Driwan Stem Cell Therapy Infprmation Center(continiu)

Dr Iwan Suwandy,MHA
more info contact
iwansuwandy@ gmail.com
all free of charge
this info to all human in the world


There are many cell of blood and immune body are continuously produced throughout life from hemopoietic stem cells residing in the bone marrow.

The ability of these cells to perform this function is why bone-marrow transplants can be used to treat leukemia and other blood or immune disorders.

Researchers in the Stem Cell Physiology Research Unit, located at The Biomedical Research Centre at UBC, are studying the biology of bone marrow stem cells and the immune system.

They are focusing on understanding the molecular mechanisms that control how bone-marrow stem cells self-renew and how they differentiate into and function as specific types of blood cells.

Their long-term goal is to understand how defence, repair, and regeneration are regulated and how this knowledge can be exploited to benefit health and offer new treatments for disease.

The Biomedical Research Centre’s researchers recently made important discoveries about the ways bone marrow stem cells differentiate into various types of cells that can fuse with cells in other tissues – such as brain or muscle – to contribute genes.



Mesenchymal stem cells: the ‘other’ bone marrow stem cells

Last updated: 

20 Jun 2012

Mesenchymal stem cells: the 'other' bone marrow stem cells

Mesenchymal stem cells (MSCs) can make several types of cells belonging to our skeletal tissues, such as cartilage, bone and fat. Scientists are investigating how MSCs might be used to treat bone and cartilage diseases. Some MSC research is also exploring therapies for other diseases, but the scientific basis for these applications has not yet been established or widely accepted.

Did you know?

Mesenchymal stem cells make up about 0.001-0.01% of all the cells in your bone marrow

Human mesenchymal stem cells grown in a single layer on the bottom of a flask; 4x magnificationHuman mesenchymal stem cells grown in a single layer on the bottom of a flask; 4x magnification

Human mesenchymal stem cells grown in a single layer on the bottom of a flask; 10x magnificationHuman mesenchymal stem cells grown in a single layer on the bottom of a flask; 10x magnification

Bone cells made from MSCs; the colour is from a stain used to mark the bone cells (von Kossa stain) Bone cells made from MSCs; the colour is from a stain used to mark the bone cells (von Kossa stain)

Fat cells made from MSCs; the colour is from a stain called Nile red O that marks fat cells red Fat cells made from MSCs; the colour is from a stain called Nile red O that marks fat cells red

Cartilage cells made from MSCs; cartilage cells are stained red using the dye Safranin O Cartilage cells made from MSCs; cartilage cells are stained red using the dye Safranin O

Cartilage cells made from MSCs; the cartilage cells are marked brown by a process called immunostainingCartilage cells made from MSCs; the cartilage cells are marked brown by a process called immunostaining

What can mesenchymal stem cells do?

Mesenchymal stem cells (MSCs) are an example of tissue or ‘adult’ stem cells. They are ‘multipotent’, meaning they can produce more than one type of specialized cell of the body, but not all types. MSCs make the different specialized cells found in the skeletal tissues. For example, they can differentiate − or specialize  −  into cartilage cells (chondrocytes), bone cells (osteoblasts) and fat cells (adipocytes). These specialized cells each have their own characteristic shapes, structures and functions, and each belongs in a particular tissue.

Some early research suggested that MSCs might also differentiate into many different types of cells that do not belong to the skeletal tissues, such as nerve cells, heart muscle cells, liver cells and endothelial cells, which form the inner layer of blood vessels. These results have not been confirmed to date. In some cases, it appears that the MSCs fused together with existing specialized cells, leading to false conclusions about the ability of MSCs to produce certain cell types. In other cases, the results were an artificial effect caused by chemicals used to grow the cells in the lab.

Mesenchymal stem cell differentiation: MSCs can make fat, cartilage and bone cells. They have not been proven to make other types of cells of the body.

Mesenchymal stem cell differentiation: MSCs can make fat, cartilage and bone cells. They have not been proven to make other types of cells of the body.

Where are mesenchymal stem cells found?

MSCs were originally found in the bone marrow. There have since been many claims that they also exist in a wide variety of other tissues, such as umbilical cord blood, adipose (fat) tissue and muscle. It has not yet been established whether the cells taken from these other tissues are really the same as, or similar to, the mesenchymal stem cells of the bone marrow.

The bone marrow contains many different types of cells. Among them are blood stem cells (also called hematopoietic stem cells; HSCs) and a variety of different types of cells belonging to a group called ‘mesenchymal’ cells. Only about 0.001-0.01% of the cells in the bone marrow are mesenchymal stem cells.

It is fairly easy to obtain a mixture of different mesenchymal cell types from adult bone marrow for research. But isolating the tiny fraction of cells that are mesenchymal stem cells is more complicated. Some of the cells in the mixture may be able to form bone or fat tissues, for example, but still do not have all the properties of mesenchymal stem cells. The challenge is to identify and pick out the cells that can both self-renew (produce more of themselves) and can differentiate into three cell types – bone, cartilage and fat. Scientists have not yet reached a consensus about the best way to do this.

Developing new treatments using mesenchymal stem cells

No treatments using MSCs are yet available. However, several possibilities for their use in the clinic are currently being explored.

Bone and cartilage repair
The ability of MSCs to differentiate into bone cells called osteoblasts has led to their use in early clinical trials investigating the safety of potential bone repair methods. These studies are looking at possible treatments for localized skeletal defects (damage at a particular place in the bone).

Other research is focussed on using MSCs to repair cartilage. Cartilage covers the ends of bones and allows one bone to slide over another at the joints. It can be damaged by a sudden injury like a fall, or over a long period by a condition like osteoarthritis, a very painful disease of the joints. Cartilage does not repair itself well after damage. The best treatment available for severe cartilage damage is surgery to replace the damaged joint with an artificial one. Because MSCs can differentiate into cartilage cells called chondrocytes, scientists hope MSCs could be injected into patients to repair and maintain the cartilage in their joints. Researchers are also investigating the possibility that transplanted MSCs may release substances that will tell the patient’s own cells to repair the damage.

