Differentiation of Peripheral Sympathetic Neurons

(1)

Silja Salokorpi

DIFFERENTIATION OF PERIPHERAL SYMPATHETIC NEURONS

Faculty of Medicine and Health

Technology

Bachelor thesis

April 2021

(2)

ABSTRACT

Silja Salokorpi: Differentiation of Peripheral Sympathetic Neurons Bachelor thesis

Tampere University

Faculty of Medicine and Health Technology April 2021

This thesis is a literature review of the methods that have been used to differentiate sympathetic neurons in vitro from human origin stem cells. Information from the previous studies was collected and different methods used to differentiate sympathetic neurons were compared and discussed.

Focus was also on the possibilities to use sympathetic neurons in co-cultures with cardiomyocytes to model human body functions.

More effective sympathetic neuron differentiation methods have been developed over the years.

The aim of the first studies performed with human stem cells was to differentiate neural crest stage cells. At first, embryonic stem cells were utilized, and the methods were based on co-cultures with stromal cell lines and use of few specific factors to guide the differentiation. The methods have been based on mimicking the signaling pathways of neuronal development during embryonic development. Later methods counted more on so called small molecule methods and induced pluripotent stem cells have been preferred over embryonic stem cells. Generation of sympathetic neurons from stem cells takes on average one month of cell culture. The yields of sympathetic neurons and their characterization has been improved with modern methods.

The most important factors in the sympathetic neuron differentiation were bone morphogenic proteins and molecules or drugs that can regulate for example Wnt- and SHH-signaling pathways.

In the most recent and the most effective differentiation studies, three small molecule inhibitors and different variations of them were used. Precise growth conditions are needed to achieve and maintain cultures of differentiated peripheral neurons. Sometimes cell sorting by specific markers was used during differentiation steps to enhance the differentiation efficiency, but to simplify the methods, it is not preferred. The markers that were most commonly used to characterize sympathetic neurons are peripherin, tyrosine hydroxylase, dopamine beta hydroxylase and PHOX2B gene expression.

In addition, this work presented how differentiated sympathetic neurons are already used in co- cultures with cardiomyocytes. Thus, it is possible to confirm neuron functionality and to model human body interactions of these cells. Results of this work clarified well the literature concerning sympathetic neuron differentiation and the most effective methods for the differentiation were recognized as an outcome.

Keywords: Stem cells, neural crest, neural development, autonomic nervous system, sympathetic neurons, cell culture, cardiomyocyte co-culture

The originality of this thesis has been checked using the Turnitin OriginalityCheck service.

(3)

TIIVISTELMÄ

Silja Salokorpi: Ääreishermoston sympaattisten hermosolujen erilaistaminen Kandidaatintyö

Tampereen yliopisto

Lääketieteen ja terveysteknologian tiedekunta Huhtikuu 2021

Tämä kandidaatintyö on kirjallisuuskatsaus menetelmiin, joilla ihmisperäisiä kantasoluja voidaan erilaistaa sympaattisiksi hermosoluiksi. Työssä tutustuttiin aiempien tutkimusten menetelmiin ja niitä vertailtiin. Yksi painopiste katsauksessa oli erilaistettujen sympaattisten neuroneiden hyödyntäminen yhteisviljelmissä sydänlihassolujen kanssa.

Tutkitun kirjallisuuden perusteella erilaistusmenetelmät ovat kehittyneet ja tulleet tehokkaammiksi vuosien varrella. Ensimmäisten ihmissoluilla tehtyjen erilaistusten tavoitteena oli tarkastella hermostopienan kehittymistä kantasoluista. Näissä ensimmäisissä tutkimuksissa hyödynnettiin alkioperäisiä pluripotentteja kantasoluja. Menetelmät perustuivat yhteisviljelmiin stromaalisten solulinjojen kanssa sekä tiettyjen kasvua ohjaavien molekyylien käyttöön. Menetelmien perustana on toiminut tavoite jäljitellä ihmisalkion kehitystä ohjaavia signalointireittejä. Tuoreimmissa moderneissa erilaistusmenetelmissä on suosittu niin kutsuttujen pienten molekyylien menetelmiä sekä indusoitujen pluripotenttien kantasolulinjojen käyttöä. Jotta kantasolut erilaistuvat sympaattisiksi neuroneiksi, on tarvittu noin kuukauden kestävä soluviljely. Sympaattisten hermosolujen saannit tutkimuksissa ovat parantuneet modernien menetelmien myötä.

Tärkeimpiä sympaattisten neuronien erilaistumisen säätelyssä ovat olleet luun morfogeneettiset proteiinit ja molekyylit, joilla voidaan säädellä Wnt- ja SHH-signalointireittejä. Tuoreimmissa tutkimuksissa on hyödynnetty niin kutsuttua kolmen molekyylin menetelmää ja sen erilaisia variaatioita. Soluviljelmissä on saavutettava ja ylläpidettävä tarkkoja kasvuolosuhteita.

Erilaistamisen tehostamiseksi on voitu käyttää viljelyn aikana solujen lajittelua tiettyjen markkereiden avulla, mutta menetelmien yksinkertaistamiseksi tästä lajitteluvaiheesta on pyritty eroon. Yleisimmät markkerit, joita sympaattisten hermosolujen karakterisointiin käytetään, ovat peripheriini, tyrosiini hydroksylaasi, dopamiini-beeta-hydroksylaasi sekä PHOX2B-geenin ekspressio.

Lisäksi katsauksessa havaittiin, että erilaistettuja sympaattisia neuroneita käytetään jo yhteisviljelmissä sydänlihassolujen eli kardiomyosyyttien kanssa. Tällä tavoin voidaan varmistaa saatujen neuroneiden toiminnallisuus ja mallintaa näiden solutyyppien todellisia vuorovaikutuksia ihmiskehossa. Työn tuloksena selvisi hyvin sympaattisten neuronien erilaistukseen liittyvä kirjallisuus sekä tehokkaimmat erilaistusmenetelmät.

Asiasanat: Kantasolut, sympaattiset neuronit, hermoston kehittyminen, soluviljely, yhteisviljelmät Tämän julkaisun alkuperäisyys on tarkastettu Turnitin OriginalityCheck -ohjelmalla.

(4)

LIST OF SYMBOLS AND ABBREVIATIONS

BDNF Brain-derived neurotrophic factor

BMP Bone morphogenic protein

cAMP Cyclic adenosine monophosphate

CNS Central nervous system

CNTF Ciliary neurotrophic factor

DBH Dopamine beta-hydroxylase

DDC Dopa decarboxylase

DMEM Dulbecco’s Modified Eagle Medium EGF Epidermal growth factor

FGF Fibroblast growth factor

FSK Forskolin

GDNF Glial cell line-derived growth factor hPSC Human pluripotent stem cell hESC Human embryotic stem cell

hiPSC Human induced pluripotent stem cell

KRS Knockout replace serum

MEA Multielectrode array

m3i Modified three small molecule inhibitors protocol

NGF Nerve growth factor

NMVM Neonatal mouse ventricular myocyte

NT-3 Neutrotrophin-3

PHOX2B Paired like homeobox 2B gene

PNS Peripheral nervous system

PRPH Peripherin

PSC Pluripotent stem cell

RA Retinoic acid

RT-PCR Reverse transcription polymerase chain reaction SDIA Stromal inducing activity

SHH Sonic hedgehog protein

SMAD Protein group named according to Caenorhabditis elegans Sma and Drosophila Mad (Mothers against decapentaplegic)

TGF-b Transforming growth factor beta

TH Tyrosine hydroxylase

(6)

1 INTRODUCTION

This work is a literature review of the methods that have been used to differentiate sympathetic neurons in vitro from human origin stem cells. The work was focussed on the methods to differentiate peripheral sympathetic neurons, and the possibilities to utilize them in studies. The goal of this review was to collect information from previous studies and compare different methods that have been used to differentiate sympathetic neurons.

