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The Haemopoietic Stem Cell

17 May

The Discovery of Haemopoietic Stem Cell

 

Blood was regarded as an important tissue, but till the mid 1800s it was not known how blood cells were made. Three discoveries lead to the modern concept of haemopoietic stem cell.

  1. Neumann and Bizzozero separately, in 1868, proposed that the bone marrow was the site of blood production throughout the postnatal life.
  2. Improvement in staining techniques lead to the discovery of a spectrum of cells in the bone marrow.  Pappenheimer organized these cells into a tree with the most mature cells being at the leaves. He proposed that at the trunk was a cell that was so primitive that it could not be typed into any lineage. Ehrlich and Schilling differed from Pappenheimer in the existence of a single precursor for all blood cells. They proposed that there were two (Ehrlich) and three (Schilling) different precursor for blood cells respectively.
  3. The stem cell defies morphological definition. It is possible to define populations that are rich in stem cells but not all the cells thus defined are stem cells. The next chapter in the definition of haemopoietic stem cell was written when the focus shifted from what stem cells looked to what they could do.  Two discoveries, both by scientists not intending to looking for the haemopoietic stem cell, laid the foundation for the modern concepts of haemopoietic stem cells
    1. Lineage is important in cattle. It provides assurance to a farmer that the calf he is rearing will give a good amount of milk. Calves of good lineage command a high price. In the 1930s and 40s blood groups seemed to be reliable way of determining paternity of an animal. In 1944 a breeder reported twin calves of different sexes but identical blood groups. In 1945 Ray Owen in an attempted to resolve why calves that appeared to have two fathers had identical groups. He discovered that the calves actually had two erythrocyte populations (Genetics 1996; 144:855-59).  Each calf had blood from both the calves. The cattle uterine anatomy allows communication between extra-embryonic circulation of twins.  The only explanation for two types of erythrocytes in each calf was that

i.     Cells capable of producing both types of blood cells were present in the bone marrow of both the calves

ii.     The bone marrow of each calf had been seeded by cells from calls that could produce blood cells and arose from the other calf.

iii.     These cells could produce blood indefinitely

  1. Till and McCoulloch were interested in developing an assay for radiosensitivity of marrow cells. Lethally irradiated animals that survive acute radiation sickness succumb to bone marrow failure. Till and McCoulloch transplanted lethally irradiated mice with bone marrow cells exposed to different doses of radiation. They discovered the following

i.     Animals survive the radiation and develop islands of hematopoiesis in the spleen in the form of nodules.

ii.     The number of the nodules is related to the dose of bone marrow cell infused.

iii.     The colonies contain cells of erythroid and myeloid. The cells show different degree of maturity

iv.     Using chromosomal markers the colonies were shown to arising from a single cell

v.     When cells from the colonies were transplanted to another lethally irradiated mouse cells some colonies were able to establish haemopoietic splenic colonies.
These studies eastablished that the bone marrow had cells that that could self-renew, have a high capacity to proliferate and have capacity to differentiate into multiple lineages defining a haemopoietic stem cell.

Stem-Cell Renewal

 

Identification of the Pleuripotent haemopoietic

Isolation of a pure population of stem cells has not been possible.

The definition of stem cell is a functional definition. The stem cell is defined as a cell that has the capacity to

  1. Self-renewal: Self-renewal is the phenomena of stem cells giving rise to more stem cells. Self-renewal is essential to maintain the stem cell pool and ensure adequate blood production. The stem cells population can be maintained at a constant levels only if half the cell produced as a result mitosis retain the characters of a stem cell and the other half differentiate into blood cells.  If more than half the stem cells differentiate, the stem cell pool will eventually deplete. If less than half the stem cells differentiate there would be an unnecessary accumulation of stem cells. The details of the self-renewal and commitment processes are not completely understood.
  2. Indefinite proliferation: The bone marrow stem cells maintain haematopoiesis for a lifetime.
  3. Capacity to differentiate into many cell types: The haemopoietic stem cell gives rise to the erythrocytes, myelocytes, lymphocytes, monocytes, megakaryocytes and the dendritic cells.