Many hurdles remain before this kind of treatment can become a reality. For example, when MSCs are transplanted, most of them are rapidly removed from the body. Researchers are working on new techniques for transplanting the cells, such as developing three-dimensional structures or scaffolds that mimic the conditions in the part of the body where the cells are needed. These scaffolds will hold the cells and encourage them to differentiate into the desired cell type.

Heart and blood vessel repair
Some studies in mice suggest that MSCs can promote formation of new blood vessels in a process called neovascularisation. MSCs do not make new blood vessel cells themselves, but they may help with neovascularisation in a number of ways. For example, they may release proteins that stimulate the growth of other cells called endothelial precursors – cells that will develop to form the inner layer of blood vessels. Such studies on animals have led researchers to hope that MSCs may provide a way to repair the blood vessel damage linked to heart attacks or diseases such as critical limb ischaemia. A number of early stage clinical trials using MSCs in patients are currently underway but it is not yet clear whether the treatments will be effective.

Inflammatory and autoimmune diseases
Several claims have been made that MSCs are able to avoid detection by the immune system and can be transplanted from one patient to another without risk of immune rejection by the body. However, these claims have not been confirmed by other studies. It has also been suggested that MSCs may be able to slow down the multiplication of immune cells in the body to reduce inflammation and help treat transplant rejection or autoimmune diseases. Again, this has yet to be proven and much more evidence is needed to establish whether MSCs could really be used for this kind of application.

Current research and the future

Research into therapies using MSCs is still in its infancy. A great deal more work is needed before such therapies can be used routinely in patients. Questions remain about how the cells can be controlled, how they will behave when transplanted into the body, how they can be delivered to the right place so that they work effectively and so forth. By studying how these cells work and interact within the body, researchers hope to develop safe and effective new treatments in the future..


Primary Investigator: 

Stem Cell Physiology and Pathophysiology

 Diabetes  and Hemopoetic Stem Cell
Why Diates impairs the interactions between long-term hematopoietic stem cells ?
to answer this question pleae read carefulyy the abstrct of this reseach

Diabetes impairs the interactions between long-term hematopoietic stem cells and osteopontin-positive cells in the endosteal niche of mouse bone marrow.


Hematopoietic stem cells (HSCs) are maintained, and their division/proliferation and quiescence are regulated in the microenvironments, niches, in the bone marrow.

Although diabetes is known to induce abnormalities in HSC mobilization and proliferation through chemokine and chemokine receptors, little is known about the interaction between long-term



osteopontin-positive (OPN) cells


endosteal niche.

To examine this interaction, LT-HSCs and OPN cells were isolated from streptozotocin-induced diabetic and nondiabetic mice.

In diabetic mice, we observed a reduction in the number of LT-HSCs and OPN cells and impaired expression of Tie2, β-catenin, and N-cadherin on LT-HSCs and β1-integrin, β-catenin, angiopoietin-1, and CXCL12 on OPN cells.

In an in vitro coculture system,

LT-HSCs isolated from nondiabetic mice exposed to diabetic OPN cells showed abnormal mRNA expression levels of Tie2 and N-cadherin. Conversely,

in LT-HSCs derived from diabetic mice exposed to nondiabetic OPN cells, the decreased mRNA expressions of Tie2, β-catenin, and N-cadherin were restored to normal levels.

The effects of diabetic or nondiabetic OPN cells on LT-HSCs shown in this coculture system were confirmed by the coinjection of LT-HSCs and OPN cells into bone marrow of irradiated nondiabetic mice.

Our results

provide new insight into

the treatment of

diabetes-induced LT-HSC abnormalities

and suggest that

the replacement of OPN cells may represent a novel treatment strategy.

to understan this situation we must understand

the physiology of normal cell


  • The typical cell comprises a nucleus and cytoplasm, separated by the nuclear membrane
  • The cytoplasm is separated from interstitial fluid by the cell membrane
  • The different substances making up a cell are termed its “protoplasm” and include:
    • water (usually 70%-85%)
    • electrolytes – chiefly potassium, magnesium, phosphate, sulphate, bicarbonate, and a little sodium, chloride and calcium
    • proteins (usually 10%-20%) – structural and globular, including enzymes
    • lipids (usually 2%) – particularly phospholipids, cholesterol, triglycerides, neutral fats
    • carbohydrates (usually 1%) – usually as glycogen
  • The cell and its organelles are surround by membranes composed of lipids and proteins
    • these include the cell membrane, nuclear membrane, and membranes of the ER, mitochondria, lysosomes and Golgi apparatus
    • each prevents free movement of water and water-soluble substances between cell compartments
  • The cell membrane is a lipid bilayer with inserted proteins
    • the lipid bilayer is mostly phospholipids and cholesterol
    • it is permeable to lipid-soluble substances, but is a major barrier to water-soluble substances such as ions
    • integral proteins protrude through the membrane, while peripheral proteins are attached to the inner surface
    • many integral proteins form structural channels, or carrier proteins
    • peripheral proteins are usually enzymes
    • the membrane is studded with glycoproteins, which are thought to repel other negatively charged molecules, allow attachment to some other cells and act as receptors for binding hormone
  • The ER synthesizes substances in the cell
    • aided by a large surface area
    • materials made include proteins, carbohydrates, lipids, lysosomes, peroxisomes, and secretory granules
    • lipids are made within the ER wall
    • proteins are made by mRNA attaching to ribosomes on the outer surface of the ER
  • The Golgi apparatus is prominent in secretory cells
    • synthesis of lysosomes, secretory vesicles and other cytoplasmic inclusions
    • transport vesicles are pinched-off bits of ER which then fuse with the Golgi apparatus
  • Lysosomes provide the cell with a digestive system
    • small round vesicles containing digestive enzymes
    • usually cordoned-off by a non-reactive membrane
  • Mitochondria release energy in the cell
    • via the citric acid cycle and oxidative enzymes, producing ATP
    • self-replicative and almost certainly originally symbiotic
  • The nucleus acts as a control centre of the cell and contains large amounts of DNA
    • separated from the cytoplasm by a nuclear envelope, pierced by several thousand pores
    • most also contain one or more nucleoli, which do not have membranes; these contain large amounts of RNA, and are enlarged in cells actively synthesizing proteins