Stem cells are undifferentiated cells, that can differentiate into various cell types during embryonic development or later in human body. They can be harvested to differentiate them towards desired cell type in laboratory environment. Stem cells can be utilized, for example, in tissue engineering research and applications, and there are various research opportunities for them. To accomplish that, specific methods are developed. The suitable method depends on the origin of the stem cells, desired final cell type and how they develop in real life. (Fenchel et al., 2013) Differentiated cells can be applied in research for example, in cell and tissue models, pharmacological studies, co-cultures, and possible cell therapies.

In the current literature review, focus was on human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC). Those have the differentiation potential covering all the cell types in the human body. Sympathetic neurons rise from a multipotent cell population called neural crest and develop in a complex process during early embryonic development. (Schoenwolf et al., 2015) Sympathetic neurons are a part of the human peripheral nervous system and they are responsible, as a part of the autonomic nervous system, for the regulation of unvoluntary vital functions such as heart beating rate and blood pressure. (Noback et al., 2007)

Symphathetic neurons differentiated in vitro offer possibilities to, for example, body-on- chip applications and to form co-cultures with cardiomyocytes to model real body functions. Cell models of neural crest and sympathetic neuron progenitors could also be used for developmental studies and disease modelling, or for improvements in regenerative medicine (Kirino et al., 2018). There are some inherited disorders that are originating from autonomic nervous system and are not yet completely understood (Takayama et al., 2020) and for example, neuroblastoma originating from neural crest cells, which give rise to the sympathetic nervous system are the most common childhood solid tumors

(7)

outside central nervous system. More knowledge about them could also give possibilities to enable the development of different cell therapy approaches to their treatment. (Carr- Wilkinson et al., 2018) .

(8)

2 SYMPATHETIC NEURONS

Overview of Human Nervous System

The human nervous system coordinates the actions and signals in all parts of the human body. The nervous system can be divided anatomically into two main parts, the central nervous system (CNS) and the peripheral nervous system (PNS). The latter is divided functionally into the somatic nervous system and the autonomic nervous system. The somatic nervous system consists of the sensory and motor nerves. The autonomic nervous system can be divided into sympathetic and parasympathetic ganglia. The cells that form the nerves and the nervous system are neurons, and there are various types of these. All neurons work as a response to some specific stimulus occurring inside or outside the body. The responses are determined by molecules called neurotransmitters.

These bind to receptors in a cell’s membrane and signals are transmitted around the body as electrical impulses called action potentials. (Noback et al., 2007) Anatomical division of the nervous system and neuron structure are presented in Figure 1.

Figure 1. Schematic images of human nervous system on the left and of neuron structure on the right. Human nerv- ous system consists of neurons that form CNS and PNS. CNS consist of the brain and spinal cord and PNS somatic and autonomic nerves. Neurons are branched cells that consist of dendrites, cell body, axon and axon terminals. Neu-

ronal signal come from the dendrites to the neuron and travels along the axon. The signal exits the cell from the axon terminals, and it is transmitted there through synapse structures to next neurons or other tissues. (Noback et al., 2007,

Figure modified from images.theconversation.com and sciencetrends.com)

Anatomically the CNS consists of the brain and the spinal cord. The PNS consists of the cranial nerves emerging from the brain and spinal nerves from the spinal cord. The peripheral nerves form connections between the CNS and every other part of the body.

They convey messages from the sense organs and sensory receptors inward to the CNS

(9)

and from the CNS outward to the muscles and glands of the body. The autonomic nervous system includes neural structures that are responsible for processing sensory signals from the visceral organs, like the cardiovascular system, and for the motoric controls of smooth muscles in the glands of the viscera and cardiac musculature. (Noback et al., 2007)

The sympathetic nervous system directs involuntary rapid responses and is active during stress or emergency situations. It is responsible for the so-called “fight or flight” responses including an accelerated heart rate and stronger heartbeats, an increase in blood pressure and a higher sugar concentration in the blood. In contrast, the parasympathetic nervous system works on the functions that maintain and renew body resources.

These functions include lowering the heart rate and activating gastrointestinal functions for food digestion and absorption. (Noback et al., 2007)

The actions of the sympathetic and parasympathetic systems are not antagonistic. Ra- ther, they are synergetic and behave differently under different physiological conditions.

Autonomic responses are diverse because of all the different kinds of receptors for the neurotransmitters. The sympathetic system has receptors for norepinephrine and the parasympathetic system has them for acetylcholine. (Noback et al., 2007)

Since the cardiovascular system provides some the most important functions of the human body, including the transportation of fluids and nutrients to all the cells in the body, it is largely controlled by the nervous system. For example, blood pressure and heart- rate are controlled through the balanced functioning of both the sympathetic and the parasympathetic nervous systems. (Noback et al., 2007)

Development of the Neural Crest and Sympathetic Neurons In Vivo

The human nervous system develops with the embryo. Embryogenesis is a complex process occurring immediately after a human ovum is fertilized as it is fused with a sperm. The resulting zygote starts to go through mitotic divisions into multiple cells. The cellular differentiation eventually leads to the development of a multicellular embryo.

(Schoenwolf et al., 2015)

In the third week after fertilization, after the embryo has already gone through several stages of its early development, the cells form a trilaminar disc. These three germ layers are the ectoderm, the mesoderm and the endoderm and their formation process is called gastrulation. During this process each layer forms a source for its own specific cell types and structures. Trilaminar disc stage is described in Figure 2. (Schoenwolf et al., 2015)

(10)

Figure 2. Schematic image of trilaminar disc stage during embryonic development. (Modified from Schoenwolf et al., 2015)

The next stage of embryogenesis takes place in the fourth week, and is called neurula- tion. The differentiated part in mesoderm called notochord, and this induces changes in the ectoderm layer cells by secreting the sonic hedgehog protein (SHH) along with bone morphogenic protein inhibitor called noggin, and the formation of a neural plate from ectoderm. The neural plate turns and then forms a neural tube. Neural crest cells then arise at the border of neural and non-neural ectoderm. Neural crest formation is showed in Figure 3. (Schoenwolf et al., 2015)

Figure 3. Neural crest development during embryonic development. (Modified from Shoenwolf et al., 2015)

The neural crest is an important population of multipotent embryonic cells. They migrate from the neural tube in the very early stages of human development and give rise to large variety of different cell types, including the neurons and glial cells of peripheral nervous system, sympathoadrenal cells, melanocytes and most of the cells for the bone and cartilage of the face and head. It is thought that there are some similarities in the development of the neural crest cell line between different species and that the cell line is unique to vertebrates. The neural crest development is induced by several transcription factors and specific genes. Major signalling pathways are known to be induced by bone mor- phogenetic proteins (BMPs), Wnt proteins, fibroblast growth factors (FGFs), notch proteins and, for example, retinoic acid (RA). (Stuhlmiller, García-Castro, 2012)

