.
From 2008 through August 2011, Sylvester Zugrav gave Mendez seven in-person cash payments ranging from $500 to $10,000 and purchased for him[?] a laptop computer and software package worth over $2,900.
During the course of the corrupt scheme, Mendez opened a foreign bank account so that Sylvester Zugrav could pay Mendez larger bribe payments.
Mendez asked his cousin in Mexico to open an account there.
After the account was opened by Mendez's cousin, Maria Zugrav made wire transfers to the bank account located in the name of Mendez's cousin to avoid detection of the larger bribe payments by law enforcement.
From 2008 through August 2011, Maria Zugrav sent to the Mexico account 10 wire transfers ranging from $350 to $26,700.
Court records also describe additional steps taken to conceal the bribery scheme, including creating and using covert e-mail accounts, using encrypted documents, adopting false names and using code words.
For instance, to avoid detection of their e-mail communications, Sylvester Zugrav and Mendez established e-mail accounts to be used only to communicate requests and offers for bribe payments.
Sylvester Zugrav and Mendez also created password-protected documents for e-mail communications and used code words and false names.
Within the encrypted documents, Mendez adopted the moniker "Chuco" and Sylvester Zugrav used the codename "Jugo."
They referred to cash as "literature."
The case was investigated by the FBI and the Air Force Office of Special Investigations.
The case is being prosecuted by Trial Attorneys Marquest J. Meeks and Edward P. Sullivan of the Criminal Division's Public Integrity Section, Assistant U.S. Attorney Carlos A. Esqueda of the District of Utah, and Trial Attorney Deborah Curtis of the National Security Division's Counterespionage Section.
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text:
Mario Kart 8 drives Wii U hardware sales up 666% in the UK
After nearly two years of being on the ropes, Nintendo's troubled console has finally gotten a pick-me-up. And a major one at that. With the release of Mario Kart 8, which secured the #2 spot in the UK for most sales during the week, Wii U purchases have increased exponentially.
As reported by CVG, retail monitor Chart-Track says these Wii U sales managed to outperform the console's debut in 2012, which was previously its highest selling point. Moreover, sales of Mario Kart 8 marked the series' second strongest debut, right behind the 3DS' Mario Kart 7.
It's no surprise that Mario Kart 8 would do well, as its predecessors were top sellers on other platforms -- however it's extremely surprising that it not only outsold the competition on a struggling system, but it still managed to become the second highest debut in series history. It's an impressive start.
Mario Kart 8 boosts UK Wii U hardware sales 666% [CVG]
In light of this good news, I now have an excuse to post this great GIF.
Hidden 'Totaka's Song' discovered in Mario Kart 8
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My daughter could probably beat you at Mario Kart 8
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Japan is getting those Mario Kart 8 McDonalds toys too
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Mario Kart 8's TV app no longer broken
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Good lord, there are a lot of Links in Mario Kart 8
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Go download the Mario Kart 8 DLC now
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Here is literally everything in the first Mario Kart 8 DLC pack
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Yes! The first Mario Kart 8 DLC pack hits next week
4:30 PM on 11.05.2014
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LONDON -- Contractors working for a Shell unit have been arrested in Nigeria as alleged accomplices in the theft of oil that led to a pipeline fire. One of the contractors, however, denies his staff was involved in the theft.
Shell Nigeria's joint-venture, Shell Petroleum Development, has previously said the Trans-Niger Pipeline, which carries 150 Mbpd of oil through the Niger Delta to the Bonny terminal, was shut after a fire caused by oil theft.
A spokesman for the Nigerian army's Joint Task Force said that the army, upon arriving at the site of the fire, found eight people whom he said are employees of two contractors hired by SPDC to fix broken pipelines. He said they were arrested and "are helping our investigations as suspected accomplices."
A Shell spokesman said its Nigeria unit had been notified of the arrest of people employed by SPDC contractors "at a work site in the Bodo area" where the fire took place.
We understand that the JTF is investigating the case, and we will cooperate with the investigations as necessary, he said.
A manager at one of the contractors named by the army, Steve Integrated Technical Services, confirmed that some of his staff was arrested but denied the allegations. My staff is not part of the crude oil theft, said the manager, who declined to be named.