Ingestion by the Cell – Endocytosis

  • Small particles enter the cell through diffusion or active transport. Very large particles enter by endocytosis, either pinocytosis or phagocytosis
  • Pinocytosis is the ingestion of small globules of extracellular fluid, forming minute vesicles in the cytoplasm
  • Phagocytosis is the ingestion of large particles such as bacteria, cells, and bits of degenerating tissue
  • Substances so absorbed are digested in the cell by lysosomes. The products of digestion are small amino acids, lipids, glucose, phosphate

Synthesis of Cellular Structures

  • The synthesis of most structures begins in the ER. The rough ER is so named because large numbers of ribosomes attach to the outer surface. Small ER vesicles continually break off from the smooth ER and migrate to the Golgi apparatus.

Extraction of Energy by Mitochondria

  • Almost all oxidative reactions occur inside mitochondria and the energy released is in the form of ATP. ATP has two very labile high-energy phosphate bonds (12,000 calories per mole)
  • Most of the ATP produced in cells is made by mitochondria. About 5% is made by glycolysis, the rest by the citric acid cycle. The process is called the “chemosmotic mechanism”
  • ATP is used for many cellular functions, particularly membrane transport (Na-K-ATPase), synthesis of chemical compounds, and mechanical work

Locomotion and Ciliary Movements by Cells

  • Amoeboid locomotion is the movement of an entire cell in relation to its surroundings (eg. a white blood cell through tissues). It begins with protrusion of a pseudopodium from one end of the cell, with continual exocytosis, and continual endocytosis of the membrane at the mid and rear portions of the cell.
  • Two effects are essential to forward movement: attachment of the pseudopodium, and adequate energy for the movement. In the cytoplasm are molecules of the protein actin, which polymerize to form a filamentous network that contracts on binding to another protein such as myosin.
  • Amoeboid movement is initiated usually by a chemotactic factor. The process is called chemotaxis.
  • Ciliary movement is a whip-like movement of cilia on the surface of cells (only in the respiratory tract, and in the uterine tubules). The mechanism is not known.

Genetic Control of Cell Function

  • Cell genes control protein synthesis. Each gene is a double-stranded helical molecule of DNA that controls the formation of RNA. RNA spreads through the cell to control the formation of a specific protein.
  • Nucleotides are organized to form two strands of DNA bound loosely to each other. There are three building blocks: phosphoric acid, deoxyribose (a sugar), and four nitrogenous bases (two purines: adenine and guanine; two pyrimidines, thymine and cytosine). Adenine always bonds with thymine, and guanine always bonds with cytosine.
  • The genetic code consists of triplets of bases. Each is called a code word. These determine the sequence of amino acids added to the protein.
  • DNA code is transferred to RNA code by the process of transcription. It occurs in the nucleus, where each DNA code word forms a complementary RNA triplet called a codon. The basic building blocks are almost the same except ribose replaces deoxyribose, and uracil replaces thymine.
  • The next step is the activation of the nucleotide, with the addition of two phosphate radicals derived from ATP. It makes available large amounts of energy to promote the reactions that add each new RNA nucleotide to the end of the RNA chain.
  • The DNA strand is used as a template to assemble the RNA molecule from activated nucleotides, under the influence of the enzyme RNA polymerase.
  • There are three different types of RNA:
    • mRNA, carries the genetic code to the cytoplasm to control the formation of proteins
    • rRNA, which along with proteins forms the ribosomes, the molecules in which proteins are actually assembled
    • tRNA, which transports activated amino acids to the ribosomes – 20 types for 20 amino acids
  • The operons of the DNA strand control biochemical synthesis and are activated by the promoter. The operon is controlled by a repressor and an activator operator, and through negative feedback by the cell product. I have no idea what this paragraph means.

The DNA Genetic System

  • With the exception of some very long-lived cells (eg. nerve cells), most cells need to be able to reproduce their own cell type.
  • Reproduction begins with replication of the DNA. As they are replicated, the DNA strands are repaired and proofread. Mistakes (mutations) rarely escape enzymes that cut out defective bits and replace them with appropriate complementary nucleotides.
  • Entire chromosomes (46, 23 pairs) are replicated. In addition to the DNA, there is a fair bit of protein as histones, molecules around which the DNA is coiled to pack it in. The two newly-formed chromosomes remain temporarily attached to each other at a point called the centromere, near the centre. The duplicated but still-attached chromosomes are called chromatids.


  • This is the process by which the cell splits into two new daughter cells.
  • Two pairs of centrioles, small structures close to one pole of the nucleus, begin to move apart. Microtubules grow radially away from each of the centriole pairs, forming a spiny star called the aster at each end of the cell. The complex of microtubules between the pairs is called the spindle, and the entire set of microtubules plus the pairs of centrioles the mitotic apparatus.
  • Prophase is the beginning of mitosis – while the spindle is forming, the chromosomes become condensed into defined chromosomes
  • Prometaphase is the stage at which microtubular spines puncture and fragment the nuclear envelope, and microtubules become attached to the chromatids at the centromere
  • Metaphase is the stage at which the two asters are pushed farther and farther apart
  • Anaphase is the stage at which the two chromatids of each chromosome are pulled apart at the centromere. All 46 pairs are separated, forming two sets of 46 daughter chromosomes.
  • Telophase is the stage at which the two sets of daughter chromosomes are pulled completely apart. Then the mitotic apparatus dissolves and a new nuclear membrane develops around each set of chromosomes.



Pada sel saraf

selubung Mielin 

Neuron-no labels.png

adalah lapisan fosfolipid 

yang membungkus akson secara konsentrik. 