(11)

The in vivo induction of neural crest cells begins at the level of midbrain and continues extending progressively caudally towards the tail. Neural crest cells are born along the length of neuraxis. These can be classified into groups based on their axial level of origin:

the cranial, cardiac, vagal, sacral and trunk neural crests. All the groups have their specific migration pathways and differentiation capacities. For example, the cranial neural crest gives rise primarily to bones, cartilage and connective tissues, while the trunk neural crest cells give rise to sensory, glial and sympathetic progenitor cells. (Bhatt, Diaz &

Trainor, 2013) Thus, sympathetic neurons are one of the cell types rising from the trunk neural crest cells. When neural crest cells migrate during embryogenesis and arrive at the dorsal aorta, they begin to express enzymes called tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH). After that, BMPs can be secreted and they induce specification of neural crest cells into autonomic noradrenergic cells. When the cells are part of PNS they are also expressing protein called peripherin (PRPH). (Carr-Wilkinson et al., 2018) When the sympathetic neurons have then successfully developed in vivo, they can create connections with different tissues and organs around the body.

(12)

3 STEM CELLS AND DIFFERENTIATION

Stem Cell Classification

Stem cells are type of cells that can proliferate indefinitely and differentiate into various cell types. They are present during embryogenesis and also at some stages in adult humans. They ensure tissue regeneration. Stem cells are divided into different types by their differentiation capacity. The egg cell can become any cell type, so it is called totip- otent. Next type is pluripotent, and they are cells that can develop cells of all different human body tissues, but not the cells of the placenta or fetal membranes. Human pluripotent stem cells (hPSCs) can be divided into hESCs and hiPSCs. The first ones are present only during embryonic development, after the zygote develops to blastocyst during the first week, and they can be collected from excessive or low-quality embryos from infertility treatments. iPSCs can be generated from person’s somatic cells, like fibro- blasts, with different technologies. Adult stem cells can proliferate only to cells of specific organ and they are classified as multipotent stem cells. Human pluripotent stem cells can be used in in vitro studies to examine development and function of specific tissues.

They give a promising platform also for neuronal studies and future applications. (Fen- chel et al., 2013)

Principles of Sympathetic Neuron Differentiation In Vitro

Stem cell differentiation into specific cell types in vitro usually mimics the known phases of tissue development in vivo. The current methods for differentiation of sympathetic neurons are based on a default hypothesis of neural induction in vivo. According to that, neural tissue forms spontaneously, when there is no BMP signalling during the early phases of gastrulation, while the existence of BMP would cause epidermal fates to the cells. (Muñoz-Sanjuán, Brivanlou, 2002) During development dorsoventral patterning is controlled by SHH and BMPs and therefor BMP inhibition molecules, growth factors or SHH signal activators are used in cell culture to guide differentiation in vitro. (Lanza, Langer & Vacanti, 2013)

Though there are specific variations in differentiation routes between neuronal subtypes, there are three main strategies for induction of PSCs into neural cells in vitro. The options are using the default hypothesis of differentiation into neural fates, systems based on

(13)

embryonic bodies and stromal feeder-cell mediated induction or they can be based on dual SMAD signalling inhibition. SMAD stands for the class of transcription factors, named according to Caenorhabditis elegans Sma and Drosophila Mad (Mothers against decapentaplegic), where those have been first recognized. These proteins have an important role in regulating TGF-b (transforming growth factor beta) signal pathway.

(Derynck, Zhang & Feng, 1998)

Embryonic bodies are floating spheres, that are formed by aggregating embryonic stem cells in suspension cultures. Those are able to form interactions that are mimicking the normal steps of embryo development and gastrulation. Embryonic bodies can be then exposed on the specific inducing factors. For example, the exposure of RA can induce strong neural differentiation. RA is a vitamin A derivate, that is naturally released by mesodermal cells during embryo development. Neural selective growth conditions can be achieved by serum free culture system. (Lanza et al., 2013)

Induction of neural differentiation can be mediated by stromal cell feeder layer. Stromal cells that can be used in this method, are usually derived from bone marrow and used in culture typically at preadipocytic stage of differentiation. Activity of this method resides mostly on the surfaces of the stromal cells. Default hypothesis -based protocol of differentiating neural cells utilizes direct induction, without cells signaling with each other. Ec- todermal cells can adopt neural fates without exposure to BMPs. (Lanza et al., 2013) Usually, if the goal of differentiation is to induce sympathetic-like cells, specific neural crest differentiation methods are needed first. It has been found that neural crest -like structures can be achieved with controlled Wnt signaling and later exposure to BMPs, and these signaling pathways can be utilized in methods for cell cultures.

Dual SMAD inhibition is based on two inhibitors noggin and SB431542 and it is possible to differentiate either CNS neuron progenitors or neural crest progenitors depending on the cell density. That method was first developed by Chambers and colleagues in 2009.

SB431542 is a drug that inhibits receptors of TGF-! pathway and therefore enhances neural cell induction and mammalian noggin7 is important neural inducer and recombinant noggin can mimic those effects in vitro. (Chambers et al., 2009) SMAD inhibition following with early exposure to RA, FGF and SHH can result neural progenitors that can be further differentiated with BDNF (brain-derived neurotrophic factor), ascorbic acid, GDNF (glial cell line-derived growth factor), TGF-!3 and cAMP (cyclic adenosine mono phosphate).(Chambers et al., 2009) Neurons differentiated from hPSCs with that method can co-express Tuj1 and TH, that indicates possible sympathetic-like characteristics.

Later in 2012 Chambers and colleagues have published an article where they used improved combined small molecule method of three inhibitors to differentiate hPSCs into

(14)

nociceptors. They found that neuralization effect could be achieved by using LDN193189 to replace noggin in dual SMAD inhibition and a combine them with three inhibitors (3i) SU5402, CHIR99021 (Wnt signaling activator) and DAPT (g-secretase inhibitor that blocks notch signaling). The method guided the cells more towards PNS identity rather than CNS compared to previous dual SMAD method. (Chambers et al., 2012) Modern methods to differentiate sympathetic neurons are based on small molecules that inhibit signalling pathways and small molecules can be used together with other methods.

3.2.1 Role of Bone Morphogenic Proteins in Sympathetic Neuron Differentiation

BMPs are thought to have a role in development of neural crest and later sympathetic neurons in embryo. Controlling of those signals can be used to differentiate cells into direction that result sympathetic-like cells. Some first methods to control the signals of differentiating hPSCs into neural crest cells have used SMAD inhibition that results block- ing BMP and TGF-! signaling pathways (Menendez et al., 2013). SMAD pathway directs the cell proliferation towards neural progenitor cell types that are not neural crest -like, so differentiation towards neural crest cells is prompted by inhibition of that pathway.