Stem cells are identified by their ability to reconstitute haematopoiesis in a lethally irradiated animal.

Haemopoietic stem cell transplant became a reality in 1968.  Initially the bone marrow was the source of stem cells. Today the peripheral blood and occasionally the cord blood serve as the source of stem cells. The number of stem cell infused determines the success of the transplant. Animal transplantation take too long to be clinically useful.

CD34 is a trans-membrane protein expressed on haemopoietic progenitors till the stage of committed progenitor cells. CD34 expression can rapidly be assessed by flow cytometery. CD34+ cells show the characters of haemopoietic stem cells

  1. Autologous CD34 enriched population has been shown to protect against myeloablative doses of radiation and chemotherapy. CD34- negative cell fail to do so.
  2. Allogenic CD34+ cell are able to establish haematopoiesis. This chemerism remains stable over a prolonged period of time.

Haemopoietic stem cell is a CD34+ cell that lacks lineage commitment markers. These include

  1. T cells: CD2
  2. B cells: CD19
  3. Monocytes: CD14
  4. Granulocytes: CD15
  5. NK Cells: CD16
  6. Erythroid cells: Glycophorin A
  7. Others: CD24, CD56 and CD66b, DR

Not all the CD34+ cells are pleuripotent stem cell but CD34 is a useful marker for estimating the number of stem cells infused during a haemopoietic stem cell transplant.

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Normoblast Maturation

7 Mar Normoblast Maturation

IMG_0354 Maturing Normoblast

The image above shows three polychromatophilic normoblasts. The one on the left is the least mature and the one on the right the most mature. Maturation is associated with

  1. Decrease in the size of the cell and nucleus: This is obvious.
  2. Clumping of chromatin: This is obvious.
  3. Increasing cytoplasmic acidophilia and decreased basophilia: The cells are flanked on each side by erythrocytes. The cytoplasm of the least mature cell (left most cell) is basophilic compared to that of the adjacent erythrocyte. The cytoplasm of the most mature cell (the right most cello) has lost basophilia and is almost the colour of the cytoplasm of the erythrocyte. 

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Orthochromatophilic Normoblast

7 Mar IMG_0286 - Orthochromatophilic Normoblast

Image

Orthochromatophilic normoblast

An orthochromatophilic normoblast is the most mature of nut;elated red cell precursors. The cytoplasm has lost almost all it’s basophilia and is the same colour as that of the erythrocyte. The nucleus is small pyknotic with densely condensed chromatin.

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Embryonic Haemoglobins

24 Oct

Embryonic haemglobins are haemoglobins produced in the yolk sac stage of erythropoiesis. There are three embryonic haemoglobins Gower I, Gower II and Portland. As shown in the figure below, embryonic haemoglobins like their adult counterparts are tetramers made of two α-like chains and two β-like chains. The α-chain is a part of haemoglobins from the embryonic stage and complete loss of α-globin leads to anaemia from early gestation.

Haemoglobin is a tetramer of two α-like and two β-like globin chains. The genes of the α-like globins are found on chromosome 16 (16p13.3) and those for the β-like genes are found on chromosome 11 (11p15.5). The α-like has seven genes ζ2, Ψζ1, μ, Ψα, α2, α1, θ. ζ2 encodes for the ζ chain found in embryonic haemoglobins , α2, α1 encode for alpha chains, Ψζ1 and  Ψα are pseudogenes and the function of μ and θ is unknown. the β-like gene cluster consists of six genes ε, Gγ, Aγ, Ψβ, δ and β. ε gene encodes for the ε chain of haemoglobin Gower I and Gower II, Gγ and Aγ encode for γ chains of HbF, Ψβ is a pseudogene and δ and β encode fro chains found in HbA2 and HbA respectively.

The earliest evidence of erythropoiesis is found in the extra-embryonic yolk sac in the form of blood islands. The erythrocytes produced by the yolk sac are larger, nucleated and contain embryonic haemoglobins (see N Engl J Med 1999; 340-617 for images of embryonic erythropoiesis). The yolk sac is the site for haematopoiesis from 19 days through week 8 of gestation.