Myelin Membrane Assembly Is Driven by a Phase Transition of Myelin Basic Proteins Into a Cohesive Protein Meshwork

  • Shweta Aggarwal,
  • Nicolas Snaidero,
  • Gesa Pähler,
  • Steffen Frey,
  • Paula Sánchez,
  • Markus Zweckstetter,
  • Andreas Janshoff,
  • Anja Schneider,
  • Marie-Theres Weil,
  • Iwan A. T. Schaap,
  • Dirk Görlich,
  • Mikael Simons


Rapid conduction of nerve impulses requires coating of axons by myelin. To function as an electrical insulator, myelin is generated as a tightly packed, lipid-rich multilayered membrane sheath. Knowledge about the mechanisms that govern myelin membrane biogenesis is required to understand myelin disassembly as it occurs in diseases such as multiple sclerosis. Here, we show that myelin basic protein drives myelin biogenesis using weak forces arising from its inherent capacity to phase separate. The association of myelin basic protein molecules to the inner leaflet of the membrane bilayer induces a phase transition into a cohesive mesh-like protein network. The formation of this protein network shares features with amyloid fibril formation. The process is driven by phenylalanine-mediated hydrophobic and amyloid-like interactions that provide the molecular basis for protein extrusion and myelin membrane zippering. These findings uncover a physicochemical mechanism of how a cytosolic protein regulates the morphology of a complex membrane architecture. These results provide a key mechanism in myelin membrane biogenesis with implications for disabling demyelinating diseases of the central nervous system.

Author Summary

Myelin is a specialized membrane that covers axons and serves as an insulator to enable the fast conduction of the action potentials.

The importance of myelin membrane is highlighted in demyelinating diseases such as multiple sclerosis, which lead to severe neurological disability.

Here, we describe a physicochemical mechanism of how myelin is generated and assembled.

We find that myelin basic protein (MBP) molecules undergo a phase transition into a cohesive meshwork at the membrane interface, which drives structural changes in the membranes.

We provide evidence that the interaction of myelin basic proteins with the inner leaflet of the myelin bilayer results in charge neutralization and triggers self-association of the protein into larger polymers.

Interactions between MBP molecules are mediated by hydrophobic phenylalanine residues and amyloid-like association.

We propose that phase transition of MBP from a cytoplasmic soluble pool into a cohesive functional amyloid-like assembly is one of the key mechanisms in myelin membrane biogenesis

Sel Schwann

merupakan sel yang membentuk selubung

pada sistem saraf tepi,

sedangkan oligodendrosit merupakan sel yang membentuk selubung yang sama pada sistem saraf pusat.

Selubung mielin merupakan karakteristik dari vertebrata (gnathostome), tetapi juga diangkat oleh evolusi pararel beberapa invertebrata.[1] 

Mielin ditemukan oleh Louis-Antoine Ranvier pada tahun 1878.


LT-HSCs derived from diabetic

Cellular Injury and Adaptation


  • Tissue injury starts with molecular or structural damage to cells. When faced with injury, cells can either adapt, sustain reversible injury, or die. Cellular adaptation occurs when stressors result in a new but altered state that preserves viability of the cell (eg. hypertrophy, atrophy)
  • There are two morphologic patterns of cell death: apoptosis and necrosis
    • apoptosis is characterized by chromatin condensation and fragmentation, occurs singly or in small clusters only, and results in the elimination of unwanted cells during embryogenesis and some physiologic and pathologic states
    • necrosis is characterized by swelling, denaturation of proteins, breakdown of cellular organelles, and cell rupture

Causes of Cellular Injury

  • Hypoxia
    • ischaemia (loss of supply)
    • inadequate oxygenation (respiratory failure)
    • loss of oxygen-carrying capacity of the blood (anaemia, CO poisoning)
  • Physical agents
    • trauma
    • thermal insult
    • radiation
    • electric shock
  • Chemical agents and drugs
    • therapeutics
    • non-therapeutic agents
  • Infectious agents
    • viruses
    • rickettsiae
    • bacteria
    • fungi
    • parasites
  • Immunologic reactions
  • Genetic derangements
  • Nutritional imbalances

General Mechanisms of Cell Injury

  • Oxygen-derived free radicals are produced in many conditions and cause damage to cell structure and function
  • Loss of calcium homeostasis, with increased intracellular calcium – this activates phospholipases, proteases, ATPases and endonucleases
  • ATP depletion
  • Defects in membrane permeability

Ischaemic and Hypoxic Injury

  • The key is early loss of oxidative phosphorylation and ATP generation by mitochondria
  • Decreased ATP stimulates aerobic glycolysis; glycogen is quickly depleted and lactate and inorganic phosphate produced, dropping intracellular pH
  • Manifests histologically as swelling, from:
    • loss of Na-K-ATPase, causing sodium to enter the cell, potassium to leave, and an osmotic gain of water
    • increased intracellular osmotic load from the accumulation of lactate and inorganic phosphate
    • polarized membranes lose their polarity
  • All of these are reversible if oxygenation is restored
  • The sentinel event for irreversible injury seems to be damage to membranes
  • Calcium is probably the most important mediator of biochemical and morphologic alterations leading to cell death
  • Reperfusion injury
    • mechanism is not well understood, but clinically important particularly to myocardial and cerebral infarction
    • definition would be a loss of cells in addition to those irreversibly damaged, by restoration of oxygenation at the end of an ischaemic episode
    • bathing compromised cells in high concentrations of calcium when they can’t fully regulate their internal ionic environment?
    • local recruitment of inflammatory cells, releasing reactive species?
    • incomplete oxygen reduction by damaged mitochondria yielding reactive species?

Free Radical-Induced Injury

  • Free radicals are reactive, unstable species that interact with proteins, lipids and carbohydrates. They are created by absorption of radiant energy, oxidative metabolism, or enzymatic conversion of exogenous chemicals.
  • Important free radicals are superoxide (from oxygen), hydrogen peroxide, hydroxyls and nitric oxide.
  • They damage cells through peroxidation of lipids, cross-linking of proteins by forming disulfide bonds, inactivation of sulfhydryl enzymes, and induction of direct DNA damage
  • Free radical termination is by antioxidants (vitamin E, glutathione, ceruloplasmin, transferrin), or enzymes (superoxide dismutase, catalase, glutathione peroxidase)

Chemical Injury

  • Direct injury. Mercury is a good example, binding to sulfhydryl groups of cell membrane proteins, increasing permeability and inhibiting ATPase-dependent transport.
  • Indirect injury. Conversion to toxic metabolites (carbon tetrachloride).