It has been evaluated, if the inhibition of BMP can be used to generate neural crest cells from human pluripotent stem cells. (Huang et al., 2016) During that study, it was noticed that using a protocol that did not inhibit BMP signals, derived cells expressed more CNS markers, than those cells that were derived with temporal BMP inhibition. Downregula- tion of CNS markers is typical to neural crest cells. After that the cells were mostly expressing cranial neural crest markers and less trunk neural crest marker PHOX2B, but when treating the cells with retinoid acid, it drove the differentiation towards trunk neural crest cells. RA showed results in cell differentiation in 2-3 days. Later addition of BMP increased expression of markers that are typical for sympathoadrenal lineage of the cells like TH. (Huang et al., 2016)

3.2.2 Combination of Stromal Inducing Activity and Controlled Bone Morphogenic Protein Exposure

Stromal inducing activity (SDIA) can be used to support neural differentiation of stem cells. It was first studied and confirmed already in 2000 by Kawasaki et al., that different stromal cell lines can produce factors to induce neuronal differentiation in co-cultures.

They focused especially on PA6 cell line that is derived from mouse skull bone marrow.

The method was first examined with mouse and primate cells, and results showed that

(15)

the method is simple from a technical point of view, fast, resulting neuronal marker expression already at 4-5 days, and effective. (Kawasaki et al., 2000) In these same studies, the effects of BMPs for SDIA treated cells were also evaluated and importance of inhibition of especially BMP4 was already recognized.

Controlled BMP exposure has been used after that in the SDIA co-cultures of hPSCs.

One optimized method with SDIA and BMP4 is described in the article by Carr-Wilkinson et al. from 2018. They were able to differentiate neural crest cells and sympathetic progenitor cells in co-culture of hESCs and PA6. They tested different culture mediums, BMP4 exposure times and concentrations, and also BMP2 and BMP4 exposure together.

According to their conclusions, the best solution was to use MACS neuronal media and BMP4 alone together with B27 supplement. (Carr-Wilkinson et al., 2018)

(16)

4 SYMPATHETIC NEURON CHARACTERIZATION

To confirm that the differentiation of sympathetic neurons has resulted to desired cell types, cells need to be thoroughly characterized. Methods to characterize the cells include immunocytochemistry, cell sorting and genome analysis. Sympathetic neurons can be also evaluated with functional assays including electrophysiology and co-culture assays with relevant cell types e.g., cardiomyocytes.

Immunocytochemistry can be done by staining cell specific markers and use fluorescence microscope to visualize them. Typical markers for sympathetic neurons and their progenitors are PRPH, TH, and dopamine beta-hydroxylase (DBH). One option is to detect the expression of PHOX2B gene. Human embryonic development is guided by ge- netic instructions and transcription factors. One transcription factor that has been noticed to be expressed during the whole process of sympathetic neuron development is PHOX2B. It is a homeodomain protein, that is expressed in neurons and during neuronal development being corresponding to expression of DBH that is a noradrenaline synthe- sis enzyme. (Tiveron, Hirsch & Brunet, 1996) Cell lines can be modified to express some marker directly or cells can be later treated with detectable markers. Flow cytometry analyses are one option to detect cells and it can be used to either sort the cells or perform counting of cells.

Cell sorting methods can be used to evaluate differentiation result but also to improve the efficiency of cell differentiation. It means that cells are sorted according the specific markers or genes they express, and therefore the right type of cells can be picked for further treatments. Method to sort cells can be for example fluorescent activated cell sorting (FACS). For example, Kirino et al. started their method by first creating a PHOX2B knocked-in reporter lines of hPSCs for that gene to detect the particular gene during the differentiation process to sort the cells that, but then showed that their method is as effective without gene expression-based cell sorting. (Kirino et al., 2018) Cell sorting of reporter lines have been necessary step in many studies to purify neural crest cells that express PHOX2B. Other neural crest markers that have been used for cell sorting or cell type detection in the methods described earlier are for example, p75 (Carr-Wil- kinson et al., 2018), SOX10 and CD49D (Kirino et al., 2018) (Wu, Zeltner, 2020). Flow cytometry analyses can be used to either sort the cells or perform counting of cells.

(17)

Different analysis of gene expression can also be performed for single cells or for whole cell population. RT-PCR method is a reverse transcription polymerase chain reaction that can be used to detect the RNA transcription factors that are expressed in the cells with the help of fluorescence markers. That method can be performed quantitative when it is called RT-qPCR. These methods were utilized in many of the here described methods to differentiate sympathetic neurons from hPSCs. (Huang et al., 2016; Jiang et al., 2009; Kirino et al., 2018; Wu & Zeltner, 2020)

Functional analysis can be done to verify that yielded neurons have also electrophysiological activity. Commonly used methods are for example multielectrode array (MEA) or patch-clamp technique. MEA is also called microelectrode array and it is a device which includes small electrodes that can be used to record neural signals. Patch clamp can be used to measure currents and potential differences over neuron cell membranes. (Frith et al., 2018) Functionality can be evaluated also in co-cultures. For example, in the case of sympathetic neurons in the co-cultures with cardiomyocytes. Neurons can be stimu- lated or treated with drugs, neurotransmitters or other suitable signals and modulation of functions can be recorded with these techniques.

(18)

5 SYMPATHETIC NEURON CULTURE METHODS

Different studies have shown methods to differentiate neural crest cells and sympathetic neurons from human pluripotent stem cells or from embryonic stem cells. These methods are described in following chapter. There are not many studies yet about especially sympathetic neuron differentiations. In some studies where neural crest induction has been performed by using hPSCs and their guided differentiation, sympathetic-like cells have been characterized. To differentiate hPSCs into sympathetic neurons they are usually first identified as neural crest stage cells to confirm their later proliferation into PNS cells and into sympathetic neurons.

Literature on Sympathetic Neuron Differentiation

First research about generating neural crest or sympathetic-like cells were performed using a specific method for mouse and primate embryonic stem cells. (Mizuseki et al., 2003) The method was to use SDIA induction using PA6 mouse stromal line co-culture.

Next studies with hESCs were based on that same cell culture method (Pomp et al., 2005). MS-5 stromal cell line has also been used in a culture with hESCs and some sympathetic neurons were also found as a result (Lee et al., 2007). PA6 stromal cell line was used to differentiate neural crest cells and peripheral sensory neurons, with some sympathetic like neurons were also observed (Brokhman et al., 2008). SDIA of PA6 fi- broblasts was used with some modifications and achieved better yield (Jiang et al., 2009). Most of these earliest studies were focusing on generation of neural crest cells and not especially sympathetic neurons and the methods started with feeder cell co- cultures and developing to the modern methods that are based on small molecules and control of signaling pathways. All the most relevant articles about the methods from the last decade are presented in Table 1.

(19)

Authors Year Title of the paper Cell line Method Characteriza- tion

Functionality

evaluation Yield Time

Huang et

al. 2016 Generating trunk neural crest from human

pluripotent stem cells H1, WTC iPSC

CHIR99021, SB431542, RA, BMP2, BMP4, BMP7. DMEM/F12 medium + N2, NGF, GDNF, ascorbic acid, cAMP

PRH, TH, DBH,

PHOX2B No Not deter-

mined

11 days (NC) + 14

days

Oh et al. 2016

Functional coupling with cardiac muscle promotes maturation of hPSC derived

sympathetic neurons H9, 01582

SMAD inhibition m3i, CHIR99021, KRS, N2 medium and neurobasal me-

dium + B27 and N2, BMP4, purmorphamine, co-culture with NMVMs

PRPH, TH,

DBH, GATA3 Yes Not deter-

mined

14 days + maturation in co-cul-

ture

Kirino et al. 2018

Efficient derivation of sympathetic neurons from human pluripotent stem cells with de-

fined condition

KhES1, KhES3

CHIR99021, BMP4, RA, Essential 6 Medium supplemented with FGF2,

Purmorphamine

PRH, TH, DBH,

PHOX2B No 75-80 % 31 days

Carr-Wil- kinson et

al.

