Talk:ANAT2341 Lab 11

From Embryology

2017

Wnt/β-catenin signaling promotes differentiation, not self-renewal, of human embryonic stem cells and is repressed by Oct4.

<pubmed>22392999</pubmed>

In studies of human embryonic stem cells (hESCs), conflicting reports claim Wnt/β-catenin signaling promotes either self-renewal or differentiation. We use a sensitive reporter to establish that Wnt/β-catenin signaling is not active during hESC self-renewal. Inhibiting this pathway over mul- tiple passages has no detrimental effect on hESC maintenance, whereas activating signaling results in loss of self-renewal and induction of mesoderm lineage genes. Following exposure to pathway agonists, hESCs exhibit a delay in activation of β-catenin signaling, which led us to postulate that Wnt/β-catenin signaling is actively repressed during self-renewal. In support of this hypothesis, we demonstrate that OCT4 represses β-catenin signaling during self-renewal and that targeted knockdown of OCT4 activates β-catenin signaling in hESCs. Using a fluorescent reporter of β-catenin signaling in live hESCs, we observe that the reporter is activated in a very heterogeneous manner in response to stimulation with Wnt ligand. Sorting cells on the basis of their fluorescence reveals that hESCs with elevated β-catenin signaling express higher levels of differentiation markers. Together these data support a dominant role for Wnt/β-catenin signaling in the differentiation rather than self-renewal of hESCs.

  • Oct4 - https://www.omim.org/entry/164177
  • Transcription factors containing the POU homeodomain
  • Yamanaka factor - Induced pluripotent stem (iPS) cells can be generated
  • CD9 - transmembrane 4 superfamily, also known as the tetraspanin family. https://www.ncbi.nlm.nih.gov/gene/928
  • mesoderm marker expression - how was this measured?
  • why measure beta catenin expression?

Speaker 1

  • Self-renewal vs differentiation, surface markers, previous research

Speaker 2

  • activation of pathway
  • not required for self renewal
  • mesoderm marker expression

Speaker 3

  • BAR - beta catenin activated reporter
  • Wnt3A stim or GSK3 inhibitor

Speaker 4

  • delay in activating
  • Oct4 represses wnt


OMIM: OCT4 | WNT3A | AXIN1

The Retinoblastoma pathway regulates stem cell proliferation in freshwater planarians

<pubmed>23123964</pubmed>

Freshwater planarians are flatworms of the Lophotrochozoan superphylum and are well known for their regenerative abilities, which rely on a large population of pluripotent adult stem cells. However, the mechanisms by which planarians maintain a precise population of adult stem cells while balancing proliferation and cell death, remain to be elucidated. Here we have identified, characterized, and functionally tested the core Retinoblastoma (Rb) pathway components in planarian adult stem cell biology. The Rb pathway is an ancient and conserved mechanism of proliferation control from plants to animals and is composed of three core components: an Rb protein, and a transcription factor heterodimer of E2F and DP proteins. Although the planarian genome contains all components of the Rb pathway, we found that they have undergone gene loss from the ancestral state, similar to other species in their phylum. The single Rb homolog (Smed-Rb) was highly expressed in planarian stem cells and was required for stem cell maintenance, similar to the Rb-homologs p107 and p130 in vertebrates. We show that planarians and their phylum have undergone the most severe reduction in E2F genes observed thus far, and the single remaining E2F was predicted to be a repressive-type E2F (Smed-E2F4-1). Knockdown of either Smed-E2F4-1 or its dimerization partner Dp (Smed-Dp) by RNAi resulted in temporary hyper-proliferation. Finally, we showed that known Rb-interacting genes in other systems, histone deacetylase 1 and cyclinD (Smed-HDAC1; Smed-cycD), were similar to Rb in expression and phenotypes when knocked down by RNAi, suggesting that these established interactions with Rb may also be conserved in planarians. Together, these results showed that planarians use the conserved components of the Rb tumor suppressor pathway to control proliferation and cell survival.

  • A planarian is one of many flatworms of the Turbellaria class.
  • Retinoblastoma 8 Tumor suppressor protein,
    • can both positively and negatively regulate transcription. (PMID: 10517868)
    • positively - ability to promote differentiation
    • negatively - ability to regulate entry into S-phase.
  • RNA interference - how does this work?
  • ISH - measures what?