 Abnormal cell injury in diabetic

Abnormal Cell Injury In Dibetes

Myelin splitting,

Schwann cell injury

and demyelination

in feline diabetic neuropathy



Nerve biopsy samples from two cats with spontaneously occurring diabetes were examined.

The predominant nerve fiber abnormalities observed were restricted to the myelin sheath and Schwann cell.

Reactive, degenerative and proliferative Schwann cell changes were evident but the most striking abnormality encountered was splitting and ballooning of the myelin sheath.

These observations highlight

the significance of Schwann cell injury in the pathogenesis of diabetic neuropathy


Dr Angela Barbour. Discipline of Pathology

University of Adelaide


This material has been reproduced and communicated to you by or on behalf of Adelaide University pursuant to Part

VB of the Copyright Act 1968 (the Act).






Large arteries (macroangiopathy): atherosclerosis and related complications



Arterioles: hyaline arteriolosclerosis



Microangiopathy/microvascular: capillary basement membrane thickening






Diabetic nephropathy



Osteoblasts and Bone Marrow Mesenchymal Stromal Cells Control Hematopoietic Stem Cell Migration and Proliferation in 3D In Vitro Model

  • Ana Paula D. N. de Barros,
  • Christina M. Takiya,
  • Luciana R. Garzoni,
  • Mona Lisa Leal-Ferreira,
  • Hélio S. Dutra,
  • Luciana B. Chiarini,
  • Maria Nazareth Meirelles,
  • Radovan Borojevic,
  • Maria Isabel D. Rossi



Migration, proliferation, and differentiation of hematopoietic stem cells (HSCs) are dependent upon a complex three-dimensional (3D) bone marrow microenvironment. Although osteoblasts control the HSC pool, the subendosteal niche is complex and its cellular composition and the role of each cell population in HSC fate have not been established. In vivo models are complex and involve subtle species-specific differences, while bidimensional cultures do not reflect the 3D tissue organization. The aim of this study was to investigate in vitro the role of human bone marrow–derived mesenchymal stromal cells (BMSC) and active osteoblasts in control of migration, lodgment, and proliferation of HSCs.

Methodology/Principal Findings

A complex mixed multicellular spheroid in vitro model was developed with human BMSC, undifferentiated or induced for one week into osteoblasts. A clear limit between the two stromal cells was established, and deposition of extracellular matrix proteins fibronectin, collagens I and IV, laminin, and osteopontin was similar to the observed in vivo. Noninduced BMSC cultured as spheroid expressed higher levels of mRNA for the chemokine CXCL12, and the growth factors Wnt5a and Kit ligand. Cord blood and bone marrow CD34+ cells moved in and out the spheroids, and some lodged at the interface of the two stromal cells. Myeloid colony-forming cells were maintained after seven days of coculture with mixed spheroids, and the frequency of cycling CD34+ cells was decreased.


Undifferentiated and one-week osteo-induced BMSC self-assembled in a 3D spheroid and formed a microenvironment that is informative for hematopoietic progenitor cells, allowing their lodgment and controlling their proliferation.

Infection -> acute and chronic pyelonephritis



Atherosclerosis related including infarction and renal artery stenosis












Diabetic retinopathy









Macular oedema






Peripheral nerves



Autonomic nerves






Diabetic polyradiculopathy






Ulcers: multifactorial



Impaired sensation due to neuropathy



Predisposition to infection



Impaired blood supply due to atherosclerosis and microangiopathy impairing healing



Necrobiosis lipoidica diabeticorum (rare)






Predisposition to infection (e.g. pulmonary, urinary tract, skin) related to hyperglycaemia and impaired function of

phagocytes and other inflammatory cells




Liver: non-alcoholic steatosis, steatohepatitis and cirrhosis



Acute metabolic complications



Diabetic ketoacidosis (primarily in type 1)



Non-ketotic hyperosmolar coma (type 2)



Hypoglycaemia from too much insulin or hypoglycaemics


Nephropathy, atherosclerosis, neuropathy, ocular complications


Are late complications



Risk increases in relation to duration of hyperglycaemia



Usually become apparent in 2nd decade of hyperglycaemia, may be present at time of diagnosis of type 2 which often

has a long asymptomatic period




Better blood glucose control reduces risk



Other undefined factors modulate the risk e.g. genetic



Nephropathy, neuropathy, retinopathy are largely related to microangiopathy



Connective tissue


Cells include: fibroblasts, myofibroblasts, mast cells, adipocytes, macrophages, undifferentiated mesenchymal cells



Extracellular matrix






Collagen, including reticulin






Ground substance: Glycosaminoglycans bound to proteoglycans



Structural glycoproteins e.g. laminin, fibrillin



Tissue fluid

Connective tissue proper: loose and dense


Specialised connective tissue e.g. adipose tissue, bone, cartilage



Basement membranes (BM)


Epithelial, endothelial and mesothelial cells sit on a BM



Similar material surrounds Schwann cells and various other connective tissue cells (e.g. adipocytes, smooth muscle

cells) – termed basal lamina







Specialised extracellular matrix



Central electron dense layer (lamina densa) with less distinct electron lucent layers (lamina rara) on either side (all

3 together sometimes termed basal lamina)




Delicate network of predominantly type IV collagen in a matrix of glycoproteins (e.g. laminin) and other

extracellular components (e.g. heparan sulphate proteoglycan) largely produced by the overlying cells




+/- reticular layer: collagen type III originating from underlying connective tissue cells



Often too thin to be seen distinctly on H&E with light microscopy except in areas where it is thicker (e.g. trachea)

but can be seen with special stains on light microscopy







Bonds cells to underlying connective tissue



Provides framework for cell development and regeneration



Freely permeable to small molecules but impedes passage of macromolecules



Layers (general):