2018

Differentiation of Human Embryonic Stem Cells to Sympathetic Neurons: A Potential Model for Understanding Neuroblastoma

Pathogenesis

H9

Coculture with PA6, MACS neuronal media +B27, exposure to BMP4, NGF,

cAMP

PRH, TH /

DBH, PHOX2B No 20 % /9,4

% +- 5,5% 28 days

Frith et al. 2018 Human axial progenitors generate trunk neural crest cells in vitro

Shef4, H9, Mas- tershef7, MIFF- 1, NCRM1

CHIR99021, FGF2, BMP4, BDNF, GDFN, NGF, BrainPhys neuronal me-

dium + B2, N2, recombinant SHH, purmorphamine

PRPH, TH,

PHOX2B, Yes

PHOX2B 73,5%, other not determined

19-22 days

Wu & Zelt- ner 2020

Efficient differentiation of postganglionic sympathetic neurons using human pluripo-

tent stem cells under feeder-free and chemically defined culture conditions

WA09

CHIR99021, BMP4, Neurobasal medium/B2, BDNF, GDFN, NGF, RA / no

RA. (protocol article)

PRPH, TH,

DBH, Yes Not deter-

mined 20-35 days

Winbo et al. 2020

Functional coculture of sympathetic neu- rons and cardiomyocytes derived from hu-

man-induced pluripotent stem cells

hiPSCs

m3i: CHIR99021, DAPT, PD173074.

SHH C211, purmorphamine, B27TM Plus Neuronal Culture System, BMP4,

BDNF, GDFN, NGF

PRPH, TH Yes

PRPH:

99%, TH:

89%

12 + 11-51 days

Takayama et al. 2020

Selective Induction of human autonomic neurons enables precise control of cardio-

myocyte beating

201B7, 253G1, H1

CHIR99021, dorsomorphin, SB431542, IWR1, SANT1, BMP4, FSK, BDNF, GDNF, NGF, NT-3, KRS/N2 medium, Neuronal differentiation medium, CNTF

TH, DDC, DBH Yes

35,6 % (31 of 87 neu-

rons)

13 days + maturation at least 21

days

information about cell lines and methods that have been used in specific studies.

B27 = B27 supplement, BDNF = Brain-derived neurotrophic factor, BMP2= Bone morphogenic protein 2, BMP4 = Bone morphogenic protein 4, BMP7 = Bone morphogenic protein 7, cAMP = Cyclic adenosine monophosphate, CNTF = Ciliary neurotrophic factor, DBH = Dopamine beta-hydroxylase, DDC = Dopa decarboxylase, DMEM = Dulbecco’s Modified Eagle Medium, FGF2 = Fibroblast growth factor 2, FSK = forskolin, GDNF = Glial cell line-derived growth factor, KRS = Knockout replacement serum, m3i = Modified three small molecule inhibitors protocol, MACS = MACS neuronal media, N2 = N2 supplement, NGF = Nerve growth factor, NMVMs= Neonatal mouse ventricular myocytes, NT-3 = Neutrotrophin-3, PHOX2B= Paired like homeobox 2B gene, PRPH = Peripherin, RA = Retinoid acid, SHH = Sonic hedgehog protein, SMAD = Group of proteins named according to Caenorhabditis elegans Sma and Drosophila Mad (Mothers against decapentaplegic), TH = Tyrosine hydroxylase

(20)

SDIA method was utilized in the earliest studies where sympathetic neurons were detected. During the time, methods have developed, and new procedures have been tested. Most of the earliest studies were performed by using hESC lines and stromal cell feeder layers. Most of the later studies used methods including Wnt signal activation by CHIR99021 compound and other molecules to control signalling pathways. All in all, methods have developed towards controlled signal inhibition by small molecules and using more hiPSCs instead of hESCs.

There are earlier studies done, that are not mentioned in Table 1. The first results from using human stem cells in differentiation of neural crest and sympathetic-like cells was done by Pomp et al. in 2005. They cultured hESCs with PA6 cells using gelatin-coated 13 mm coverslips in 24 well dishes, placing about 1000 hESCs to every well. During the 3-week differentiation process, they used BHK-21 medium or Glasgow modified Eagle’s medium with N2 supplement, and the culture was performed without exposure to BMPs.

(Pomp et al., 2005) In the later article from same authors, in 2007, they performed co- culture with PA6 cells. Approximately 10 000 neurosphere cells were placed in each well and Dulbecco’s modified eagle medium and nerve growth factor exposure was used.

According to this work, neural crest cells were present already in 1-week culture and those differentiate further in co-culture in 3 weeks. The yield of sympathetic-like cells was not determined in this study, but markers PRPH and TH were detected, from where can be concluded, that some of the resulted cells were sympathetic-like. (Brokhman et al., 2008)

Lee et al. studied in 2007 method for directed differentiation of neural crest cells, using co-culture of hESCs and MS5 stromal cell line. They placed 5000 - 10 000 hESCs on radiated MS5 in 60mm cell culture plates. They isolated cells that were positive for p75 and HNK1 by fluorescence activated cell sorting to get neural crest derivates. Serum replacement medium was used during the first 16 days and N2 medium after that for the rest of the 28 days differentiation culture and culture included exposure to FGF2 and epidermal growth factor (EGF). They performed also an in vivo transplantation of derived neural crest cells to chick embryos and adult mice to examine cell survival and neural crest differentiation towards peripheral neurons or smooth muscle in vivo. They used markers PRPH, DBH and TH to characterize 1-2% of the cells to be sympathetic-like.

(Lee et al. 2007)

Jiang et al. work from 2009 focused on derivation of neural crest stem cells using again PA6 co-culture. They used hESC colonies that were treated with collagenase and then mechanically separated into clumps that were then inserted into 3 cm dishes in co-culture with PA6, with density up to 1000 colonies per dish. BHK-21 medium/Glasgow modified

(21)

Eagle’s medium with N2 supplement was used in the culture. Peripheral neurons were detected after 3-4 weeks of co-culture. (Jiang et al. 2009)

The first more recent study that is mentioned in Table 1 was reported by Huang et al. in 2016. Their focus was on differentiation of trunk neural crest cell lines from hPSC lines.

