Information about Hematopoietic Stem Cells

Hematopoietic stem cells (HSC) are stem cells which give rise to all the blood cell types including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells) and lymphoid lineages (T-cells, B-cells, NK-cells). The definition of hematopoietic stem cells has undergone considerable revision in the last two decades. The hematopoietic tissue contains cells with long term and short term regeneration capacities and committed multipotent, oligopotent and unipotent progenitors. Recently, long-term transplantation experiments point towards a clonal diversity model of hematopoietic stem cells. Here, the HSC compartment consists of a fixed number of different types of HSC, each with epigenetically preprogrammed behavior. This contradicts older models of HSC behavior, which postulated a single type of HSC that can be continuously molded into different subtypes of HSC.

Source

HSC are found in the bone marrow of adults, which includes femurs, hip, ribs, sternum, and other bones. Cells can be obtained directly by removal from the hip using a needle and syringe, or from the blood following pre-treatment with cytokines, such as G-CSF (granulocyte colony stimulating factors), that induce cells to be released from the bone marrow compartment. Other sources for clinical and scientific use include umbilical cord blood, placenta, molilized peripheral blood. For experimental purposes, fetal liver, fetal spleen and AGM (Aorta-gonad-mesonephros) of animals are also useful sources of HSCs.

Functional Characteristics

Multipotency and self-renewal

As stem cells, they are defined by their ability to form multiple cell types (multipotency) and their ability to self-renew.

Multipotency: Individual HSC have the ability to give rise to any of the end-stage blood cell types. During differentiation, daughter cells derived from HSC undertake a series of commitment decisions, retaining differentiation potential for some lineages while losing others. Intermediate cells become progressively more restricted in their lineage potential, until eventually lineage-committed end stage cells are generated.

Self-Renewal: Some kinds of stem cells are thought to undertake asymmetric cell division, generating one daughter cell that remains a stem cell and one daughter cell that differentiates. For Hematopoietic Stem Cells, however, whether asymmetric cell division occurs during self-renewal is not known with certainty. It is instead possible that hematopoiesis occurs via symmetrical divisions, that sometimes give rise to two daughter HSC, and that at other times give rise to progeny that are committed to differentiate. The balance between self-renewal versus differentiation would therefore be regulated by the control of these two kinds of symmetrical cell division.

It is known that a small number of HSC can expand to generate a very large number of progeny HSC. This phenomenon is used in bone marrow transplant when a small number of HSC reconstitute the hematopoietic system. This indicates that at least during bone marrow transplant, symmetrical cell divisions that give two progeny HSC must occur, as expansion in HSC numbers seen during bone marrow transplant cannot occur in any other way.

Stem cell self-renewal is thought to occur in the stem cell niche in the bone marrow, and it is reasonable to assume that key signals present in this niche will be important in self-renewal. There is much interest in the environmental and molecular requirements for HSC self-renewal, as understanding the ability of HSC to replenish themselves will eventually allow the generation of expanded populations of HSC ex vivo that can be used therapeutically.

Lineage-Bias

Christa Muller-Sieburg's group at the Sidney Kimmel Cancer Center was the first in 2002 to work out a rationale for the generation of diversity (GOD) in the HSC compartment ^{[1] [2] [3]}. Using limiting dilution strategies combined with other streamlined experimental and statistical methods for examining HSC at the clonal level, it was shown that HSC fall into three distinct clusters. These are quantitatively defined by the ratio ρ of lymphoid to myeloid cells that HSC generate upon differentiation (which makes ρ a peripheral predictor for the clonal association of a reconstituted hematopoietic system). Balanced HSC repopulate peripheral white blood cells in the same ratio of myeloid to lymphoid cells as seen in unmanipulated mice (on average about 15% myeloid and 85% lymphoid cells, or 3≤ρ≤10). Myeloid-biased (My-bi) HSC give rise to too few lymphocytes resulting in ratios 0<ρ<3, while lymphoid-biased (Ly-bi) HSC generate too few myeloid cells which results in lymphoid-to-myeloid ratios of 10<ρ<oo. All three types are normal HSC in that they have self-renewal capacity and can regenerate all hematopietic lineages (pluripotency). Strikingly, the lineage-bias is preserved through multiple rounds of serial transplantation: balanced HSC self-renew to give rise to daughter HSC that are also balanced, My-bi HSC give rise to My-bi daughter HSC, and Ly-bi produce Ly-bi daughter HSC. There is no precursor-progeny relationship between the three types of HSC and they do not represent stages of differentiation. Rather, these are three classes of HSC, each with an epigenetically fixed differentiation program. <br>