The manager said he had not been notified that they were suspected of being complicit in the crime, but the army spokesman said he was not obliged to inform the contractor of the allegation.
The manager said his staff was in tug boats near barge carrying oil recovered from an illegal tapping point when an explosion occurred. He said thieves had tried to pump from the barge at night as his staff slept.
Despite the challenges faced in the area, Shell said that it had given a green light to investments worth $3.9 billion, including a loop line of the Trans-Niger Pipeline.
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Marshawn Lynch, your "punishing" 67 yard Touchdown run against the World Champion New Orleans Saints on Saturday was one of the Best Football Plays of the entire NFL season! It's too bad we didn't see it when you were a BILL, the best run you had was on Chippewa Street!
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Life With My Littles
**Giveaway Rules**
NBFluffNStuff Giveaways!!
Current Giveaways
♥Mama of the Littles♥
Tuesday, November 11, 2014
Tula Baby Carrier giveaway {US and Canada}
I'm running a Tula baby carrier giveaway in my Bumgenius B/S/T and chat group!
It started today and will end in 2 weeks!
here is the link if you'd like to join: http://tinyurl.com/l9bjyn6
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Thursday, October 9, 2014
Back to School with Santa Cruz organic review and #GIVEAWAY {US only}
It's back to school time here... and you know what that means? Packing lunches for my littles. I was so excited when Santa Cruz reached out to me and asked me if I'd be interested in trying out some of their products! If you know me, then you know that eating local and organic foods is high on my list of priorities!
I was sent a 4 pack of apple sauce in a squeezable pouch, a jar of strawberry fruit spread, and a jar of peanut butter!
This is what I received:
I cannot tell you how yummy they were! They are awesome! My kids loved them even more! I really appreciate that their peanut butter had no added sugar. Ingredients 'organic roasted peanuts, *may contain 1% or less of salt.'
I sent the applesauce with my 4 yo to preschool. It is so easy for little ones to open and eat! No need to send a cup of applesauce with a spoon (which never makes their way home to me anyway)!
Santa Cruz offers a wide variety of awesomely organic products. To see what all they have to offer, visit their
. Oh and you may also want to check out their
'recipe' page
for a list of delicious recipes!!
Santa Cruz has generously offered one of you the chance to receive these same items I received (open to residents of the US only)!
To enter, simply click on the link below!
Good luck!!
~Mama of the Littles~
**Disclosure. I received the item(s) mentioned above free of charge in exchange for my review. I was not compensated monetarily for my review. These opinions are my own and may differ from your own**
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Tuesday, June 17, 2014
Tots Bots Winner and summer break!
Congratulations to Laura C.
You are the winner of the Tots Bots diaper.
You have been e-mailed and have 48 hours to respond to claim your prize before another winner is drawn!
**Summer time is here and the kids are out of school. We are moving at the beginning of July!
The blog has been neglected for awhile but I've found it great to be able to spend some quality time with my family while taking a break!
I have an awesome review and giveaway for UV skins coming up very soon. You won't want to miss out!
Stay tuned!
~Mama of the Littles~
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Friday, April 11, 2014
#GIVEAWAY Tots Bots New binky diaper review and #GIVEAWAY {US/Canada}
I am so thrilled to introduce to you my latest
I mean go-to diaper!!
Now I may be partial to this diaper because it's made in the UK (Scotland to be exact) and my father was born in England. Or maybe it's the absolutely adorable prints that these diapers come in
These are the new prints ^^ that have come out in the new V4 version!
The diapers pictured below are a part of their 'story book collection' and come in the older (V3 version)! If you are stateside, you can still get the V3 version (I saw a few in snaps only) from retailers such as
Thanks Mama!
Or perhaps it's the way these diaper work, the high quality of production, or maybe it's a matter of all of the above combined!
Tots Bots has a few different versions of their diapers to choose from. There is the
(which is an all in one (AIO) diaper designed to fit babies from 5-12 lbs; the
(also an AIO), and a one size diaper, designed to fit babies from 8-35 lbs; and lastly the
. The Bamboozle is a stretchy bamboo fitted diaper, which comes in two sizes. Size one designed for babies weighing from 6-18 lbs and size 2 designed for babies weighing 9-35 lbs! Tots Bots also offers swim diapers and training pants!