They tested methods for both embryonic and induced pluripotent stem cells. They used Wnt pathway activation via CHIR99021 and inhibition of TGF- " signaling pathway via TGF-b inhibitor SB431542 to induce neural crest cells, and exposure to RA and BMPs to induce differentiation towards sympathetic-like cells. For the culture media, Dalton modified media was used (described in Menendez et al.). Media was composed of Dul- becco’s Modified Eagle Medium (DMEM) with F12 nutrients and N2 supplement, nerve growth factor (NGF), GDNF, ascorbic acid and cyclic AMP (cAMP). They were able to confirm the presence of sympathoadrenal cell markers in their immunofluorescence ex- aminations but did not specify the yield of their method. (Huang et al., 2016)

Kirino et a. reported in their article from 2018, an effective method to derivate sympathetic neurons from hESCs or hiPSCs. As Huang et al., they also utilized CHIR99021 together with BMP4 and RA. The medium was Essential 6 Medium supplemented with FGF2 and they used Purmorphamine, that can activate SHH signalling. They planted hPSCs on 96-well Ultra-Low Attachement Surface multiwell plates, 10 000 cells per well.

At day 10, achieved cell aggregates were seeded on 10 cm dishes at a cell density of 250 000 cells/ml. Multiwell plates or same dishes were used for neuronal maturation.

Authors demonstrated that the method was as effective whether a cell sorting for PHOX2B expressing cells was done or not. Sympathetic direction neural crest cells were achieved at 40% efficiency and those were inducted into sympathetic progenitors, with exposure of EGF, FGF and BMP4, and maturated with addition of NGF, brain-derived neurotrophic factor (BDNF) and GDNF. The final yield of sympathetic neurons with this method was then determined to be 70-80% when they performed immunostaining of the cells for PHOX2B, TH, PRPH and DB. (Kirino et al. 2018)

There is another publication from 2018 listed in Table 1. It is from Carr-Wilkinson et al.

and they studied a method to differentiate sympathetic neurons from hESCs using PA6 cells together with BMP4. They got most promising results when combination of MACS neuronal media with B27 supplement, N2 supplement and nerve growth factor was used.

They transferred 500-800 hESCs to 12- or 24-wellplates on 10 000 or 5000 PA6 cells and culture was maintained for 28 days and they used cell sorting for neural crest stage cells at day 8. They reported yield of sympathetic neurons according to positivity for PRPH and TH to be 20% and, according to DBH and PHOX2B positivity to be 9,4±5,5%.

(Carr-Wilkinson et al., 2018)

(22)

Also published in 2018, Frith et al described a method to generate trunk neural crest via axial progenitors. They used at least three different cell lines of those mentioned in Table 1 in their differentiation experiments. hPSCs were first plated at density 55 000 cells/cm² on vitronectin or fibronectin coated 12-wellplates. They used neuromesodermal progenitor inducing media containing CHIR99021, FGF2 and Y-27632 ROCK inhibitor.

LDN193189 was used to inhibit BMP and at day 3 they dissociated those resulted axial progenitors and replated them on Geltrex-coated plates at density 30 000 cells/cm². Then they used neural crest inducing medium that contained DMEM/F12, CHIR99021, N2 supplement, amino-acids, Glutamax, TGF-" inhibitor SB431542, DMH1, BMP4 and Y-27632 until the day 5. To differentiate sympathetic progenitors, they again replated the cells on Geltrex at a density of 200 000-300 000 cells/cm². At this point they used medium that contained BrainPhys neuronal medium, B27 and N2 supplements, non-essential amino acids, Glutamax, and BMP4, SHH and purmorphamine. After day 12 of differentiation, they changed medium to one that contained ascorbic acid, NGF, BDNF and GDNF instead of those last three latter factors earlier. They determined that they were able to induce progenitors that express PHOX2B and also GATA3 that is a sympathetic neuron regulator, yield of those being about 40%. They also told that “high proportion” of their cells expressed TH and DBH and that PRPH was “widely expressed” in their cultures, but more specific yield was not determined. They used patch-clamp recordings and detection of dopamine and norepinephrine to confirm sympathetic neuron physiology and functionality. (Frith et al., 2018)

In 2020, also Wu & Zeltner published a protocol-article, where they described their group’s method to differentiate postganglionic sympathetic neurons. They have optimized a protocol with two different options after day 10 of differentiation. They combined TGF-" inhibition with BMP4 and Wnt signalling during first 2 days and detected AP2a, that is an early neural crest marker. Later from day 4 to day 10 they detected marker SOX10 and CD49D showing that the cells were at neural crest stage. The yield of CD49 positive cells was reported to be 80% and they recommended cell sorting if that was not achieved. After that the neural crest cells were plated on dishes coated with polyorni- thine, laminin and fibronectin using neurobasal medium with B27 supplement. In option 1 they used first ultra-low attachment plates during the days 10 to 14. They exposed cells to FGF2 and CHIR first for days 10 to 14 in option one, and from day 10 to 28 in option two. In option two neural crest cells were maintained longer. The later maturation was induced then by using cAMP, ascorbic acid, BDNF, GDNF and NGF until days 20-30 in option one and until day 35 in option two. They showed that fold changes increased for

(23)

sympathetic factors like PHOX2B, PRPH, TH,, DBH in the cultures compared to undifferentiated hPSCs, but more defined final yield was not provided. They performed functionality characterization by immunofluorescence and detecting sympathetic markers and norepinephrine transporter NET1 in cultured cells. MEA was also used to detect action potentials under nicotine and propranolol treatments. (Wu, Zeltner, 2020)

Functional Coupling with Cardiomyocytes

Functionality is an important property for sympathetic neurons in vivo, and it has been discovered, that also stem cell -derived sympathetic neurons can functionally connect with cardiomyocytes. Furthermore, coupling with cardiomyocytes can promote neuron differentiation to sympathetic direction. The first study showing this was done by Oh et al. in 2015. They differentiated different hPSC lines towards neural crest and sympathetic lineages using similar methods as described earlier. Their method focused on regulation of Wnt and SHH signaling pathways using SMAD inhibition including SB431542 and LDN193189, modified three inhibitors (m3i) CHIR99021, DAPT and SU5402, purmorphamine and BMP4 treatment from day 10. Cells were sorted according to PHOX2B expression at day 14 and those were then cultured 4-15 days with growth factors. After that sympathetic neurons were co-cultured with mouse ventricular myocytes and neuronal maturation and functional coupling was detected. (Oh et al., 2016)

More advanced methods have been under interest thereafter. Takayama et al. studied the induction of both sympathetic and parasympathetic neurons from hiPSCs and achieved better functionality when coupled with cardiomyocytes in their article from 2020.

Their method was also based on controlled exposure to small molecules. To be able to generate both types of autonomic nervous system cells, it would be possible to regulate cardiomyocytes function by either accelerating or slowing down their beating rate. Para- sympathetic neurons raise mainly from cranial neural crest instead of trunk neural crest in vivo, but their approach was based on theory that those cell types could be generated at the same time. First, neural crest induction step was performed by transferring embryonic bodies to inhibition of BMPs with dorsomorphin, SB431542 for 7 days using KSR and N2 medium. That was followed by Wnt signaling inhibitor IWR1, SHH inhibitor SANT1 and BMP4 for next 6 days. At day 13, cells were plated on poly-L-ornithine and laminin coated plates and neuronal differentiation medium was used. Immunofluores- cence, RNA sequencing and gene ontology analysis were performed at day 13 and they saw upregulation of autonomic and sympathetic lineage genes. That indicated that the method promoted differentiation of the cells towards autonomic specification. Those resulted progenitors were maturated at least for three weeks by forskolin, ascorbic acid,

(24)