The embryonic haemoglobin show co-operative oxygen binding but to a lesser extent than the adult haemoglobin. This results in a high affinity which reflect in a lower P50. The P50 of haemoglobin Gower I is 4mm, Gower II is 12mm and Portland is 6mm. The values for foetal haemoglobin is 19mm and adult haemoglobin is 26mm.

The ζ and ε are involved in large deletions resulting in thalassaemia. These deletions manifest as α or β thalassaemias respectively. There are no symptoms referable to the ζ and ε genes. Deletions of genes for embryonic haemoglobins is fatal. These embryos are presumed to be lacking haemoglobin and die of hypoxia. Detection of ζ chains in the blood has a role in diagnosis of α thalassaemia.

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Megaloblasts

25 Jul Megaloblasts

Development of erythrocytes involves coordinated changes in the nucleus and the cytoplasm of erythroid processors (see Morphology of Erythroid Precursors) . The proerythroblast is a large cell with a fine chromatin, the earliest forms not being different from other blasts. As the cell matures the chromatin becomes more clumped and the nucleus reduces in size and the cytoplasm becomes acidophilic. Finally a dark pyknotic nucleus is extruded from the orthchromatophilic normoblast to give a reticulocyte.

Figure 1. Basophilic Normoblast

A group of basophilic normoblast are shown above. The cytoplasm is basophilic and the chromatin more clumped than a proerythroblast. The two cells on the right are less mature than the two on the left.

Figure 2. A group of orthochromatophilic normoblasts

The figure above show a group of  orthochromatophilic normoblasts. The cells of the left are more mature. The nucleus is reduced to a dense body in the more mature forms. The cytoplasm still has a blue tinge. which contrasts from the megaloblasts shown below.

Figure 3. Basophilic, polychromatophilic and orthochromatophilic normoblasts

The figure above shows three stages of erythroid maturation. The cell on the top right is a basophilic normoblast, bottom right is a orthochromatophilic normoblast and the bottom right is an orthochromatophlic normoblast. Note the evolution of nuclear and cytoplasmic changes.

Figure 4. Megaloblasts

The figure above shows a group of megaloblasts. Cells in the right lower corner have a cytoplasm which is fully haemoglobinized and resembles the mature erythrocyte. These cell still have a nucleus. The cell on the left upper corner has a cytoplasm resembling a orthochromatophilc normoblast (see figure 2 and 3) but nuclear features resembling a basophilic normoblast (figures 1 and 2). Megaloblastic anaemia results from conditions that hamper DNA synthesis (B12 deficiency, folate deficiency, Chemotherapy drugs). The nucleus of a megaloblast thus matures slower than the cytoplasm resulting in cells having a nuclear morphology resembling a previous stage. This, known as nucleo-cytoplasmic dissociation, is the characteristic feature of megaloblastic anaemia.

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Basophilic and Polychromatophilic Normoblasts

22 May Baso-Poly-Normo-250px-IMG_0507

ImageThe adjacent image shows three erythroid cells, two polychromatophilic normoblasts on the sides and a basophilic normoblasts between the two cells.

The color of cytoplasm of erythroid cells is balance between blue staining due to ribosomes and pink-red staining of haemoglobin. With maturation haemoglobin increases and the number ribosome numbers fall and the cytoplasm changes from blue to pink-red. Cytoplasm of the basophilic normoblasts is blue, adjacent erythrocytes pink-red and that of a polychromatophilic normoblast is a combination of blue and pink-red.

In keeping with nuclear maturation in the erythroid cells the nucleus of the polychromatic normoblasts is smaller and has a chromatin  that shows a greater degree of clumping than that of basophilic normoblast.