Functional Assays

• Cobble stone area forming Cell (CAFC) assay: This is a cell culture based empirical assay. When plated onto a confluent culture of stromal feeder layer, a fraction of HSCs creep between the gaps (even though the stromal cells are touching each other) and eventually settle between the stromal cells and the substratum (here the dish surface) or trapped in the cellular processes between the stromal cells. Emperipolesis is the in vivo phenomenon in which one cell is completely engulfed into another (eg thymocytes into thymic nurse cells); on the other hand, when, in vitro, lymphoid lineage cells creep beneath nurse like cells it is called pseudoemperipolesis. This similar phenomeonon is more commonly known in HSC field by the cell culture terminology cobble stone area forming cells (CFAC) which means areas of cluster of cells which look dull cobblestone-like under phase contrast microscopy, compared to the other HSCs which are refractile. This happens because the cells which are folating loosely on top of the stromal cells are spherical and thus refractile. However, the cells which creep beneath the stromal cells are flattened and thus not refractile. The mechanism of pseudoemperipolesis is only recently coming to light. it may be mediated by interection through CXCR4 (CD184) the receptor for CXC Chemokines (eg SDF1) and α4β1 integrins.^[4].

  Physical characteristics

  Hematopoietic stem cells morphologically resemble lymphocytes. They are non-adherent, rounded, rounded nucleus, and low cytoplasm to nucleus ratio. Since PHSC can not be isolated as a pure population, it is not possible to identify them in a microscope. The above description is based on the morphological characteristics of a heterogeneous population of which PHSC are a component.

  Markers

  Hematopoeitic stem cells are phenotypically identified by their small size, lack of lineage (lin) markers, low staining (side population) with vital dyes such as rhodamine 123 (rhodamine^DULL, also called rho^lo) or Hoechst 33342, and presence of various antigenic markers on their surface many of which belongs to the cluster of differentiation series, like: CD34, CD38, CD90, CD133, CD105, CD45 and also c-kit- the receptor for stem cell factor. The hematopoietic stem cells are negative for the markers which are used for detection of lineage commitment and are thus called Lin-, and during their purification by FACS, a bunch of up to 13 to 14 different mature blood-lineage marker eg CD13 & CD33 for myeloid, CD71 for erythroid, CD19 for B cells, CD61 for megakaryocytic etc for humans; and, B220 (murine CD45) for B cells, Mac-1 (CD11b/CD18) for monocytes, Gr-1 for Granulocytes, Ter119 for erythroid cells, Il7Ra, CD3, CD4, CD5, CD8 for T cells etc for mice) antibodies are used as a mixture to deplete the lin+ cells or late multipotent progenitors (MPP)s.
  
  There are a lot of differences between the human and mice hematopoietic cell markers for the commonly accepted type of hematopoietic stem cells.[1].
  • Mouse HSC : CD34^lo/-, SCA-1⁺ , Thy1.1^+/lo, CD38⁺, C-kit⁺, lin^-
  • Human HSC : CD34⁺, CD59⁺, Thy1/CD90⁺,CD38^lo/-, C-kit^-/lo, lin^-
  However not all stem cells are covered by these combinations which nonetheless have become pupular. In fact even in humans there are hematopoietic stem cells which are CD34^-/CD38^-. ^[5][6]. Also some later studies suggested that earliest stem cells may lack c-kit on the cell surface^[7]. For human HSCs use of CD133 was one step ahead as both CD34⁺ and CD34^- HSCs were CD133⁺.
  