BDNF, Glial-cell derived neurotrophic factor GDNF, NGF, recombinant human neurotro- phin 3 and recombinant human ciliary neurotrophic factor CNTF. Cells were plated using densities of 5000-20 000 cells/cm². Neurotrophic growth factors and low cell density were used to induce sympathetic neurons. During the maturation, their cells formed spontaneously ganglion-like structures that were interconnected on day 52. Co-culture of those progenitors with hiPSCs-derived cardiomyocytes was also performed after day 13 and functionality of those co-cultures was examined after 3 weeks using MEA and stimulation of neurons with nicotine or blue light. Axon elongation was examined by plating day 13 progenitors into microtunnels including microfabricated devices. They characterized peripheral neurons according to positivity for TUIJ1 and PRPH and that 48%

of those were positive for TH. On the other hand, their characterization by single-cell RNA sequencing resulted 31 sympathetic neurons among 87 analyzed neurons (35,6%) and some neurons that expressed also parasympathetic markers. Functionality of the neurons was examined also by calcium imaging and electrophysiological analysis. (Ta- kayama et al., 2020)

Winbo et al. have also published co-culture study with hiPSC derived sympathetic neurons and cardiomyocytes. They started the induction by plating hiPSCs on Geltrex coated 12-wellplates and culturing them in StemFlex medium. Series of small molecule inhibitors were used during the first two weeks. From day 4 the medium was changed gradually to N2 supplement enriched B-27 plus neuronal culture system. On day 12 the cells were passaged and resuspended in N2 media and plated on Geltrex-coated 12 mm glass coverslips in 24-wellplates at the density of 1 000 000 – 2 000 000 cells/well. They used neuronal medium containing B-27 plus neuronal culture system, L-glutamine, N2 supplement, ascorbic acid, db-cAMP, NGF, BDNF and BMP4. Small molecules that were used were LDN193189 and SB431542 on days 0-4, m3i molecules CHIR99021, DAPT and PD173074 (fibroblast growth factor inhibitor) during days 2-7, after those SHH C25II and purmorphamine during days 3-12, and BMP4 on days 10-12 and 0-2 days after re- plating. Cardiac myocytes were also generated from hiPSCs and one confluent plate of them was seeded with mature derived sympathetic neurons on coverslips. Co-cultures were maintained at least for 7 days before imaging and characterization of electrophysiological properties. They performed immunohistochemical staining, whole-cell patch- clamp recording, measurements of norepinephrine concentrations in culture medium and evaluation of coupling in co-culture. They reported that TH was expressed in 86% of nucleated sympathetic neurons and PRPH expressed in 99%. They detected presence of nicotinic acetylcholine receptors in the cells and they noted electrophysiological maturation occurring over time and that they were mature at days 48-76. Cell potentials were

(25)

detected in patch-clamp experiments. In neurocardiac co-cultures, they detected synaptic connections between cardiomyocytes and neurons, and some changes, like that action potential kinetics and upstroke velocity enhanced and shorter decay times compared to sympathetic neuron monoculture. Co-cultures were maintained for over 15 days.

(Winbo et al., 2020) These kinds of co-cultures could serve as novel human neurocardiac models and ideally, they would include both sympathetic and parasympathetic neurons.

(26)

6 CONCLUSIONS

There are in total 12 relevant studies presented here, where sympathetic-like neurons have been characterized and different variations of the differentiation methods have been used. Eight of these studies and their key properties are collected in Table 1. Those can be thought to be the most relevant and effective methods to date. The first studies mentioned, were concentrated on differentiating neural crest cell line from hPSCs (Lee et al., 2007; Jiang et al, 2009; Huang et al. 2016) and one focused on generating sensory neurons (Brokhman et al. 2008), but later studies have been more concentrated on sympathetic-like cells. The methods have been developed to be more effective over the years. At first, the most common method was the use of SDIA co-culture approach, since it was used in five of all the studies presented here and the most commonly used stromal cell line was PA6, but there is only one method based on stromal cells co-culture presented in Table 1, indicating that the methods have developed more towards small molecule inhibition methods. All small molecule methods used, were based on the inhibition protocols. They all included use of CHIR99021 molecule to activate Wnt signaling, use of BMP4 and growth factors. Other development that has occured in the methods was in the need of cell sorting. In the latest methods, cell sorting was not a necessary step.

There were some differences in culture times and times of exposures of specific factors.

The required time for neuronal maturation varied between 23 and 63 days. Specific yield of sympathetic neurons was not described in three of the studies in Table 1.

Characterization of the cells was done by using at least markers for PRPH and TH in all of the studies presented in Table 1. Another commonly detected marker was DBH and expression of PHOX2B was also detected in half of the characterizations. Functional analysis can confirm cell maturation, but functionality was not evaluated in all of the studies. According to the yields that are told in the articles, Kirino et al. (2018) have reported the most effective method since their yield of sympathetic neurons was 75-85% and they did their evaluation by using all of the four markers. But it should be considered that they did not evaluate neuron functionality. With neuronal cell experiments electrophysiological activity is important. Winbo et al. (2020) reported very high yields of both PRPH and TH positive neurons, but they did not report double positivity of those cells. However, their cells were functionally active in co-culture with cardiomyocytes and culture was maintained for the longest time. Takayama et al. (2020) have also promising results about

(27)

neuron functionality and their method included differentiation of parasympathetic neurons, but their yield was not as high as in other studies where the cells were functionally tested.

Stem cell technologies have developed during the recent decade and use of hiPSCs in in vitro studies have become more common. That can be seen in the development of the methods described here. There are ethical issues related to use of hESCs since they can be collected only from human embryos. Other challenges in the methods are related into need of long culture times and need of very specific culture conditions. Small molecules need to be changed in particular days and different medium compositions need to be used. Methods can therefore be expensive and difficult to maintain. If the goal is to model adult tissues instead of neonatal tissues even longer times for proper maturation is needed and according to results of these methods, long enough maintenance is not yet achieved.

In the future, even more attention will probably be paid on the co-cultures of sympathetic neurons with cardiomyocytes or any other cell type that autonomic nervous system can regulate. That is the way to get closer to real body functions and eventually model those in improved body-on-chip applications. The co-culture systems can also include device based microfluidic systems. Better maturation of sympathetic neurons is also needed to get more relevant and applicable information. With more research and improved effective differentiation methods, it will be possible to achieve better understanding of neuronal differentiation steps during embryonic development. It will also make it possible to gather more information about different autonomic nervous system -based disorders and their origins.

(28)

REFERENCES

[1] Bhatt, S., Diaz, R. & Trainor, P.A. 2013, "Signals and switches in mammalian neural crest cell differentiation", Cold Spring Harbor Perspectives in Biology, vol.

5, no. 2, pp. a008326.

[2] Brokhman, I., Gamarnik-Ziegler, L., Pomp, O., Aharonowiz, M., Reubinoff, B.E. &

Goldstein, R.S. 2008, "Peripheral sensory neurons differentiate from neural pre- cursors derived from human embryonic stem cells", Differentiation, vol. 76, no. 2, pp. 145-155.

[3] Carr-Wilkinson, J., Prathalingam, N., Pal, D., Moad, M., Lee, N., Sundaresh, A., Forgham, H., James, P., Herbert, M., Lako, M. & Tweddle, D.A. 2018, "Differenti- ation of Human Embryonic Stem Cells to Sympathetic Neurons: A Potential Model for Understanding Neuroblastoma Pathogenesis", Stem cells international, vol. 2018, pp. 4391641.