Speaker 1

  • Rb, E2F, DP

Speaker 2

  • loss of Cyclin E

Speaker 3

  • phylogenic data
  • piwi-1
  • RNA interference

Speaker 4

  • cell proliferation changes with RNAi

Speaker 5

  • pathway evolution
  • stem cell lineage development

Cell Cycle

Single Adult Kidney Stem/Progenitor Cells Reconstitute 3-Dimensional Nephron Structures in Vitro

<pubmed>25422083</pubmed>

(Group 2)

The kidneys are formed during development from two distinct primordial tissues, the metanephric mesenchyme and the ureteric bud.

Morphological analyses revealed that these kidney-like structures contained every substructure of the kidney, including glomeruli, proximal tubules, the loop of Henle, distal tubules, and collecting ducts, but no vasculature. Our results demonstrate that a cluster of tissue stem/progenitor cells has the ability to reconstitute the minimum unit of its organ of origin by differentiating into specialized cells in the correct location. This process differs from embryonic kidney development, which requires the mutual induction of two different populations of progenitors, metanephric mesenchymal cells and ureteric bud cells.

What could they NOT do?

The kidney-like structures could not show the vascularization and make urine.

Speaker 1

  • kidney embryology
  • nephron structure
  • KS cells

Speaker 2

  • characterise KS cells
  • KS markers
  • growth factors for diff - GDNF, b-FGF, HGF, EGF and BMP-7

Speaker 3

  • 3d culture morphology
  • distal tubule ciliated

Speaker 4

  • molecular markers of components

Speaker 5

  • conclusions


In vivo rescue of the hematopoietic niche by pluripotent stem cell complementation of defective osteoblast compartments

<pubmed>28741855</pubmed>

Bone-forming osteoblasts play critical roles in supporting bone marrow hematopoiesis.

embryonic stem cells (ESCs) and induced PSCs (iPSC), are capable of differentiating into osteoblasts.

Moreover, PSC contribution to long bones successfully restored bone marrow hematopoiesis.

In summary, we demonstrate that fractional contribution of PSCs in vivo is sufficient to complement and reconstitute an osteoblast-deficient skeleton and hematopoietic marrow. Further investigation using genetically modified PSCs with conditional loss of gene function in osteoblasts will enable us to address the specific roles of signaling mediators to regulate bone formation and hematopoietic niches in vivo.


Osteoblast embryonic origin

mesenchymal progenitors to osteoblast lineage

Bone is constructed through 3 processes: osteogenesis, modeling, and remodeling. All these processes are mediated by osteoblasts, which work in tight cooperation with bone-resorbing osteoclasts, together constituting a “bone multicellular unit”


(Group 3)

Speaker 1

  • intro slide - lots of text
  • PCS

Speaker 2

  • methodology
  • KO technique

Speaker 3

  • ESC and iPC on mineralisation rescue
  • OSX kill off

Speaker 4

  • discussion

Adipose-derived stem cells attenuate pulmonary microvascular hyperpermeability after smoke inhalation

<pubmed>28982177</pubmed>

(group 5)

Pulmonary edema is a hallmark of acute respiratory distress syndrome (ARDS). Smoke inhalation causes ARDS, thus significantly increasing the mortality of burn patients. Adipose-derived stem cells (ASCs) exert potent anti-inflammatory properties. The goal of the present study was to test the safety and ecfficacy of ASCs, in a well-characterized clinically relevant ovine model of ARDS

Female sheep


ASCs infusion was well tolerated. The results suggest that intravenous ASCs modulate pulmonary microvascular hyper-permeability and prevent the onset of ARDS in our experimental model.


Pulmonary edema - is an abnormal buildup of fluid in the lungs. This buildup of fluid leads to shortness of breath.


6. Stem Cell Therapy for Craniofacial Bone Regeneration: A Randomized, Controlled Feasibility Trial 2013 (Cell Transplantation)


Stem cell therapy offers potential in the regeneration of craniofacial bone defects; however, it has not been studied clinically.


Tissue repair cells (TRCs) isolated from bone marrow represent a mixed stem and progenitor population enriched in CD90- and CD14-positive cells.


Transplantation of TRCs for treatment of alveolar bone defects appears safe and accelerates bone regeneration, enabling jawbone reconstruction with oral implants.


CD90 - Thy 1 major cell surface glycoprotein characteristic to T cells


CD14 - MONOCYTE DIFFERENTIATION ANTIGEN CD14 is a single-copy gene encoding 2 protein forms: a 50- to 55-kD glycosylphosphatidylinositol-anchored membrane protein (mCD14) and a monocyte or liver-derived soluble serum protein (sCD14) that lacks the anchor.