  Traditional purification method used to yield a reasonable purity level of mouse hematopoietic stem cells generally requires a large(~10-12) battery of markers, most of which were surrogate markers with little functional significance and thus partial overlap with the stem cell populations and sometimes other closely related cells which are not stem cells. Also some of these markers 9eg Thy1) are not conserved across mouse species, and use of markers like CD34^- for HSC purification requires mice to be at least 8 weeks old. Alternative methods which could give rise to similar or better harvest of stem cells is a hot area of research and are presently emerging. One such method uses a signature of SLAM family of cell surface molecules. SLAM (Signaling lymphocyte activation molecule) family is a group of >10 molecules whose genes are mostly located tandemly in a single locus on chromosome 1 (mouse), all belonging to a subset of immunoglobulin gene superfamily, and originally thought to be involved in T-cell stimulation. This family includes CD48, CD150, CD244 etc, CD150 being the founding member thus also called slamF1 ie SLAM family member 1. However, this method still needs to be confirmed by other researchers.
  
  The signature SLAM code for the hemapoietic higherchy are:
  • Hematopoietic stem cells (HSC) : CD150⁺CD48^-CD244^-
  • Multipotent progenitor cells (MPPs) : CD150^-CD48^-CD244⁺
  • Lineage-restricted progenitor cells (LRPs) : CD150^-CD48⁺CD244⁺
  For HSCs CD150⁺CD48^- was sufficient instead of CD150⁺CD48^-CD244^- because CD48 is a ligand for CD244 and both would be positive only in the activated lineage restricted progenitors. This code was more efficient than the more tedious earlier set of the large number of markers and are also conserved across the mouse strains.^[8]. CD150⁺CD48^- gave stem cell purity comparable to Thy1^loSca-1⁺lin^-c-kit⁺ in mice.^[9]
  
  Irving Weissman's group at Stanford University that was the first to isolate mouse hematopoietic stem cells in 1988, was also the first to work out the markers to distinguish the mouse long term (LT-HSC) and short term (ST-HSC) hematopoietic stem cells (self renew capable), and the Multipotent progenitors (MPP, low or no self renew capability — the later the developmental stage of MPP, the lesser the self renewal ability and the more of some of the markers like CD4 and CD135):
  • LT-HSC : CD34^-, SCA-1⁺ , Thy1.1^+/lo, C-kit⁺, lin^-, CD135^-, Slamf1/CD150⁺
  • ST-HSC : CD34⁺, SCA-1⁺ , Thy1.1^+/lo, C-kit⁺, lin^-, CD135^-, Slamf1/CD150⁺, Mac-1 (CD11b)^lo
  • Early MPP : CD34⁺, SCA-1⁺ , Thy1.1^-, C-kit⁺, lin^-, CD135⁺, Slamf1/CD150^-, Mac-1 (CD11b)^lo, CD4^lo
  • Late MPP : CD34⁺, SCA-1⁺ , Thy1.1^-, C-kit⁺, lin^-, CD135^high, Slamf1/CD150^-, Mac-1 (CD11b)^lo, CD4^lo

  Nomenclature of hematopoietic colonies and lineages

  Between 1948 and 1950, the Committee for Clarification of the Nomenclature of Cells and Diseases of the Blood and Blood-forming Organs issued reports on the nomenclature of blood cells.^[10][11] An overview of the terminology is shown below, from earliest to final stage of development:
  • [root]blast
  • pro[root]cyte
  • [root]cyte
  • meta[root]cyte
  • mature cell name
  The root for CFU-E is "rubri", for CFU-GM is "granulo" or "myelo" and "mono", for CFU-L is "lympho" and for CFU-Me is "megakaryo". According to this terminology, the stages of red blood cell formation would be: rubriblast, prorubricyte, rubricyte, metarubricyte and finally erythrocyte. However, the following nomenclature seems to be, at present, the most prevalent:
  
  