[4] Chambers, S.M., Fasano, C.A., Papapertrou, E.P., Tomishima, M., Studer, L. &

Sadelain, M. 2009, "Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling", Nature biotechnology; Nat Biotechnol, vol.

27, no. 3, pp. 275-280.

[5] Chambers, S.M., Qi, Y., Mica, Y., Lee, G., Zhang, X., Niu, L., Bilsland, J., Cao, L., Stevens, E., Whiting, P., Shi, S. & Studer, L. 2012, "Combined small-molecule inhibition accelerates developmental timing and converts human pluripotent stem cells into nociceptors", Nature biotechnology, vol. 30, no. 7, pp. 715-720.

[6] Derynck, R., Zhang, Y. & Feng, X. 1998, "Transcriptional Activators of TGF-β Re- sponses: Smads", Cell, vol. 95, no. 6, pp. 737-740.

[7] Fenchel, T., Lanza, R.P. & Atala, A. 2013, Handbook of stem cells, 2nd edn, Elsevier Academic Press, London.

[8] Frith, T.J.R., Granata, I., Wind, M., Stout, E., Thompson, O., Neumann, K., Stav- ish, D., Heath, P.R., Ortmann, D., Hackland, J.O.S., Anastassiadis, K., Gouti, M., Briscoe, J., Wilson, V., Johnson, S.L., Placzek, M., Guarracino, M.R., Andrews, P.W. & Tsakiridis, A. 2018, "Human axial progenitors generate trunk neural crest cells in vitro", eLife, vol. 7.

[9] Huang, M., Miller, M.L., McHenry, L.K., Zheng, T., Zhen, Q., Ilkhanizadeh, S., Conklin, B.R., Bronner, M.E. & Weiss, W.A. 2016, "Generating trunk neural crest from human pluripotent stem cells", Scientific reports, vol. 6, no. 1, pp. 19727.

(29)

[10] images.theconversation.com, Available in: https://images.theconversation.com/files/251037/original/file-20181217-185240-at9go0.png?ixlib=rb- 1.1.0&q=45&auto=format&w=1000&fit=clip, Accessed: 11.3.2021

[11] Jiang, X., Gwye, Y., McKeown, S.J., Bronner-Fraser, M., Lutzko, C. & Lawlor, E.R. 2009, "Isolation and Characterization of Neural Crest Stem Cells Derived From In Vitro–Differentiated Human Embryonic Stem Cells", Stem Cells and De- velopment, vol. 18, no. 7, pp. 159-1071.

[12] Kawasaki, H., Mizuseki, K., Nishikawa, S., Kaneko, S., Kuwana, Y., Nakanishi, S., Nishikawa, S. & Sasai, Y. 2000, "Induction of Midbrain Dopaminergic Neurons from ES Cells by Stromal Cell–Derived Inducing Activity", Neuron, vol. 28, no. 1, pp. 31-40.

[13] Kirino, K., Nakahata, T., Taguchi, T. & Saito, M.K. 2018, "Efficient derivation of sympathetic neurons from human pluripotent stem cells with a defined condition", SCIENTIFIC REPORTS, vol. 8, no. 1, pp. 1-11.

[14] Lanza, R., Langer, R. & Vacanti, J.P. 2013, Principles of Tissue Engineering:

Fourth Edition, .

[15] Lee, G., Kim, H., Elkabetz, Y., Al Shamy, G., Panagiotakos, G., Barberi, T., Ta- bar, V. & Studer, L. 2007, "Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells", Nature biotechnology, vol.

25, no. 12, pp. 1468-1475.

[16] Menendez, L., Kulik, M.J., Page, A.T., Park, S.S., Lauderdale, J.D., Cunningham, M.L. & Dalton, S. 2013, "Directed differentiation of human pluripotent cells to neural crest stem cells", Nature Protocols, vol. 8, no. 1, pp. 203-12.

[17] Mizuseki, K., Sakamoto, T., Watanabe, K., Muguruma, K., Ikeya, M., Nishiyama, A., Arakawa, A., Suemori, H., Nakatsuji, N., Kawasaki, H., Murakami, F. & Sasai, Y. 2003, "Generation of neural crest-derived peripheral neurons and floor plate cells from mouse and primate embryonic stem cells", Proceedings of the National Academy of Sciences, vol. 100, no. 10, pp. 5828-5833.

[18] Muñoz-Sanjuán, I. & Brivanlou, A.H. 2002, "NEURAL INDUCTION, THE DE- FAULT MODEL AND EMBRYONIC STEM CELLS", Nature Reviews.Neurosci- ence, vol. 3, no. 4, pp. 271-80.

[19] Noback, C.R., Ruggiero, D.A., Demarest, R.J. & Strominger, N.L. 2007, The Hu- man Nervous System : Structure and Function, Sixth edn, Humana Press, To- towa, NJ.

[20] Pomp, O., Brokhman, I., Ben-Dor, I., Reubinoff, B. & Goldstein, R.S. 2005, "Gen- eration of peripheral sensory and sympathetic neurons and neural crest cells from human embryonic stem cells", Stem cells (Dayton, Ohio), vol. 23, no. 7, pp.

923-930.

(30)

[21] Schoenwolf, G.C., Bleyl, S.B., Brauer, P.R., Francis-West, P. & Larsen, W.J.

2015, Larsen's human embryology, 5th edn, Elsevier/Churchill Livingstone, Phila- delphia, PA.

[22] sciencetrends.com, Bolano A. / Pixabay, Available in: https://sciencetrends.com/labeled-neuron-diagram/, Accessed: 12.3.2021

[23] Stuhlmiller, T.J. & García-Castro, M.I. 2012, "Current perspectives of the signaling pathways directing neural crest induction", Cellular and Molecular Life Sci- ences, vol. 69, no. 22, pp. 3715-3737.

[24] Takayama, Y., Kushige, H., Akagi, Y., Suzuki, Y., Kumagai, Y. & Kida, Y.S. 2020,

"Selective Induction of Human Autonomic Neurons Enables Precise Control of Cardiomyocyte Beating", Scientific reports, vol. 10, no. 1, pp. 9464.

[25] Tiveron, M., Hirsch, M. & Brunet, J. 1996, "The Expression Pattern of the Tran- scription Factor Phox2 Delineates Synaptic Pathways of the Autonomic Nervous System", The Journal of neuroscience; J Neurosci, vol. 16, no. 23, pp. 7649- 7660.

[26] Winbo, A., Ramanan, S., Eugster, E., Jovinge, S., Skinner, J.R. & Montgomery, J.M. 2020, "Functional coculture of sympathetic neurons and cardiomyocytes derived from human-induced pluripotent stem cells", American journal of physiol- ogy.Heart and circulatory physiology; Am J Physiol Heart Circ Physiol, vol. 319, no. 5, pp. H927-H937.

[27] Wu, H.F. & Zeltner, N. 2020, "Efficient Differentiation of Postganglionic Sympa- thetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions", Journal of Visualized Experiments, , no.

159.

Differentiation of Peripheral Sympathetic Neurons

Silja Salokorpi