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The Erythropoietin Receptor Signalling

23 Dec

Erythropoietin (EPO), a 34kD 166 amino acid polypeptide, is the main regulator of erythrocyte production. It acts via the erythropoietin receptor (EPOR). EPO signalling pathway appears to be critical for survival as no inactivating mutations of EPO or the EPOR are known. Mice with deletion of EPO or EPOR gene die of anaemia at a gestational age of 12-13 days. EPO is anti-apoptotic. The target cell is a stage between CFU-E and pronormoblast. These cells can not survive in the absence of EPO. It has been suggested that EPO has proliferative effects but the evidence is less compelling. The non-erythroid targets of EPO for which therapeutic efficacy of EPO is under evaluation include the heart and the brain.

 


 

Control of EPO Production
Erythropoietin is produced in response to hypoxia by the interstitial fibroblasts of the kidney. Hypoxia increases the levels of factors known as hypoxia inducible factors (HIF). There are three HIFs, HIF-1, HIF-2 and HIF-3. HIF-2 controls erythropoietin production.. HIF-2 has two units, α and β. The levels of the β subunit are constant. The levels of the α vary inversely with oxygen availability and determine the HIF 2 concentration. The α subunit is continuously being synthesized and destroyed. This apparently futile exercise is aimed at mounting a rapid response to hypoxia. Hypoxia prevents destruction of the α subunit allowing an almost instantaneous rise in concentration of HIF 2.

HIF 2α is destroyed by a proteolytic system known as proteasome. Proteasome destroys any peptide tagged for destruction. The tagging is done by transferring multiple molecules of a protein ubiquitin (polyubiquitination) by an enzyme complex known as ubiquitin E3 ligase. This complex consists of pVHL (product of the von Hipple-Lindau tumour suppressor gene), elongins B and C, cullin 2 and ring box 1 (Rbx1). pVHL identifies targets giving it specificity for HIF-1 and HIF-2. HIF-2α is needs to be hydroxylated at proline residues before polyubiquitination. The hydroxylation is brought about at proline residues by HIF prolyl hydroxylase (PHD) with oxygen as one of the substrates. In hypoxic conditions hydroxylation and the subsequent polyubiquitination does not take place. Proteasomal destruction stops and the levels of HIF-2α levels rapidly rise. The molecule translocates to the nucleus where it combines with HIF-2β. The heterodimer (HIF 2) acts on segments of DNA, known as hypoxia response elements, flanking the erythropoietin gene and promotes erythropoietin synthesis. HIF 2 along with HIF 1, which is regulated by mechanisms identical to those regulating HIF 2, promotes the expression of multiple genes of proteins involved in response to hypoxia.


Mechanism of Action

EPO acts via the EPO receptor. The EPO receptor is a homodimer (a dimer made of two similar units). A cascade of molecules that links the receptor to its action. Phosphorylation of tyrosine or serine/threonine residues on the peptides plays an important role receptor signalling. EPO receptor signals by phosphorylation of tyrosine residues. EPO receptor, unlike other receptors like insulin receptor, lacks tyrosine kinase activity. EPO receptor overcomes this limitation by associating with a cytoplasmic kinase, Janus kinase 2 (JAK2). Each peptide of EPO receptor is associated with a JAK2 molecule. Binding of EPO brings about a conformational change in the receptor bringing the two JAK2 moleclues in proximity. The proximity allows transautophosphorylation and activation of the JAK2 kinases. Activated JAK2 phosphorylates tyrosine residues on the receptor forming docking sites for molecules resulting in activation of the following pathways.

  1. Dimerization and translocation of STAT5 to the nucleus where it induces transcription of genes involved in proliferation and cell survival. STAT pathway appears to be the most important pathway for EPO action.
  2. Phophoinositide-3-kinase (PI3-K) mediated induction of several anti-apoptotic proteins e.g. Bcl-2 abd BclX,
  3. Activation of Ras/extracellular-signal-regulated kinase mitogen-activated protein (RAS/Erk/MAP) kinase pathway that sustains proliferation.

The EPO signalling is short lasting and the activation of EPOR returns to normal levels in 30-60 minutes. The cytoplasmic portion of the receptor is polyubiquitinated and degraded by proteasome. The extracellular portion bound to EPO is internalized and degraded. Regulators of cytokine activity can inhibit EPOR.