Intima: endothelium (simple squamous), basement membrane, (+ small amount of connective tissue/elastin in some),

internal elastic lamina (except in smallest arterioles)




Media: variable amounts of smooth muscle and extracellular matrix, predominantly elastin; in some vessels an external

elastic lamina is also present




Adventitia: connective tissue including abundant elastin, vasa vasorum in larger vessels





Large arteries (aorta and it’s main branches): abundant elastic tissue



Medium (distributing arteries) and small (<2mm) arteries: predominantly smooth muscle



Arterioles (diam 100um or less): several layers of smooth muscle cells only

Smooth muscle cells produce the extracellular matrix including elastin




Complex and not fully understood, different pathways that interact. Genetic factors in the patient also have an important



Hyperglycaemia ->



Formation of advanced glycation end products (AGE) – glucose binds irreversibly to protein amino groups. AGE may

also form on lipids, nucleic acids.




AGE modified extracellular matrix components



Lead to protein cross-linking



Are resistant to proteolytic digestion



Trap other proteins (e.g. plasma proteins)



May promote cell damage



Non-enzymatic glycation of haemoglobin: HbA1c – serves as marker of glycemic control



May alter intracellular signalling, gene expression and oxygen derived free radical formation



Increase in aldose reductase pathway



In cells not requiring insulin for glucose uptake e.g. Schwann cells, retinal pericytes, lens of eye



Metabolism of excess intracellular glucose -> excessive glucose metabolites (e.g. sorbitol) with reduced synthesis of

an important antioxidant -> osmotic cell injury and increased susceptibility to oxidative injury




Production of reactive oxygen species ->



Upregulation of growth factor expression e.g. VEGF



Alteration of proteins and lipoproteins



Cell damage



Increased activation of protein kinase C (PKC) signal transduction pathway



May be related to AGE formation



Activation of signal transduction pathways for extracellular matrix protein production



Overactive renin-angiotensin system



Angiotensin II stimulates production of important growth factors e.g. TGF-beta that plays a role in extracellular

matrix formation in the renal mesangium and VEGF is important in proliferative retinopathy




Important in nephropathy and retinopathy

Understanding pathogenesis helps in the development of potential treatments e.g. use of inhibitors of AGE formation,


antioxidants, angiotensin II inhibitors


The simplified version




Increased amounts of extracellular matrix with altered structure and function due to:



Upregulation (various mechanisms) of production of various growth factors and cytokines -> increased extracellular

matrix production




AGE modified extracellular matrix proteins -> cross-linking, plasma protein trapping, reduced proteolytic digestion



Cell damage e.g. via aldose reductase pathway and osmotic injury, activation of reactive oxygen species

The microangiopathy (capillary basement membrane thickening), hyaline arteriolar changes and mesangial thickening in


diabetes are caused by the above processes. Hypertension may play a role in the development of hyaline arteriolosclerosis.



Normal renal glomerular structure


Filtration barrier



Endothelial cells, epithelial cells and basement membrane






Fenestrated with large pores that only prevent blood cells and platelets from passing through



The diaphragm that typically spans fenestrations between endothelial cells in certain other vascular beds is absent



Epithelial cells (podocytes)



Extensive array of processes that interdigitate on outside of capillary with slit like spaces (filtration slits) between



Each filtration slit is spanned by a thin filtration slit diaphragm or membrane



Glomerular capillary basement membrane (BM) of central importance



Made by epithelial and endothelial cells



Composed of collagen type IV, glycoproteins and proteoglycans



No reticular component



Size barrier: molecules larger than about 69,000 daltons unable to cross



Charge barrier: proteoglycan component is anionic (negatively charged), therefore repels negatively charged





Albumin has a molecular weight of 69,000 daltons and is negatively charged






Mesangial cells and extracellular matrix (various collagens and ground substance)






Supports capillary loops



Cells have phagocytic function and clean the basement membrane



May regulate blood flow in capillaries


Diabetic nephropathy


A clinical syndrome in patients with diabetes mellitus characterised by persistent microalbuminuria, worsening proteinuria,

hypertension and progressive deterioration in renal function. The pathological correlate of diabetic nephropathy is diabetic


glomerulosclerosis characterised by a progressive increase in extracellular matrix in the mesangium and capillary basement






Approximately 25-35% of patients with longstanding diabetes (types 1 and 2) develop diabetic nephropathy and renal failure.



Diabetic retinopathy often also present, especially in type 1 diabetes



Clinical and pathological features and progression






Initial increased intraglomerular pressure with hyperfiltration, potentially at least partly related to vasoconstrictor

effect of angiotensin II on efferent arterioles, associated with enlarged glomeruli, commencing within first few years


of onset




Microalbuminuria appears 5–10 years after the onset of diabetes






Onset of overt proteinuria (10-15 years or more after diabetes onset) +/- nephrotic syndrome +/- hypertension,

beginning deterioration in renal function.




Diffuse and nodular diabetic glomerulosclerosis






Diffuse thickening of glomerular capillary BMs (not seen on light microscopy until very thickened)



Diffuse increase in mesangial matrix +/- mild proliferation of mesangial cells



Nodular (Kimmelstiel-Wilson lesion)



Localised nodular areas of increased mesangial matrix with few cells



Less common than diffuse and generally superimposed on diffuse lesion



Insudative glomerular lesions: Eosinophilic nodular accumulations of plasma constituents in capillary loops or in

Bowmans capsule




Hyaline arteriolosclerosis in both afferent and efferent arterioles



Tubular basement membrane thickening



Immunofluorescence: non-specific (not immunologic) trapping of plasma albumin and IgG in tubular and capillary

basement membranes




Most of the changes are not specific for diabetes



Mesangial and glomerular capillary BM thickening related to excessive and altered extracellular matrix:

pathogenesis involves accumulation of AGE products, upregulation of growth factors




Glomerular changes -> narrowing of capillary lumina and glomerular ischaemia, hyaline arteriolosclerosis

contributes to glomerular ischaemia.