The new TotsBots easy fit V4 has a bamboo, cotton and minky blend fabric booster!
Here is some more information on this diaper (taken from the
TotsBots website):
The perfect daytime nappy
Pop in booster comes as standard for increased absorbency.
Fast drying.
Integral waterproof outer means there is no need for a separate cover.
Hook & loop fasteners enable quick and easy changes.
Wrap-around stretch for ultimate comfort and fit.
Super absorbent bamboo interior is so soft next to baby's skin.
Made in Scotland.
Fits from 8lbs to 35lbs.
Oekotex certified.
I was thrilled to have been sent the brand new V4 TotsBots "binky" in the Hickory Dickory Dock print in hook and loop closure! For those of you that have been a reader of my blog for any amount of time, know that I am a
snaps (poppers) girl!! Read on to find out what I discovered while using this diaper!!
This is what I received:
This print is so much more vibrant and rich in real life!!
Clocks, and mice and cheese!!! When I was stationed in Germany... I flew home for Christmas the first year I was there and I bought my parents a cuckoo clock from the Black Forest! It was awesome! This diaper kind of brings back memories of that!
The inside of the diaper:
A good look at the print!
Ok, here is the inside. It is indeed an all in one diaper with a pocket near the back that you stuff the inners into! As you can see in this picture, the snap in soaker which is bamboo, cotton, minky blend (aka binky) fabric gives this diaper superb absorbency and is GREAT when used at night!!
The soaker simply snaps to the inside of the diaper with 2 snaps:
Here is the diaper with soaker attached and unstuffed:
You simply fold the soaker at the snap and stuff it into the back side of the diaper's pocket.
This is what your diaper will look like when it's ready to go onto baby's bum:
Let's talk hook and loop! You can see here the vibrant red strip of hook and loop material. You can also see the two rise snap settings here:
My daughter (who is about 14 lbs at 1 year old) wears this diaper on the second rise snap as shown here:
This diaper is amazing! I LOVE the prints (and solid colors of the diapers which can be found
)! I LOVE that it's an all in one (AIO) with the optional snap in soaker for heavy wetters or overnight use!
I LOVE, LOVE, LOVE the prints! I can't wait to get them ALL!
I also love the quality! These diapers are very well made and Tots Bots really does a great job with their customer service!!
Here is this diaper on my little girl:
No gaps in the legs
As you can see it is a bit 'fluffier' with the snap in soaker:
What's not to love!?
The older Easy fit Tots Bots diapers can be purchased now on Amazon.com!
The newer V4 version is not available *yet* in North America... and this new version will replace the older V3 style!
BUT wait, that's not all!! Tots Bots has graciously given one of the the opportunity to win a Tots Bots V4 right here!! Winner can choose color/print in either hook and loop or snap closure! Please note that the prize will not be mailed to the winner until the V4 version is available in North America!!
To enter, simply fill out the rafflecopter form below!!
You may have to click 'read more' in order for the rafflecopter form to fully load!
Good luck and have fun!
**Disclosure. I received the item(s) mentioned above free of charge in exchange for my review. I was not compensated monetarily for my review. These opinions are my own and may differ from your own**
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Tuesday, April 1, 2014
Another clothdiaper giveway!! 7 winners and one of the prizes is a Bumgenius Freetime in Jules print!!
We are having another giveaway event over in the Bumgenius cloth diapers b/s/t and chat group that I co-admin!!
I'd love for you all to join the page and enter to win!!
1. A Bumgenius freetime cloth diaper in JULES print {US/Canada}
2. An 8x10 nursery wall art from "The Laughing Goose" {US/Canada}
3. A package of 100 biodegradable/flushable diaper liners {US only}
4. A pair of gently used girlie baby legs {US only}
5. 1 bottle each of booty cleansing spray and one of booty balm {US only}
6. A Babee Greens size small cashmere diaper cover {US only}
7. A NIP pair of girlie babylegs {US only}
8. a $20 GC to Rump A Dump Diapers {US/Canada}
To enter, simply fill out the rafflecopter form below!
Good luck and have fun!
~Mama of the littles~
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Tuesday, March 18, 2014
Gia Nursing pillow winner!!
Congrats to Kristin F. You are the winner of the Gia breastfeeding pillow giveaway!