Speaker 1

  • intro

Speaker 2

  • methods

Speaker 3

  • catheter measurements
  • ASC effect

Speaker 4

  • results continued

Speaker 5

  • discussion
  • issues/shortfall

Stem cell therapy for craniofacial bone regeneration: a randomized, controlled feasibility trial

<pubmed>23123964</pubmed>

(group 1)

Stem cell therapy offers potential in the regeneration of craniofacial bone defects; however, it has not been studied clinically.

Tissue repair cells (TRCs) isolated from bone marrow represent a mixed stem and progenitor population enriched in CD90- and CD14-positive cells.


Transplantation of TRCs for treatment of alveolar bone defects appears safe and accelerates bone regeneration, enabling jawbone reconstruction with oral implants.

CD90 - Thy 1 major cell surface glycoprotein characteristic to T cells

CD14 - MONOCYTE DIFFERENTIATION ANTIGEN CD14 is a single-copy gene encoding 2 protein forms: a 50- to 55-kD glycosylphosphatidylinositol-anchored membrane protein (mCD14) and a monocyte or liver-derived soluble serum protein (sCD14) that lacks the anchor.


Group switched between speakers, not in order.

Speaker 1

  • Introduction

Speaker 2

  • timeline of study

2016

Lab 10 - Stem Cell Presentations 2016
Group Mark Assessor General Comments

Group 1: 15/20

Group 2: 19/20

Group 3: 20/20

Group 4: 19/20

Group 5: 16/20

Group 6: 16/20

The students put great effort in their presentation and we heard a nice variety of studies in stem cell biology and regenerative medicine today. The interaction after the presentation was great.

As general feedback I would like to advise students to:

  • Never discuss M&M as a separate section in journal clubs. I gave this advice prior to the lab, but still most groups did talk through the M&M section.
  • Do not use your slides as cheat sheets, avoid text on slides, know what messages you need to get across, use images to illustrate these
  • Engage with your slides. Talk through them. Point at panels. Gauge your audience’s understanding by making eye contact with them
  • Avoid using abbreviations. Most people do not readily understand these and will lose track

Introduction

This is the last practical class of the course.

  1. Practical class will cover fetal, through to birth and the neonate period of development.
  2. Theory exam example questions.
  3. Revision of any course content.

Human development timeline graph 02.jpg


Textbooks

Embryology

Logo.png Hill, M.A. (2012) UNSW Embryology (12th ed.). Sydney:UNSW.
Fetal Links: fetal | Week 10 | Week 12 | second trimester | third trimester | fetal neural | Fetal Blood Sampling | birth weight | Developmental Origins of Health and Disease | fetal growth restriction | macrosomia | BGD Practical | Medicine Lecture | Science Lecture | Lecture Movie | preterm birth | birth | Category:Human Fetus | Category:Fetal
Historic Embryology  
1940 Fetus Physiology
Carnegie Fetal: 95 | 96 | 142 | 145 | 184 | 211 | 217 | 300 | 362 | 448 | 449 | 538 | 590 | 607 | 625 | 662 | 693 | 847 | 858 | 922 | 928 | 948 | 972 | 1318 | 1388 | 1455 | 1591 | 1597b | 1656 | 1686 | 2250a | 2250b | 3990 | 5652 | 6581 | 7218
Fetal Graphs: Crown-Rump Length (CRL) | Third trimester CRL | Head Circumference | Head Circumference 2nd Trimester | Liver Weight | Pancreas Weight | Thymus Weight | Small Intestine Length | Large Intestine Length | Length and Weight Changes | Fetal Development

The Developing Human: Clinically Oriented Embryology

The Developing Human, 9th edn.jpg Citation: The Developing Human: clinically oriented embryology 9th ed. Keith L. Moore, T.V.N. Persaud, Mark G. Torchia. Philadelphia, PA: Saunders, 2011. (links available to UNSW students)

Larsen's Human Embryology

Larsen's human embryology 4th edn.jpg Citation: Larsen's human embryology 4th ed. Schoenwolf, Gary C; Larsen, William J, (William James). Philadelphia, PA : Elsevier/Churchill Livingstone, c2009. (links available to UNSW students)


Links: Embryology Textbooks


  • Human Embryology (3rd ed.) Larson Chapter 15: Fetal development and the Fetus as Patient p481-499
  • The Developing Human: Clinically Oriented Embryology (8th ed.) Moore and Persaud Chapter 6. The Fetal Period: Ninth Week to Birth
  • Color Atlas of Clinical Embryology (2nd ed.) Moore, Persaud and Shiota Ch3: 9th to 38th weeks of human development p50-68