Hypertension can contribute to glomerular injury.



Later still: Falling GFR: damaged glomeruli -> impaired renal function. Progressive irreversible glomerulosclerosis,

related tubules atrophy (no blood flow into efferent arterioles and peritubular capillaries) and interstitial tissue undergoes


fibrosis -> further impairment of renal function




End stage renal disease: 5-10 years after development of overt proteinuria (or 10-20 years after onset of

microalbuminuria), however, there is considerable variability between patients. Macroscopically the kidneys may be


slightly enlarged with a granular surface, or small due to co-existent changes related to hypertension or chronic






Pathogenesis of diabetic glomerulosclerosis: complex, at least partly related to microangiopathy (pathogenesis outlined



Various pathways implicated, pathways interact




Haemodynamic factors



Hyperglycaemia -> glomerular hyperfiltration and microalbuminuria, altered tubuloglomerular feedback



Vasoconstrictor effect of angiotensin II more potent on efferent arteriole -> increased glomerular capillary pressure

and filtration




Hypertension may contribute via glomerular hyperfiltration and endothelial and mesangial damage due to

haemodynamic stress




Metabolic factors e.g. AGE mechanism, activation of protein kinase C



Hormonal factors: overactive intrarenal renin angiotensin system (RAS), also contributes to development of



Effects of angiotensin II in kidney




Sodium and water retention by tubules



Can activate other cytokine pathways such as transforming growth factor-beta (TGF-beta) and platelet derived

growth factor (PDGF) systems. TGF-beta-1 stimulates an increase in mesangial matrix deposition and glomerular


basement membrane (GBM) thickening




Impairs nitric oxide function which has vasodilatory and protective role



Can downregulate nephrin, an important protein of the slit diaphragm, leading to proteinuria



Genetic influences




Progression slowed by good blood glucose control, strict blood pressure control, administration of ACE inhibitors or


angiotensin receptor blockers (ARBs) and treatment of dyslipidemia.




Proteinuria and nephrotic syndrome



Structural alterations in and loss of negative charge of glomerular capillary BM allow albumin to pass through



Albuminuria is a marker of greatly increased cardiovascular morbidity and mortality for patients with either type 1 or

type 2 diabetes




Hypertension in diabetics



From diabetic nephropathy



From chronic renal failure



Other risk factors e.g. obesity, familial



Renal artery stenosis (less common)



Chronic renal failure (CRF)



Diabetic glomerulosclerosis and hyaline arteriolosclerosis -> chronic glomerular ischaemia -> glomerular

obsolescence/sclerosis and also chronic renal ischaemia with tubular atrophy and interstitial scarring -> poorly


functioning kidneys




Approx. 30% of patients develop chronic renal failure



Chronic pyelonephritis and hypertension may also cause/contribute to CRF

Remember also that diabetics can get other renal diseases.



Ocular complications


Cataracts: from lens swelling and opacity



Glaucoma: formation of fibrovascular membranes on iris related to ischaemia (from microangiopathy) -> blockage of

outflow channels of aqueous humour




Diabetic retinopathy



Develops in about 75% within 15 years of disease onset



More prevalent among patients with type 1 diabetes than type 2



Often coexists with diabetic nephropathy, but they can also occur independently of each other



Non-proliferative: microvascular changes with pericyte and also ultimately endothelial cell loss, leads to increases in

vascular permeability, vascular weakening and alterations in retinal blood flow, including blockage of capillaries ->


exudates, haemorrhages, microaneurysms, microinfarcts




Macula oedema



Proliferative: ischaemia from microvascular changes -> proliferation of small vessels, initially on surface of retina

and later into the vitreous -> haemorrhages, fibrosis and retinal detachment.




Increased VEGF expression, contributed to by hypoxia and oxidative stress is important in pathogenesis of

proliferative retinopathy




VEGF is an angiogenic growth factor and increases vascular permeability




Distal symmetric sensory or sensorimotor neuropathy



Symmetric, motor and sensory



?from Schwann cell injury -> demyelination



?from axonal injury



?from microangiopathy impairing blood flow and nutrition of nerve



Autonomic neuropathy



-> impotence, bowel and bladder dysfunction etc



?similar pathogenesis



Diabetic polyradiculopathy



Severe disabling pain in the distribution of one or more nerve roots



May be accompanied by motor weakness



Focal or multifocal asymmetric neuropathy/mononeuropathy



Affects larger nerves



?from microangiopathy impairing blood flow and nutrition of nerve




Slow build up over years of lipids and fibrous material in the intima of large and medium sized arteries



In diabetics may extend more distally than usual



Pathogenesis in diabetes



Vascular basement membrane and endothelial changes -> increased permeability, trapping of lipids in intima









Anthony S. Fauci, Eugene Braunwald, Dennis L. Kasper, Stephen L. Hauser, Dan L. Longo, J. Larry Jameson, and Joseph

Loscalzo, Eds. Harrison’s online: Harrison’s Principles of Internal Medicine, 17th Edition. The McGraw-Hill Companies.

Ahmed N. Advanced glycation endproducts—role in pathology of diabetic complications. Diabetes Research and Clinical

Practice 2005: 67: 3–21

Caldwell RB, Bartoli M, Ali Behzadian M, El-Remessy AEB, Al-Shabrawey M. Platt DH, Caldwell RW. Vascular endothelial

growth factor and diabetic retinopathy: pathophysiological mechanisms and treatment perspectives. Diabetes Metab Res Rev

2003; 19: 442–455.

D’ Agati VD, Jennette JC, Silva FG. Non-Neoplastic Kidney Diseases. 2005. American Registry of Pathology Press.

Jawa A, Kcomt J, Fonseca VA. Diabetic nephropathy and retinopathy. Med Clin N Am 2004: 88: 1001–1036

Kumar V, Abbas AK, and Fausto N. Robbins and Cotran Pathologic Basis of Disease. 7th edition, 2005. Elsevier Saunders.