You have been e-mailed and have 48 hours to respond to claim your prize!
Don't forget to enter the multi winner giveaway
7 winners and over $150 in prizes!!
~Mama of the Littles
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Tuesday, March 11, 2014
BG B/S/T and Chat 500 member giveaway!!
500 member giveaway!
*Please click the rafflecopter link at the bottom of this page to be entered to win one of these fabulous prizes!
**You must be a member of the Bumgenius B/S/T and chat Facebook group in order to be eligible to enter to win! You can find the link to the
group HERE!
1. Starter pack (3) of LooHoo wool dryer balls {US/Canada}
2. A Bumgenius cloth diaper in winner's choice of color and closure from Cotton Babies {US only}
A 3-pack of size small Fuzzi Bunz diapers {US/Canada} in choco truffle, green apple and watermelon!
4. 1 bottle of booty cleansing spray and 1 bottle of booty balm {US only}
5. A $35 GC to All Things Diapers {US/Canada}
6. A bar of cherry almond handmade soap from Bump and Rump {US only}
7. A Bumgenius 4.0 cloth diaper in Lovelace print in hook and loop closure {US only}
Click on the Rafflecopter form below to enter for your chance to win!
Good Luck and Have fun!
~Mama of the Littles~
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text:
Katin Corp 2 T-Shirt
SALE $13.00 was $26.00 Item #48274KN1022
Sorry, this product is currently not available on buckle.com.
- Speckled front and back Katin graphic t-shirt
- 50% Cotton, 50% Polyester.
- Machine wash cold. Tumble dry low.
- Made in United States.
Have a Question?
Email Guest Services.
We changed our page. Tell us what you think.
- Chest measures 40" on a size medium.
- Center back length measures 26".


text:
OP Junior Member
Thanks Meter: 0
3 posts
Join Date:Joined: Feb 2014
I have my newly imported Nexus 7 from New Jersey and I'm having some issues when it comes to gaming. Thing is, I'm new to Android as well as tablets. But being a hardcore PC gamer, I did my homework and found the most elite racing games before I ordered my tab. Turns out, Real Racing 3 and GT Racing 2 are the best there is out there, atm. So when I received my tab, I tried downloading RR3. Now lets look at the REAL problem- I have a ****ty internet. No, really. It goes out like 10 times a day. Then I have this speed issue. I pay for a 512 kbps speed but get only 50. Why don't you change it then, you say? I live in a Central Govt. colony and all internet providers are forbidden to set foot inside the colony gate. Failure in complying with the rules will result in their licenses getting cancelled (whoa!). So I have to live with what the central Govt. provides me with.
Anyways, so I have registered here just to post this topic. Since RR3 took centuries to download, I had to uninstall it. Then, I tried GTR2 and found out that downloading it from Playstore doesn't give you resumable download facility if your internet connection dies for even a sec. Fed up, I decided to give sideloading a try. Downloaded RR3 cache via PC from a website and put it in android/data/. The file was well over 1GB but running the app shows it has downloaded only 356 MB and still needs to an extra 711 MB. Downloaded GTR2's cache and put it in obb folder. It starts up and then crashes right away saying "Unfortunately GT Racing 2 has stopped". I'm so embarrassed and kinda screwed right now. I bought N7 only to find out I can't play games on it. I bought it solely for gaming when I'm on the move and now I can't seem to download these huge data files or the game just simply refuses to run. I have got Internet Download Manager on PC so I have downloaded game caches from third party websites without a problem but as you can see, even those efforts were futile. Are there any hopes for this situation? Anyone here knowing a workaround? Suggestions are more than welcome.


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The Tread kid's waterproof walking boots are breathable with a mesh & suede upper and mesh lining. The waterproof boots are embedded with an Isodry membrane to ensure your feet remain dry. The rubber sole and cushioned inner ensures comfort and durability while out walking. The non-abrasive stainless steel lace loops will help to keep your laces in new condition.
July 08, 2012
My daughter loves these boots
July 11, 2012
Fantastic, sturdy boot.
July 12, 2012
Good product and good value
July 17, 2012
Good price and very comfortable.
August 01, 2012
Children have worn them and they are very comfy and keep their feet dry.
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