  Committee	"lympho"	"rubri"	"granulo" or "myelo"	"mono"	"megakaryo"
  Lineage	Lymphoid	Myeloid	Myeloid	Myeloid	Myeloid
  CFU	CFU-L	CFU-E	CFU-GM	CFU-GM	CFU-Me
  Process	lymphocytopoiesis	erythropoiesis	granulocytopoiesis	monocytopoiesis	thrombocytopoiesis
  [root]blast	Lymphoblast	Proerythroblast	Myeloblast	Monoblast	Megakaryoblast
  pro[root]cyte	Prolymphocyte	Polychromatophilic erythrocyte	Promyelocyte	Promonocyte	Promegakaryocyte
  [root]cyte	-	Normoblast	Eosino/neutro/basophilic myelocyte		Megakaryocyte
  meta[root]cyte	Large lymphocyte	Reticulocyte	Eosinophilic/neutrophilic/basophilic metamyelocyte, Eosinophilic/neutrophilic/basophilic band cell	Early monocyte	-
  mature cell name	Small lymphocyte	Erythrocyte	granulocytes (Eosino/neutro/basophil)	Monocyte	thrombocytes (Platelets)

  
  
  Osteoclasts also arise from haemopoietic cells of the monocyte/neutrophil lineage, specifically CFU-GM.

  Colony-forming units

  There are various kinds of colony-forming units:
  • Colony-forming unit lymphocyte (CFU-L)
  • Colony-forming unit erythrocyte (CFU-E)
  • Colony-forming unit granulo-monocyte (CFU-GM)
  • Colony-forming unit megakaryocyte (CFU-Me)
  • Colony-forming unit Basophil (CFU-B)
  • Colony-forming unit Eosinophil (CFU-Eo)
  The above CFUs are based on the lineage. Another CFU, the colony-forming unit–spleen (CFU–S) was the basis of an in vivo clonal colony formation, which depends on the ability of infused bone marrow cells to give rise to clones of maturing hematopoietic cells in the spleens of irradiated mice after 8 to 12 days. It was used extensively in early studies, but is now considered to measure more mature progenitor or Transit Amplifying Cells rather than stem cells.

  References

  1. ^ Muller-Sieburg CE, Cho RH, Thoman M, Adkins B, Sieburg HB, Deterministc regulation of hematopoietic stem cell self-renewal and differentiation. Blood. 2002; 100; 1302-9
  2. ^ Muller-Sieburg CE, Cho RH, Karlson L, Huang JF, Sieburg HB. Myeloid-biased hematopoietic stem cells have extensive self-renewal capacity but generate diminished progeny with impaired IL-7 responsiveness. Blood. 2004; 103:4111-8.
  3. ^ Sieburg HB, Cho RH, Dykstra B, Eaves, CJ, Muller-Sieburg, CE. The hematopoietic stem cell compartment consists of a limited number of discrete stem cell subsets. Blood. 2006; 107:2311-6. Epub 2005 Nov 15.
  4. ^ Burger JA, Spoo A, Dwenger A, Burger M, Behringer D. CXCR4 chemokine receptors (CD184) and alpha4beta1 integrins mediate spontaneous migration of human CD34+ progenitors and acute myeloid leukaemia cells beneath marrow stromal cells (pseudoemperipolesis).

Br J Haematol. 2003 Aug;122(4):579-89. PMID: 12899713
5. ^ Bhatia, M., D. Bonnet, B. Murdoch, O.I. Gan and J.E. Dick, A newly discovered class of human hematopoietic cells with SCID-repopulating activity, 4(9), 1038, 1998.
6. ^ Guo, Yalin , Lubbert, Michael , Engelhardt, Monika CD34- Hematopoietic Stem Cells: Current Concepts and Controversies Stem Cells 2003; 21: 15-20; First published online ; doi:10.1634/stemcells.21-1-15
7. ^ H. Doi et al. (1997) Proc. Natl. Acad. Sci. USA 94, 2513–2517
8. ^ Gary Van Zant Stem cell markers: less is more! Blood 107: 855-856.
9. ^ Kiel et al, Cell, Vol. 121, 1109–1121, July 1, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.cell.2005.05.026
10. ^ (1948) "First report of the Committee for Clarification of the Nomenclature of Cells and Diseases of the Blood and Blood-forming Organs.". Amer J Clin Pathol 18: 443-450. 
11. ^ (1950) "Third, fourth and fifth reports of the committee for clarification of the nomenclature of cells and diseases of the blood and blood-forming organs.". Am J Clin Pathol 20 (6): 562-79. PMID 15432355.