Rubin E, Gorstein F, Rubin R, Schwarting R and Strayer D. Rubin’s Pathology Clinicopathologic Foundations of Medicine. 4




edition, 2005. Lippincott Williams and Wilkins

Tsilibary E.C. Microvascular basement membranes in diabetes mellitus. J Pathol 2003; 200: 537–546.

Wilkinson-Berka JL. Angiotensin and diabetic retinopathy. The International Journal of Biochemistry and Cell Biology 2006:


Morphology of Reversible Cell Injury and Necrosis


  • Two processes cause the basic changes of necrosis: denaturation of proteins, and enzymatic digestion of organelles and cytosol.
  • Autolysis indicates digestion by lysosomal enzymes of the dead cells themselves
  • The necrotic cell is eosinophilic and glassy, maybe vacuolated. The membranes are fragmented. Nuclear changes include pyknosis (small, dense nucleus), karyolysis (faint, dissolved nucleus) and karyorrhexis (broken into bits)
  • Types of necrosis include:
    • coagulative (myocardium, kidney, liver – usually ischaemic)
    • liquefactive (brain and abscesses – autolysis and heterolysis dominate)
    • caseous (tuberculous – soft and cheesy cell debris)
    • fatty (adipose – actions of lipases that decompose triglycerides that complex with calcium to form soaps)


  • Morphological features include cell shrinkage, chromatin condensation and fragmentation, formation of cytosolic blebs and apoptotic bodies, phagocytosis of apoptotic bodies by adjacent healthy cells or macrophages, and a conspicuous lack of inflammation
  • In many instances, it is dependent on gene activation
  • Occurs in the following settings:
    • programmed cell destruction during embryogenesis
    • hormone-dependent involution (eg. endometrium)
    • cell depletion in proliferating populations
    • pathological atrophy in parenchymal organs after duct obstruction
    • cell death by cytotoxic T cells
    • cell injury from some viral diseases
    • mild traumatic insult

Cellular Adaptation to Injury

  • Atrophy = shrinkage in size by the loss of cell substance
    • causes include decreased workload, loss of innervation, diminished blood supply, inadequate nutrition, loss of endocrine stimulation, aging
    • diminished function but not dead – autophagy, reduced organelles, increased number of autophagic vacuoles; components resisting digestion are deposited as lipofuscin granules, giving a brown appearance
  • Hypertrophy = increase in the size of cells (but not their number)
    • causes include increased functional demand, hormonal stimulation
    • triggered by cell membrane interactions and mechanical factors
  • Hyperplasia = increase in the number of cells
    • can be physiologic (endometrial proliferation, hyperplasia of the liver after lobectomy) or pathologic (almost always excessive hormonal stimulation)
    • when the stimulus is removed, the hyperplasia disappears – this is a crucial difference from neoplasia
  • Metaplasia = a reversible change in which one cell type is replaced by another
    • examples include Barretts’ oesophagus, the respiratory tract of smokers
    • thought to occur from genetic reprogramming

Subcellular Responses

  • Lysosomes are involved in digestion of ingested materials through autophagy and heterophagy. It is pronounced in cells undergoing atrophy. Lysosomes with indigestible material may persist within cells or be extruded.
  • Hypertrophy of the smooth ER, such as by barbiturates, increasing the surface area available for mixed-function oxidase pathway.
  • Mitochondrial alterations, in size, number and function.
  • Cytoskeletal abnormalities, which impede transport of organelles and molecules. The basic cell architecture can be disrupted.
  • Induction of heat shock proteins. These play a role in normal intracellular protein housekeeping (eg. folding them). They are thought to be induced to refold damaged proteins or to tag them for destruction (ubiquitin).
  • Intracellular accumulations – fats (steatosis), cholesterols (atheroma), proteins (Mallory body in alcoholic hepatocytes), glycogen (diabetic renal tubular epithelium), pigments (anthracosis).
  • Pathologic calcification, which implies the abnormal deposition of calcium salts in soft tissues. Dystrophic calcification occurs in nonviable tissue, where metastatic calcification is deposition of calcium salts in vital tissues and is associated with hypercalcaemia (hyperparathyroidism, hypervitaminosis D, sarcoidosis, Addison’s disease, hyperthyroidism, bone cancers).
  • Hyaline change, intracellularly or extracellularly. This is deposition of proteinaceous material that stains a glassy-pink on H&E.
What are the  Diabetic OPN cells ?
Whate are the   LT-HSCs ?
  • Diabetes impairs the interactions between long-term hematopoietic stem cells and osteopontin-positive cells in the endosteal niche of mouse bone marrow
    Hironori Chiba, Koji Ataka, Kousuke Iba, Kanna Nagaishi, Toshihiko Yamashita, Mineko Fujimiya
    October 1, 2013
  • Human-induced pluripotent stem cell-derived cardiomyocytes for studies of cardiac ion transporters
    Michael Fine, Fang-Min Lu, Mei-Jung Lin, Orson Moe, Hao-Ran Wang, Donald W. Hilgemann
    September 1, 2013
  • Functional expression of smooth muscle-specific ion channels in TGF-β1-treated human adipose-derived mesenchymal stem cells
    Won Sun Park, Soon Chul Heo, Eun Su Jeon, Da Hye Hong, Youn Kyoung Son, Jae-Hong Ko,Hyoung Kyu Kim, Sun Young Lee, Jae Ho Kim, Jin Han
    August 15, 2013
  • Elevated SOCS3 and altered IL-6 signaling is associated with age-related human muscle stem cell dysfunction
    Bryon R. McKay, Daniel I. Ogborn, Jeff M. Baker, Kyle G. Toth, Mark A. Tarnopolsky, Gianni Parise
    April 15, 2013
  • Autophagy in endothelial progenitor cells is cytoprotective in hypoxic conditions
    Hai-Jie Wang, Dan Zhang, Yu-Zhen Tan, Ting Li
    April 1, 2013
    • in vitro model system for smooth muscle differentiation from human embryonic stem cell-derived mesenchymal cells
      Xia Guo, Steven L. Stice, Nolan L. Boyd, Shi-You Chen


The many cells that make up the blood and immune system


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