 

Mutations and Therapeutic Manipulation of EPO Signalling

Acquired (somatic) mutations of the JAK2 kinase are associated with myeloproliferative disease. The JAK2V617F mutation is seen in about 95% of the patients of polycythaemia vera and about half the patients with essential thrombocytosis and idiopathic myelofibrosis. JAK2v617F causes the JAK2 molecule to be constitutively active eliminating the erythropoietin dependence of erythropoiesis. JAK2 exon 12 mutations are found in patients with JAK2V617F negative polycythemia vera. These patients, unlike those with JAK2V617F positive patients, do not show thrombocytosis or leucocytosis. Inherited (germline) mutations in the EPOR, HIF 2α, VHL gene and the PHD gene have been associated with congenital erythrocytosis. The erythrocytosis seen in patients with EPOR mutations is primary, i.e. with low EPO levels. Other mutations result in secondary erythrocytosis, i.e. with inappropriately high EPO levels. Inhibitor of HIF prolyl hydroxylase FG-2216 and FG-4592 are under evaluation for treatment of anaemia associated with chronic kidney disease.

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The Reticulocyte Count

10 Oct

Anaemia induces production of erythropoietin which promotes differentiation of the hemopoietic stem cell along the erythroid lineage. With maturation, the erythroid precursors shrink in size, loose nucleus and haemoglobinize (see Morphology of Erythroid Precurssors). A newly released erythrocyte contains ribosomal RNA that it looses over 24-36 hours. These young erythrocytes are called reticulocytes because the ribosomal RNA gives a reticular appearance when stained by certain supravital stains like new methylene blue.

Anaemia may result from an increased destruction or impaired production of erythrocytes. The former is characterised by an increased erythrocyte production and the latter by an inappropriately low erythrocyte production. The normal life span of the erythrocyte is about 120 days. About 0.8% of the erythrocytes are destroyed everyday. If erythrocyte production ceases completely, a 10% fall in erythrocyte count (also in haemoglobin) would take about two weeks. Though a greater fall would indicate a loss of erythrocytes in the form of haemolysis or acute blood loss, the erythrocyte count is of little in diagnostic value in assessing bone marrow activity accompanying anaemia. The number of the reticulocytes indicates the bone marrow activity in a short period preceding assessment (a day or two) and allows classification anaemia into those resulting from decreased production and increased destruction.

The reticulocyte

On Romanowsky staining, other than polychromasia and a slightly larger size, there are no morphological features to differentiate reticulocytes from other erythrocytes. Differentiation on the basis of size is difficult and all reticulocytes do not show polychromasia. When reticulocytes are stained with new methylene blue or brilliant cresyl blue the ribosomes precipitate as a blue staining reticulum (hence the name reticulocytes). As the staining is done on cells that are not fixed (are live) it is knows as supravital staining. New methylene blue stains the cytoplasm blue-green obviating the need for a cytoplasmic counterstain. The density of the reticulum falls with age of the reticulocyte. The most mature reticulocytes may show only one or two dots. The majority of reticulocytes have a few dots and the precise definition of a reticulocyte has a bearing on the reticulocyte count. The definition of a reticulocyte is any non-nucleated erythroid cell containing two or more bluish–stained material corresponding to ribosomal RNA. The reticulocyte needs to be differentiated from other intracellular inclusions viz. Pappenheimer bodies, Heinz bodies, Howell-Jolly Bodies and Hb H inclusions.

