Hematopoietic Stem Cells HSC Mouse Lineagelow Sca 1

Hematopoietic Stem Cells (HSC) Mouse Lineage-/low Sca 1+ c. Kit+ flk 2 CD 34 CD 48 CD 150+ Human Lineage-/low CD 90+ CD 45 RACD 34+ CD 38 CD 49 f+ Functional definition - Repopulate Irradiated Recipients - Generate all Blood cell types (Peripheral Blood myeloid and lymphoid) - Long-term (>4 month) Adapted from {Gazit R, Weissman IL and Rossi DJ 2008} HSC MPP Self. Renew Multipotent

Stem and Progenitors Cells form a Population within the Hematopoietic system HSPC pre. T pro. B MF Tgd Mo B DC NKT GN NK T Unguided transcriptome PCA found clear clustering of cell types

HSC-specific Transcription regulators include known and novel genes – that cluster together

HSC specific Transcription Factors (and other genes) Lymphoid- and Eryth. Myeloid- Progenitors Multipotent Oligo-potent GN Mo. Mac Ost. DC HSC MPP 1 MPP 2 Glis 2 Hoxa 9 Hoxa 5 Rbpms Prdm 16 Zfp 467 Mycn Pbx 1 Irf 6 Ndn Hoxb 5 Rarb Zfp 37 Hlf Meis 1 Msi 2 Zfp 532 Nap 1 l 3 Zfp 521 Zfp 612 B cells NK Lineage-committed and effector cells T Cells

One Transcription Factor, Hlf, induce HSC-markers and sustain multilineages +Zs. Green a +HLF HSC b 100 HLF Zs. Green 75 50 25 1. 1± 0. 4 55. 4± 2. 2 MPPflk 2 - 0 100 HSC MPPflk 2 - 75 50 25 60. 2± 7. 2 2. 3± 0. 4 MPPflk 2+ 0 100 MPPflk 2+ 75 50 25 0. 8± 0. 4 56. 2± 5. 3 0 100 GMP c HSC +ds. Red control GMP 75 50 25 13. 2± 3. 0 12. 4± 12. 1 mac. CMP 0 100 CMP 75 mac. 50 25 6. 0± 2. 3 5 10 prog. CD 11 b gran. MEP 5 10 104 103 2 2 10 0 meg. 22. 1± 10. 3 2 0 10 3 10 4 10 MEP 10 0 5 10 2 0 10 CD 150 FACS at 2 weeks 63. 6± 5. 5 103 104 105 0 100 Percent Lin- CD 150+ HSC mac. +HLF 60. 8± 18. 4 MEP 75 50 25 0 0 1 2 time (weeks) 3

Eight Transcription Factors can reprogram progenitors into long-term repopulating cells 0. 83 CD 45. 1 3. 32 58. 5 40. 4 10. 9 48. 9 66. 4 30. 1 CD 45. 1 Zs. Green (Control) CD 45. 2 With 8 TFs Mac 1 CD 45. 2 0. 21 9. 51 30. 6 CD 4+CD 8+GR 1 Eto Hoxb 5 Hlf Irf 6 Msi 2 Nap 1 l 3 Rbpms Zfp 467

Knockin mouse with Fgd 5 m. Cherry label HSC Pre-gated on Live, doublet-discriminated, BM cells 103 0. 035% 102 99. 9% 0 0 50 K 100 K 150 K 200 K 250 K 104 103 c. Kit 104 105 94% FSC m. Cherry 105 102 0 0 0 102 FSC 103 104 105 98% Gating on m. Cherry+ cells identify HSCs 104 99% CD 48 105 103 102 0 0 102 103 Lineage 104 105 0 102 Sca 1 103 104 105 CD 150 10 Fgd 5 +/m. Cherry 0. 03% 104 m. Cherry+ cells are functional HSCs, and all of the HSCs. 5 100 m. Cherry+ 103 102 99. 9% 0 0 50 K 100 K FSC 150 K 200 K 250 K 100, 000 m. Cherry- Donor chimerism (%) 100 10 1 0. 1 4 8 12 16 20 Weeks Post-Transplant 24