Performing the Reticulocyte Count

The count is performed by mixing 2-3 drops of new methylene blue (or brilliant cresyl blue) with 2-4 volumes of EDTA-anticoagulated blood, allowing the mixture to stand for 15-20mins, making films on a glass slide and examining these when dry under 100X oil immersion. The number of erythrocytes and reticulocytes is counted till at least 100 reticulocytes and a total of 10 oil immersion fields are counted. The reticulocyte count is expressed as follows:

Reticulocyte count = [number of reticulocytes counted]/[number of erythrocytes counted]

The reticulocyte count may also be expressed as absolute reticulocyte count as follows:

Absolute reticulocyte count = [RBC count X Reticulocyte count]/100

The normal reticulocyte count is 0.5-2.5% and the normal absolute reticulocyte count is 50-100X109/L

Correcting the reticulocyte count for anaemia

Anaemia decreases the amount of time the reticulocyte spends in the marrow. The reticulocytes of patients with haematocrits in the range of 45% are estimmated to spend 3.5 days in the marrow and about 1 day in the peripheral blood. At a haematocrit 15% these times are 1.5 and 2.5 days respectively. Decreases RBC count gives a false elevation in reticulocyte count. The haematological parameters of four patients given in the table below highlight the limitations of a reticulocyte count.

Patient A

Patient B

Patient C

Patients D
Haemoglobin

13g/dL

5 g/dL

7.5 g/dL

7.2 g/dL
Erythrocyte Count

4.4 Million/mm3

1.2 Million/mm3

2.5 Million/mm3

3.5 Million/mm3
Haematocrit

40.48%

16.2%

23.25%

25.2%
MCH

29.55 pg

41.67 pg

30 pg

20.57 pg
MCV

92 fl

135 fl

93 fl

72 fl
MCHC

32 g/dL

31 g/dL

32 g/dL

29 g/dL
Reticulocyte Count

1.3%

3%

15%

2%
Absolute Reticulocyte count

57200 /mm3

36000/mm3

375000/mm3

70000/mm3
Corrected Reticulocyte Count

1.17

1.08

7.75

1.12
Reticulocyte Production Index

0.78

0.43

3.1

0.56
  • Low erythrocyte count can cause an false impression of reticulocytosis: The patient B has an erythrocyte count of 1.2 million/mm3, a hematocrit of 16.2% and a reticulocyte count of 3%. The absolute reticulocyte count is 36000/mm3. Patient A has a erythrocyte count of 4.4 million/mm3, a haematocrit of 40.48% and a reticulocyte count of 1.3%. The absolute reticulocyte count is 57200/mm3. Despite having a higher reticulocyte count patient B actually has a lower absolute reticulocyte count and a less active marrow than patient A. Corrected reticulocyte count corrects reticulocyte count for low erythrocyte count and is calculated as follows:

Corrected Reticulocyte Count = Reticulocyte count X (Patinets Haemotocrit/Normal Haemotocrit)

(Haemoglobin or erythrocyte count may be used for the correction instead of haematocrit)

The corrected reticulocyte count in both patients is almost identical.

  • In addition to correcting for erythrocyte count one must correct for a premature release of erythrocytes: Patinet A and patient B have almost identical corrected reticulocyte count. Does this mean they are producing the same number of erythrocytes per day? The reticulocyte spends about 2.5 days in peripheral blood in the patient A and about 1.5 days in the patient B. Only 14,400/mm3 (36,000/number of days reticulocyte spends in peripheral blood) reticulocytes are produced every day in patient B and about 38,000/mm3 produced in patient A. Despite having an almost identical corrected reticulocyte count as patient B, patients A is producing twice as many reticulocytes as patient B. The corrected reticulocyte needs to be further corrected for an early release of erythrocyte in anaemia. This gives the reticulocyte production index as follows

    Reticulocyte Production Index = Corrected Reticulocyte Count X Correction Factor

    The correction factor is 1 for haemotocrit of 40-45%, 1.5 for haemotocrit of 35-40%, 2 for haematocrit of 25-35% and 2.5 for haematocrit of 15-25%.

    A reticulocyte production index of <2 in the presence of anaemia indicates a bone marrow pathology. Patient C, a patient of haemolytic anaemia, and has a RPI of 3.1 indicating a normally responding bone marrow. Patient D is a patients with a hypochromic n=microcytic anaemia with a low RPI. This indicates a impaired erythropoiesis which in the case is most likely to be an iron deficiency anaemia.

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